Annulation of substituted anthracene-9,10-diones yields promising selectively antiproliferative compounds

Article abstract of DOI:10.1016/j.ejmech.2013.01.049

Anthraquinone derivatives are well-known antiproliferative compounds, and some are currently used in cancer chemotherapy. Some families of annulated anthraquinone analogs have also been examined for antiproliferative activity, but in this regard almost nothing is known of 1-azabenzanthrones (7H-dibenzo[de,h]quinolin-7-ones). A series of 1-azabenzanthrone derivatives, their 2,3-dihydro analogs, and congruently substituted 9,10-anthracenediones were tested against normal human fibroblasts and four human cancer cell lines. Most of the heterocyclic compounds proved to be weakly to moderately antiproliferative with IC₅₀ values extending down to 0.86 μM, and exhibited up to 30-fold selectivity between cancer and normal cells. Both 1-azabenzanthrones and 1-aza-2,3-dihydrobenzanthrones were more potent than their anthraquinone counterparts, and almost without exception, the 2,3-dihydro compounds were more potent than the fully aromatic 1-azabenzanthrones.

Full text of DOI:10.1016/j.ejmech.2013.01.049

European Journal of Medicinal Chemistry 62 (2013) 688e692
Contents lists available at SciVerse ScienceDirect
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journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Annulation of substituted anthracene-9,10-diones yields promising selectively
antiproliferative compounds
,
b
,
a
a
c
Vicente Castro-Castillo a , Cristian Suárez-Rozas , Natalia Castro-Loiza , Cristina Theoduloz ,
*
Bruce K. Cassels b
a Faculty of Basic Sciences, Metropolitan Educational Sciences University, Avenida J.P. Alessandri 774, Ñuñoa, Santiago 7760197, Chile
b Department of Chemistry, Faculty of Sciences, University of Chile, Las Palmeras 3425, Ñuñoa, Santiago 7800024, Chile
c Cell Culture Laboratory, Faculty of Health Sciences, University of Talca, Talca, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Anthraquinone derivatives are well-known antiproliferative compounds, and some are currently used in
cancer chemotherapy. Some families of annulated anthraquinone analogs have also been examined for
antiproliferative activity, but in this regard almost nothing is known of 1-azabenzanthrones (7H-dibenzo
[de,h]quinolin-7-ones). A series of 1-azabenzanthrone derivatives, their 2,3-dihydro analogs, and con-
gruently substituted 9,10-anthracenediones were tested against normal human fibroblasts and four
human cancer cell lines. Most of the heterocyclic compounds proved to be weakly to moderately anti-
Received 3 December 2012
Received in revised form
18 January 2013
Accepted 22 January 2013
Available online 8 February 2013
proliferative with IC50 values extending down to 0.86 mM, and exhibited up to 30-fold selectivity between
Keywords:
Anthracene-9,10-diones
1-Azabenzanthrones
1-Aza-2,3-dihydrobenzanthrones
Synthetic oxoisoaporphines
Antiproliferative activity
cancer and normal cells. Both 1-azabenzanthrones and 1-aza-2,3-dihydrobenzanthrones were more
potent than their anthraquinone counterparts, and almost without exception, the 2,3-dihydro com-
pounds were more potent than the fully aromatic 1-azabenzanthrones.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
not been studied to a comparable extent, particularly considering the
many publications and patents related to their chemistry as in-
Antiproliferative anthraquinones such as ametantrone and its
dihydroxy derivative mitoxantrone are historically important anti-
tumor drugs whose usefulness is limited by toxicity concerns, as is also
the case with the structurally more complex anthracyclines. One
approach that has been explored to overcome such problems is the
annulation of anthraquinones or their aza analogs, which might show
reduced cardiotoxicity and at the same time be less susceptible to
multidrug resistance. Recent examples of these families of compounds,
preceded more than two decades ago by the anthrapyrazoles [1], are
the 7-oxo-7H-dibenz[f,ij]isoquinolines (3-azabenzanthrones), 7-oxo-
7H-benzo[e]perimidines (1,3-diazabenzanthrones), 3-azabenzanthra-
2,7-diones (anthrapyridones) and 1,2-diazabenzanthra-3,7-diones
(anthrapyridazones) (Fig. 1) [2e7].
