Studies on quinones. Part 47. Synthesis of novel phenylaminophenanthridinequinones as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2011.05.003

In our search for potential anticancer agents, a series of 8- and 9-phenylamino-3,4-tetrahydro-phenanthridine-1,7,10(2H)-triones with substituent variations at 6-, 8- and 9-positions were prepared using a highly efficient sequence involving: a) solar photoacylation reactions of benzoquinone with arylaldehydes, b) one-pot procedure for the synthesis of 3,4- dihydrophenanthridine-1,7,10(2H)-trione intermediates from acylhydroquinones and c) highly regiocontrolled acid-induced amination reaction of phenanthridinequinones with phenylamines. The members of this series were in vitro evaluated using the MTT colorimetric method against one normal cell line and three human cancer cell lines. The SAR analysis indicates that the location of nitrogen substituents on the quinone nucleus, the presence of methyl, phenyl, furyl and thienyl groups at the 6-position and the aromatization of the angular cycloaliphatic ring of the phenylamino-3,4-tetrahydrophenanthridine-1,7,10(2H)- trione pharmacophore play key roles in the antitumor activity.

Full text of DOI:10.1016/j.ejmech.2011.05.003

European Journal of Medicinal Chemistry 46 (2011) 3398e3409
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Studies on quinones. Part 47. Synthesis of novel
phenylaminophenanthridinequinones as potential antitumor agentsq
,
d
,
a
b
,
,d
Jaime A. Valderrama a , Andrea Ibacache , Jaime A. Rodriguez 1, Cristina Theoduloz b, Julio Benites c
*
a Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago, Chile
b Facultad de Ciencias de la Salud, Universidad de Talca, Chile
c Departamento de Ciencias Químicas y Farmacéuticas, Universidad Arturo Prat, Casilla 121, Iquique, Chile
d Instituto de Etno-Farmacología (IDE), Universidad Arturo Prat, Casilla 121, Iquique, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
In our search for potential anticancer agents, a series of 8- and 9-phenylamino-3,4-tetrahydro-phe-
nanthridine-1,7,10(2H)-triones with substituent variations at 6-, 8- and 9-positions were prepared using
a highly efficient sequence involving: a) solar photoacylation reactions of benzoquinone with arylalde-
hydes, b) one-pot procedure for the synthesis of 3,4-dihydrophenanthridine-1,7,10(2H)-trione interme-
diates from acylhydroquinones and c) highly regiocontrolled acid-induced amination reaction of
phenanthridinequinones with phenylamines. The members of this series were in vitro evaluated using
the MTT colorimetric method against one normal cell line and three human cancer cell lines. The SAR
analysis indicates that the location of nitrogen substituents on the quinone nucleus, the presence of
methyl, phenyl, furyl and thienyl groups at the 6-position and the aromatization of the angular cyclo-
aliphatic ring of the phenylamino-3,4-tetrahydrophenanthridine-1,7,10(2H)-trione pharmacophore play
key roles in the antitumor activity.
Received 19 January 2011
Received in revised form
20 April 2011
Accepted 2 May 2011
Available online 12 May 2011
Keywords:
Aminophenanthridinequinones
Regioselectivity
Cytotoxicity
Half-wave potentials
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
new N-heterocyclic quinones substituted with alkylamino- and
arylamino groups on the quinone ring. In the frame of this
There are several anticancer agents that contain the quinoid
moiety in their structures. Because of the presence of this electro-
active unit, these compounds can undergo a biochemical reduction
by one or two electrons that are catalyzed by flavoenzymes in the
organism using NADPH as an electron donor. This process leads to
semiquinone radical intermediates and subsequent reactions with
oxygen, all of which are believed to be responsible for most of the
drug activity [2e4].
Among the broad variety of N-heterocyclic quinones with anti-
cancer activity there are examples of naturally occurring amino-
quinones containing the isoquinolinequinone scaffold such as
cribrostatin 3 [5], caulibugulone A [6] and mansouramycin C [7]
(Fig. 1).
objective we have reported a high yield synthesis of sub-
stituted aminoisoquinoline-5,8-quinones, by acid-induced amina-
tion reaction of a substituted isoquinolinequinone with alkyl- and
arylamines [8]. The screening of these compounds on cancer cell
lines demonstrates that these aminoisoquinolinequinones express
in vitro cytotoxic activity against gastric, lung and bladder cancer
cell lines. Also, the quantitative structure-activity relationship
(QSAR) analysis of the arylaminoisoquinolinequinones reveals
that the half wave potential and the lipophilicity are important
parameter determining the antitumoral activity on gastric adeno-
carcinoma and bladder carcinoma cells. We have also reported the
synthesis of a variety of aminopyrimido[4,5-c]isoquinolinequinone
derivatives using acid-induced nucleophilic substitution reactions
of pyrimido[4,5-c]isoquinolinequinones with amines [1,9]. The
antitumor evaluation of these aminoquinones and their precursors
on cancer cells indicates, that the insertion of the nitrogen
substituents on the quinone ring of the aminopyrimido[4,5-c]iso-
quinolinequinone pharmacophore increases the cytotoxic potency
in almost all the evaluated cell lines. The structure-activity rela-
tionship (SAR) study reveal that both the nature of the nitrogen
substituent into the quinone ring and the methyl group at the
6-position play key roles in the antitumor activity [9].
Based on this structural feature we are developing a research
program directed to the synthesis and antitumor evaluation of
q
For Part 46 of this series see Ref. [1].
* Corresponding author: Pontificia Universidad Catolica de Chile, Facultad de
Química, Vicuña Mackenna, 4860 Macul, Santiago, Chile. Tel.: þ56 02 6864432;
fax: þ56 02 6864744.
E-mail address: jvalderr@uc.cl (J.A. Valderrama).
In memorial to Dr. Jaime A. Rodríguez.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.003