termediates for the manufacture of dyes and pigments. It is also of
interest to note that the 1-azabenzanthrone skeleton occurs natu-
rally in a small group of alkaloids, limited thus far to the family
Menispermaceae, where they are generally termed “oxoisoapor-
phines”. Moreover, a couple of these compounds isolated from
Menispermum dauricum DC, bearing a nitrogen substituent on the
polycyclic scaffold (daurioxoisoaporphines, Fig. 2), are toxic to sev-
eral cell lines and are claimed to be more potent against human
mammary cancer cells than the widely used etoposide or VP-16 [8].
We have recently shown that another aminooxoisoaporphine
(lakshminine, 6 from Sciadotenia toxifera Krukoff & A.C. Sm.) is
weakly toxic, as are one of its unnatural positional isomers and their
nitro precursors [9]. In connectionwith possible anticanceractivities,
the only other exception seems to be a recent paper describing the
It is somewhat surprising that the 1-azabenzanthrones (7H-
dibenzo[de,h]quinolin-7-ones), which are closely related to anthra-
cene-9,10-diones and the above-mentioned annulated analogs, have
synthesis, DNA binding and cytotoxicity of
aminoalkanamido-1-azabenzanthrones [10].
a number of 9-
As an initial attempt to fill this gap, we have synthesized a more
extensive series of 1-azabenzanthrones, their 2,3-dihydro analogs,
and 9,10-anthracenediones with congruent substitution patterns
(Fig. 3), and determined their toxicities versus normal human fi-
broblasts and four human cancer cell lines.
* Corresponding author. Faculty of Basic Sciences, Metropolitan Educational Sci-
ences University, Avenida J.P. Alessandri 774, Ñuñoa, Santiago 7760197, Chile.
E-mail addresses: vicente.castro@umce.cl, vicecastro@gmail.com (V. Castro-
Castillo).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.049

V. Castro-Castillo et al. / European Journal of Medicinal Chemistry 62 (2013) 688e692
689
H
N
OH
R
N
N
N
R
R
O
O
NH
NH
O
OH
O
N
H
ametantrone: R = H
mitoxantrone: R = OH
3-azabenzanthrones
R
anthrapyrazoles
O
R
N
O
N
N
N
N
O
O
O
1,3-diazabenzanthrones
anthrapyridones
anthrapyridazones
Fig. 1. Some structurally related families of antiproliferative drugs.
2. Results and discussion
molecular bromine in acetonitrile afforded 10, while the similar
reaction of 2 gave a mixture of 11 and 12 [13].
2.1. Synthesis
9,10-Anthraquinone 1B was obtained commercially, and
anthracene-9,10-diones 2Be9B were prepared following well-
established routes. Thus, 2-methoxyanthracene-9,10-dione (2B)
was obtained by nucleophilic substitution on the 2-chloro analog
[14], 3B and 4B were obtained by methylation of alizarin [15,16],
and the nitro and amino derivatives (5Be9B) were prepared by
similar routes to those used for the same modifications of the 1-
azabenzanthrones and their dihydro analogs [17].
The 1-azabenzanthrones and their 2,3-dihydro derivatives, mostof
them described previously, required considerably more synthetic
effort. Thus, 1A was obtained starting from N-(2-phenylethyl)phtha-
limide which was reduced and cyclized to afford 5,6,8,12b-tetrahy-
droisoindolo[1,2-a]isoquinolin-8-one. This, in turn, was air-oxidized
to its 12bhydroxyderivative, which byring-openingandesterification
with dimethyl sulfate afforded 1-(2-methoxycarbonylphenyl)-3,4-
dihydroisoquinoline, cyclized in sulfuric acid to yield 1A [11]. 1 was
prepared by Pd-catalyzed dehydrogenation of 1A with air, by analogy
with the oxidation of 2A to 2 [12]. 2A, 3A and 4A were obtained
via cyclodehydration of appropriately substituted N-phenyl-
ethylphthalimidines with polyphosphoric acid, and 2, 3 and 4 were
prepared by the above-mentioned dehydrogenation reaction [12].
Nitration of 2A with nitricesulfuric acid mixture in trifluoro-
acetic acid gave 7A and 8A. Amines 5 and 6 were prepared by
reduction of 7 and 8, respectively, with sodium sulfide [9]. Attempts
to prepare the amino-2,3-dihydro analogs 5A and 6A failed, as the
products formed initially by similar reduction of the nitrated 7A
and 8A were rapidly oxidized in contact with air.
2.2. Pharmacology
The effects of these compounds on cell proliferation were deter-
mined using the MTT reduction assay in five different human cell
lines (MRC-5: normal lung fibroblasts; AGS: gastric adenocarcinoma
cells; SK-MES-1: lung cancer cells; J82: bladder carcinoma; and HL-
60: promyelocytic leukemia cells). The concentrations of the com-
pounds inhibiting cell growth by 50% (IC50 values) were obtained
adjusting the doseeresponse curves to a sigmoidal model.
Tables 1e3 show that some of the compounds in these series
exhibit IC50 values of 4 mM or less, suggesting that they may be of
interest for further development. Their selective toxicities toward
gastric and bladder cancer cells vis-à-vis normal fibroblasts are also
promising and, in the case of bladder carcinoma, superior to that of
the reference drug (etoposide) (Table 4).
Dinitration of 2A to afford 9A required the use of sulfuric acid as
solvent. 7, 8 and 9 were obtained by dehydrogenation of the 2,3-
dihydro analogs (7A, 8A and 9A), as before. Bromination of 1 with
Comparing Tables 1 and 3, it can be seen that annulation of the
anthracene-9,10-dione skeleton to 1-azabenzanthrones generally
leads to increased antiproliferative potency, with the marked
exception of 1,3-dinitro-2-methoxyanthracene-9,10-dione (9B),
which is fairly toxic to all five cell lines, especially HL-60. Moreover,
reduction of these 1-azabenzanthrones to their 2,3-dihydro de-
rivatives (Tables 1 and 2) leads to lower IC50 values vs. cancer cells,
i.e. higher potency, in almost every case. Table 4 shows that some of
2
3
1N
N
O
11
8
4
OCH3
OCH3
10
9
H3CO
5
H3CO
OCH3
6
7
NH
O
NH2
the new compounds are not only antiproliferative with IC50 <10 mM
(Tables 1e3) but some are also more selective than etoposide vs.
normal fibroblasts. Of particular interest are the 1-aza-2,3-
dihydrobenzanthrones 1A, 2A and 3A, which exhibit comparable
potencies to etoposide in gastric adenocarcinoma and bladder
carcinoma cells, plus similar to considerably greater selectivity vs.
OH
daurioxoisoaporphine A
daurioxoisoaporphine B
Fig. 2. Structures and numbering scheme of daurioxoisoaporphines A and B.

690
V. Castro-Castillo et al. / European Journal of Medicinal Chemistry 62 (2013) 688e692
R4
N
C
O
O
N
A
R3
R2
R3
R2
R3
R2
D
B
R1
R1
O
R1
O
1
2
3
4
5
6
7
8
9
R1 = R2 = R3 = R4 = H
1A R1 = R2 = R3 = H
1B R1 = R2 = R3 = H
R1 = H; R2 = OMe; R3 = H; R4 = H
R1 = OH; R2 = OMe; R3 = R4 = H
R1 = R2 = OMe; R3 = R4 = H
2A R1 = H; R2 = OMe; R3 = H
3A R1 = OH; R2 = OMe; R3 = H
4A R1 = OMe; R2 = R3 = H
2B R1 = H; R2 = OMe; R3 = H
3B R1 = OH; R2 = OMe; R3 = H
4B R1 = R2 = OMe; R3 = H
R
1 = H; R2 = OMe; R3 = NH2; R4 = H
5A
R
1 = H; R2 = OMe; R3 = NH2
5B R1 = H; R2 = OMe; R3 = NH2
6B R1 = NH2 ; R2 = OMe; R3 = H
7B R1 = H; R2 = OMe; R3 = NO2
8B R1 = NO2 ; R2 = OMe; R3 = H
R1 = NH2; R2 = OMe; R3 = R4 = H
R1 = H; R2 = OMe; R3 = NO2; R4 = H
R1 = NO2; R2 = OMe; R3 = R4 = H
6A R1 = NH2; R2 = OMe; R3= H
7A R1 = H; R2 = OMe; R3 = NO2
8A R1 = NO2; R2 = OMe; R3 = H
R1 = NO2; R2 = OMe; R3 = NO2; R4 = H 9A R1 = NO2; R2 = OMe; R3 = NO2 9B R1 = NO2; R2 = OMe; R3 = NO2
10 R1 = R2 = R3 = H; R4 = Br
11 R1 = H; R2 = OMe; R3 = H; R4 = Br
12 R1 = H; R2 = OMe; R3 = Br; R4 = H
Fig. 3. Structures of 1-azabenzanthrones, 1-aza-2,3-dihydrobenzanthrones and similarly substituted anthracene-9,10-diones.