J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
3399
Me
by acid-induced amination reaction of phenanthridinequinones 3a
and 3b with p-nitroaniline and 4-aminoacetophenone. However,
no reaction of these weak nucleophiles with the quinones 3a and
3b were observed even after long period of reflux of the reaction
mixtures.
O
N
O
O
O
O
O
MeNH
Me
O
MeNH
Me
N
N
H2N
CO2Me
O
The structure of the new compounds was established on the
basis of their nuclear magnetic resonance (1H NMR, 13C NMR, 2D
NMR) and high resolution mass spectra (HRMS). The position of the
nitrogen substituent in these aminoquinones was determined by
means of HMBC experiments. For example, the location of the
cribrostatin 3
mansouramycin C
caulibugulone A
Fig. 1. Structures of naturally occurring isoquinolinequinones with antitumor activity.
3
nitrogen group at C-8 in compound 4a was deduced by the JC,H
Recently we have described preliminary results on the reaction
of phenanthridine-7,10-quinones 3a and 3b with amines that
provides a regioselective access to 8- and 9-amino-3,4-tetrahy-
drophenanthridine-1,7,10(2H)-triones derivatives and the signifi-
cant antitumor activity observed on some of these members [10].
These results, which suggest that derivatives of this aminoquinone
scaffold might be good candidates as antitumor compounds,
encouraged us to explore the scope of the amination reaction of
various substituted aminophenanthridine-7,10-quinones and the
SAR analysis of the series.
During the preparation of this manuscript, the synthesis
and cytotoxic evaluation of a number of anilinophenanthridine-
quinones, prepared from the proper phenanthridine-7,10-quinone,
have appeared. The reported aminoquinones display potent cyto-
toxic activity on breast (MCF-7), lung (NCI-H460, A549), brain
(SF268), prostate (DU145), and epothilone-resistant ovarian (A8)
cancer cell lines [11].
couplings between the carbon at C-7 (
d
181.42 ppm), with the
7.41). In
protons at C-6 ( 9.26), C-9 ( 6.43) and that of NH group (d
d
d
the case of aminoquinone 10, the location of the nitrogen substit-
uent at C-8 was established by 3JC,H coupling between the carbon at
C-7 (
group (
group at C-6 (
d
181.58) with the proton at C-9 (
7.69), and by 4JC,H coupling with the protons of the methyl
2.94).
d 6.38), the proton of the NH
d
d
The results show that the amination reaction of quinone 3a
provides access to a mixture of aminophenanthridinequinones
containing the nitrogen substituent at 8- and 9-position. According
to the ratio between the regioisomers, the reaction of 3a with
the amines proceeds with regioselective preference to give the
8-substituted regioisomer as the main product. Concerning the
6-substituted-aminoquinones 3bee, the results demonstrate that
the amination reaction proceed in high yields and under a regio-
specific manner to give the corresponding aminoquinones sub-
stituted at 8-position.
On the basis of the results reported by Pratt [14] on the catalytic
action of a Lewis acid, such as cerium chloride, to promote regio-
selective 6-amination reactions of quinoline-5,8-quinone, we
assume that the effect of the CeCl3$7H2O catalyst to induce
a favorable attack of the amines at C-8 in quinones 3aee may be
ascribed to coordination of the cerium ion to the heterocyclic
nitrogen atom and/or the carbonyl group at the C-10. The coordi-
nation strongly enhances the electron-withdrawing capacity of the
carbonyl group at the C-10, which is transferred to the 8-position,
leading to preferential C-8 substitution via nucleophilic attack by
the amines. The regiospecificity of the substitution reaction on
quinones 3bee can be explained assuming stereoelectronic inter-
actions between the substituent at C-6 with the C-7 carbonyl group.
These factors probable affect the electrophilicity of the C-9 atom
and the attack of the nucleophiles occurs exclusively at C-8.
The mixtures of the regioisomers arising from the amination
reaction of quinone 3a were purified by column chromatography
and the proportion between the isomers was evaluated by 1H NMR,