normal cells. The brominated 1-azabenzanthrone 11 is also note-
worthy, as it is quite active vs. stomach, lung and bladder cancer
cells in culture, again with similar or greater selectivity than
etoposide.
negligible effects on the tumor cell lines, while amination at C6 (6)
results in modest antiproliferative activity in all the tested cell lines
with no significant selectivity [9]. The 2,3-dihydro compounds 5A
and 6A were not tested because they could not be isolated in
adequate purity, as described above.
Although 1-aza-5-methoxybenzanthrone (2) is practically
inactive, its 4-nitro derivative (7) is quite potent vs. normal and
tumor cell lines, while its 6-nitro isomer (8) is less so, and the 4,6-
dinitro analog (9) exhibits intermediate activity in all cell lines. The
correspondingly nitrated 2,3-dihydro compounds (7A, 8A and 9A)
show a similar trend to the fully aromatic 7, 8 and 9. However, the
non-nitrated 2A somewhat resembles the 4-nitro 7A in its activ-
ities, with the important difference that the nitrated compound is
several-fold more toxic to normal cells. These results may be
compared with the behavior in the anthraquinone series, where the
inactive 2B acquires fairly weak activities on mononitration at C3
(7B), but not at C1 (8B). However, anthraquinone 2B becomes quite
The recently described series of antiproliferative 9-
aminoalkanamido-1-azabenzanthrones is suggested to act pri-
marily via intercalation between DNA base pairs, as is believed to be
the case with planar anthracene-9,10-diones such as ametantrone
and mitoxantrone [10]. A recent paper describes the X-ray crys-
tallographic structure of 1-aza-6-[(2-hydroxyethyl)amino]benzan-
throne, in which the tetracyclic core is quite flat and in addition
shows a stacking interaction in the crystal which mimics the
stacking interactions of molecules that intercalate between DNA
base pairs [18]. Besides, the (protonated) amino side chains of these
anthraquinones and 1-azabenzanthrones are expected to enhance
DNA binding by interacting with the negatively charged phosphate
residues of the nucleic acid [10]. Our active compounds, which are
at least as potent as the 9-substituted 1-azabenzanthrones or more
so, lack amino side chains. Besides, they do not have the perfectly
flat ring system of the wholly aromatic 1-azabenzanthrones and
anthraquinones, suggesting that DNA intercalation might not be
favored to the same extent.
Table 1
In vitro activity (IC50, mM) of 1-azabenzanthrones (boldface numbers) vs. normal
human fibroblasts (MRC-5), gastric adenocarcinoma (AGS), lung cancer (SK-MES-1),
bladder carcinoma (J82), and myelocytic leukemia (HL-60) cells. Each concentration
was tested in quadruplicate and repeated three times in separate experiments.
Values are expressed as means ꢀ S.E.M.
The 1-aza-2,3-dihydrobenzanthrone molecules are expected to
depart from strict planarity, and indeed the X-ray diffraction
structures of 2A and 3A, which are among the most potent of this
series, clearly show the nonplanarity of ring A in the crystals
[19,20]. Although this feature does not preclude DNA intercalation,
it would seem to make it less likely. Nevertheless, the similarly
nonplanar aporphine alkaloid dicentrine inhibits topoisomerase II
and is active in a DNA unwinding assay, leading to the proposal that
in complex with biological molecules this alkaloid might adopt
a slightly less stable planar conformation as an “adaptive” DNA
intercalator [21], which could also be the case with our compounds.
It may be pointed out that both 2A and 3A have been shown to
undergo reversible electrochemical reduction, via a semiquinone
radical, to their 4-aminophenol analogs [22]. Autoxidation of these
reduced forms could lead to the unsurmountable generation of
reactive oxygen species via redox cycling, a well-established
mechanism of cell toxicity [23]. If this were so, the 1-aza-2,3-
dihydrobenzanthrones might be acting as mitochondrial poisons,
interfering with electron transfer and causing the disruption of
these cellular power plants.
R4
N
R3
R2
O
R1
Compound IC50
MRC-5
41.6 ꢀ 2.9 32.9 ꢀ 1.6
(mM)
AGS
SK-MES-1
J82
HL-60
1
2
3
4
5
6
7
8
9
10
11
12
>100
>100
>100
>100
>100
93.6 ꢀ 5.7
>100
e
e
e
e
e
e
e
e
>100 >100
65.1 ꢀ 3.9 45.5 ꢀ 2.7
>100 >100
62.6 ꢀ 3.8
>100
37.0 ꢀ 1.9 >100
>100
17.9 ꢀ 0.7 78.6 ꢀ 3.9
10.2 ꢀ 0.5 4.5 ꢀ 0.3
80.0 ꢀ 4.8 47.7 ꢀ 2.9
41.8 ꢀ 2.7 36.3 ꢀ 2.5
43.3 ꢀ 3.4 37.6 ꢀ 2.6
17.1 ꢀ 1.0 25.4 ꢀ 1.3
>100
48.3 ꢀ 2.4
55.6 ꢀ 3.3 76.6 ꢀ 6.3 22.2 ꢀ 1.3
>100
>100
>100
>100
2.4 ꢀ 0.1
e
28.9 ꢀ 2.0 3.3 ꢀ 0.2
9.2 ꢀ 0.5
e
>100
3.9 ꢀ 0.2
94.9 ꢀ 6.1
73.9 ꢀ 4.4 >100
e
We had shown previously that the introduction of an amino
group at C4 (5) leads to some toxicity to normal fibroblasts, but
Etoposide
0.36 ꢀ 0.02 2.5 ꢀ 0.2
2.8 ꢀ 0.2
1.8 ꢀ 0.1

V. Castro-Castillo et al. / European Journal of Medicinal Chemistry 62 (2013) 688e692
691
Table 2
Table 4
In vitro activity (IC50
,
m
M) of 1-aza-2,3-dihydrobenzanthrones (numbers followed by
Selectivity ratios (based on IC50 values) of some 1-azabenzanthrones (boldface
numbers), 1-aza-2,3-dihydrobenzanthrones (numbers followed by letter A) and
anthracene-9,10-diones (numbers followed by letter B), and etoposide, comparing
their activities vs. gastric adenocarcinoma (AGS), lung cancer (SK-MES-1), bladder
carcinoma (J82), and myelocytic leukemia (HL-60) cells with their activities vs.
normal human fibroblasts (MRC-5). IC50 (MRC-5)/IC50 (tumor cell line) ꢂ1 is nonselective.
letter A) vs. normal human fibroblasts (MRC-5), gastric adenocarcinoma (AGS), lung
cancer (SK-MES-1), bladder carcinoma (J82), and myelocytic leukemia (HL-60) cells.
Each concentration was tested in quadruplicate and repeated three times in separate
experiments. Values are expressed as means ꢀ S.E.M.
N
Compound
IC50 (MRC-5)/IC50 (tumor cell line)
R3
AGS
SK-MES-1
J82
HL-60
1A
2A
3A
7
7A
9B
11
Etoposide
19
13
11
2.3
2.1
1.0
8.8
11
1.0
4.7
0.6
0.6
0.5
0.7
3.1
1.6
33
28
7.3
0.4
0.5
0.6
12
1.4
e
R2
e
O
R1
e
e
e
Compound IC50 (mM)
2.8
e
MRC-5
AGS
SK-MES-1 J82
HL-60
1A
2A
3A
4A
28.0 ꢀ 1.7
85.2 ꢀ 5.1
42.4 ꢀ 2.6
1.5 ꢀ 0.1
6.8 ꢀ 0.3
3.9 ꢀ 0.2
28.9 ꢀ 2.0 0.86 ꢀ 0.04
e
2.2
18.2 ꢀ 0.4
73.8 ꢀ 4.6
3.0 ꢀ 0.2
5.8 ꢀ 0.4
e
e
reaction of 11 with a cellular nucleophile might explain this un-
expected result, either due to damage to some essential component
of the cell or to the generation of a more toxic 1-azabenzanthrone.
50.5 ꢀ 3.0 62.1 ꢀ 3.7
17.2 ꢀ 0.9 33.9 ꢀ 2.4
e
5A
e
e
e
e
e
e
e
e
6A
e
e
7A
8A
11.7 ꢀ 0.6
5.7 ꢀ 0.3
23.0 ꢀ 1.4 22.9 ꢀ 1.7
>100
22.8 ꢀ 1.6
25.7 ꢀ 1.8 17.5 ꢀ 1.1
3.9 ꢀ 0.2 0.36 ꢀ 0.02 2.5 ꢀ 0.2 2.8 ꢀ 0.2
e
65.6 ꢀ 4.6 18.4 ꢀ 1.1
36.4 ꢀ 2.2 33.3 ꢀ 1.8
e
9A
Etoposide
19.5 ꢀ 1.9
1.8 ꢀ 0.1
2.3. Conclusion
In summary, our preliminary exploration of the 1-
azabenzanthrone scaffold in regard of its antiproliferative properties
has uncovered several compounds that are sufficiently potent to
warrant further study, with IC50 values in the same range as the
important anticancer drug etoposide, and, in comparison with the
normal fibroblastline, withupto30-foldselectivityfortheJ82bladder
carcinoma cell line, for which etoposide is practically unselective.
toxic to all cell lines upon introduction of two nitro groups at these
two positions (9B). These results might be related to the hypo-
thetically different ease of reduction of the nitro groups to anion
radical species, as in a number of heterocyclic nitro compounds
used or tested as antiparasitic drugs [24]. Nevertheless, lacking
electrochemical studies, such a proposal can only be tentative.
Intriguingly,
bromination
of
the
inactive
1-aza-5-
3. Experimental section
methoxybenzanthrone (2) at C3 to generate 11, but not at C4, giv-
ing 12, leads to a considerable increase in potency, affording one of
the most potent analogs, and by far the most potent (11) of the fully
aromatic compounds of this series. The lipophilicity of the isomeric
11 and 12 should be almost identical, practically ruling out the
hypothesis that differential cell penetration and/or subcellular
distribution play a role in their different activities. Therefore, the
electronic structures of these isomers might be considered. A bro-
mine atom at the same position of 1-aza-2-methoxybenzanthrone
is susceptible to nucleophilic substitution [25], and therefore
3.1. Chemistry
3.1.1. General experimental procedures
All reagents and solvents were commercially available from
SigmaeAldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany)
and were used without further purification. Melting points are un-
corrected and were determined with a Reichert Galen III hot-plate
microscope equipped with a DUAL JTEK Dig-Sense thermocouple
thermometer. 1H and 13C NMR spectra were recorded at 300 or
400 MHz and 75 or 100 MHz, respectively, on Bruker Avance 300 or
AMX 400 spectrometers, using CDCl3, D2O or DMSO-d6 as solvent.
Table 3
In vitro activity (IC50, mM) of anthracene-9,10-diones (numbers followed by letter B)
vs. normal human fibroblasts (MRC-5), gastric adenocarcinoma (AGS), lung cancer
(SK-MES-1), bladder carcinoma (J82), and myelocytic leukemia (HL-60) cells. Each
concentration was tested in quadruplicate and repeated three times in separate
experiments. Values are expressed as means ꢀ S.E.M.
The chemical shifts are reported as d (ppm) downfield from TMS for
1H NMR and relative to the central DMSO-d6 resonance (39.5 ppm)
for 13C NMR. Coupling constants (J) are given in Hz. EIMS were run on
a Thermo Finnigan MAT 95XP instrument, with electron impact
ionization at 70 eV and with perfluorokerosene as reference. Purities
of the compounds subjected to biological testing were >95% in every
case (quantitative 1H NMR or HPLC). Elemental analyses were done
with a Fisons Instruments EA-1108 apparatus. Most of the syntheses
are either classical or have recently been described in detail [13].
O
R3
R2
O
R1
Compound IC50 (mM)
3.1.1.1. 2,3-Dihydro-5-methoxy-4,6-dinitro-7H-dibenzo[de,h]quino-
lin-7-one (1-aza-5-methoxy-4,6-dinitrobenzanthrone, 9A). To a so-
lution of 2A (0.5 g, 1.9 mmol) in concentrated H2SO4 (2 mL), H2SO4/
HNO3 1:1 (5 mL) was added carefully, stirring at room temperature
for 2 h. The reaction mixture was poured into water (50 mL), made
alkaline with NaOH(aq) (pH 8e9) and extracted with CH2Cl2. The
CH2Cl2 layer was washed with water and dried over Na2SO4. The
solvent was evaporated leaving a dark brown residue that was
chromatographed on silica gel (AcOEt) to afford 9A 0.407 g (61%);
MRC-5
AGS
SK-MES-1
J82
HL-60
1B
2B
3B
4B
5B
6B
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.9 ꢀ 3.4
56.8 ꢀ 3.4 42.9 ꢀ 3.9 >100
>100 >100 >100
78.4 ꢀ 4.9 62.0 ꢀ 4.2 35.6 ꢀ 2.5
>100 >100 >100
15.4 ꢀ 1.0 18.1 ꢀ 1.5 4.1 ꢀ 0.3
0.36 ꢀ 0.02 2.5 ꢀ 0.2 2.8 ꢀ 0.2 1.8 ꢀ 0.1
>100
7B
8B
9B
Etoposide
95.0 ꢀ 5.5 82.5 ꢀ 5.8
>100 >100
11.4 ꢀ 0.7 11.7 ꢀ 0.8
3.9 ꢀ 0.2
Mp 170e171 ꢁC; 1H NMR (CDCl3)
3.97 (3H, s, OCH3), 4.21 (2H, t, J ¼ 7.8 Hz, H3 or 2), 7.80 (1H, t,
d
2.94 (2H, t, J ¼ 7.9 Hz, H2 or 3),

692
V. Castro-Castillo et al. / European Journal of Medicinal Chemistry 62 (2013) 688e692
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d, 4.16
(3H, s, OCH3), 7.58 (1H, d, J ¼ 5.9 Hz, H3), 7.73 (1H, t, J ¼ 7.8 Hz, H11),
7.89 (1H, t, J ¼ 7.6 Hz, H10 or 9), 8.33 (1H, t, J ¼ 7.1 Hz, H9 or 10), 8.89
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ꢀ
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3.2. In vitro testing
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Minimum Essential Medium, containing 2 mM
sodium pyruvate and 1.5 g/L NaHCO3.
L-glutamine, 1 mM
AGS cells were grown in Ham’s F-12 medium supplemented with
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For the assay, cells were plated in 96-well plates (100 mL/well) at
a density of 5 ꢃ 104 cells/mL. One day after seeding, the cells were
treated with the medium containing the compounds at concentra-
tions ranging from 0 to 100 mM, first dissolved in DMSO (final con-
centration of 1%), diluted with complete medium, and incubated for
72 h in a humidified incubator with 5% CO2 in air at 37 ꢁC, after which
the MTT reduction assay was performed as described previously
[26]. Etoposide (98% purity, SigmaeAldrich, St. Louis, MO, USA) was
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times in quadruplicate. The IC50 value was obtained adjusting the
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a þ ðb ꢄ aÞ=1 þ 10ðxꢄcÞ ; where c ¼ log IC50
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Acknowledgments
V.C.-C. is the recipient of a MeceSup (UMCE-0204) fellowship.
This work was supported by CONICYT grant AT-23070040 and ICM
grant P05-001-F.
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