using the signals of the quinone protons. Pure samples of the
regioisomers, for characterization and biological evaluation, were
obtained by column chromatography of the corresponding
mixtures. Attempts to isolated pure samples of regioisomers 5a and
5b by chromatography were unsuccessful.
Herein, we wish to report full details on the access to a broad
variety of 8- and 9-phenylamino-3,4-tetrahydrophenanthridine-
1,7,10(2H)-triones and the in vitro antitumor evaluation against
normal human lung fibroblasts MRC-5 and three human tumor
cells: AGS gastric adenocarcinoma, SK-MES-1 lung, and J82 bladder
carcinoma.
2. Chemistry
Phenanthridinequinones 3aee and a variety of aromatic amines
were selected as precursors of the designed phenylaminophe-
nanthridinequinones. Compounds 3aeb were prepared from the
commercially available 2,5-dihydroxybenzaldehyde 1a; 2,5-dihy-
droxyacetophenone 1b and 3-aminocyclohex-2-en-1-one 2 by
using a previously one-pot procedure [12]. Quinones 3cee were
obtained from acylhydroquinones 1cee and 2, employing the
above mentioned one-pot procedure (Scheme 1). The access to
the precursors 1cee was performed by solar-chemical photo-
Friedel-Crafts acylation of 1,4-benzoquinone with benzaldehyde,
furan-2-carbaldehyde and thiophene-3-carbaldehyde, according to
a recently reported procedure [13] (Scheme 1).
Recent results on the reaction of phenanthridine-7,10-quinones
3a and 3b with aniline indicate that 3a reacts with aniline in
ethanol at room temperature to yield
a
40:60 mixture of
In order to have evidences on the influence of the angular
cyclohexanone ring of the phenylamino-3,4-tetrahydro-phenan-
thridine-1,7,10(2H)-trione pharmacophore on the cytotoxic activity,
we attempted the preparation of compounds 19 and 20 by
aromatization of the corresponding regioisomers 7a and 7b. The
access to these phenanthridinquinones was achieved by reaction of
7a and 7b with Pd(AcO)2 in refluxing acetic acid. Compounds 19
and 20 were isolated in 69 and 63% yields, respectively (Scheme 3).
regioisomers 4a and 4b in moderate yield (Scheme 2). The presence
of CeCl3.7H2O in this reaction induces a change of the regiose-
lectivity and improves the yield of the amination reaction (Table 1).
In the case of the reaction of 3b with aniline, the aminoquinone 10
was isolated in low yield; however the use of the CeCl3$7H2O
catalyst improves the yield of the amination reaction (Table 1) and
does not change the regioselectivity (Scheme 2) [10].
In order to explore the scope of the amination reaction, phe-
nanthridinequinones 3aee were reacted with a variety of phenyl-
amines under the above acid-induced condition and the results are
summarized in Table 1. We tried to prepare members of the series
of phenylamino-3,4-tetrahydrophenanthridine-1,7,10(2H)-triones
containing electron-withdrawing substituents in the anilino group
3. Biological results and discussion
The phenylaminoquinones 4a þ 4b, 4a, 4b, 5a þ 5b, 6a, 6b, 7a,
7b, 8a, 8b, 9a, 9b, 10e18 and the parent phenanthridinequinones
3aee were evaluated for in vitro anticancer activity against normal

3400
J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
O
O
H
O
sun
aryl-CHO,C6H6
R
OH
O
=
=
=
=
=
a a.
3
,
1
1
R
R
R
R
R
H
Ag2O
.
1b 3b
,
Me
CH2Cl2
c c
.
1
3
,
phenyl
.
1d 3d
,
fur n
a
- -
2 yl
e e.
1
3
,
thiophen-3-yl
O
O
O
O
O
2
NH2
N
Ag2O,CH2Cl2
O
R
O
R
3
Scheme 1. Synthesis of phenanthridine-7,10-quinones.
human lung fibroblasts MRC-5 and three human tumor cells: AGS
gastric adenocarcinoma, SK-MES-1 lung, and J82 bladder carci-
noma, in 72-h drug exposure assays. The cytotoxicity of the
compounds was measured using a conventional microculture
tetrazolium reduction assay [15e17]. The broad variety of the
synthesized compounds was designed in order to gain insight upon
the influence of phenylamino groups at the quinone nucleus and
also the presence of methyl, phenyl, furyl and thienyl groups at the
6-position of the phenylamino-3,4-tetrahydro-phenanthridine-
1,7,10(2H)-trione pharmacophore on the biological activity. The
cytotoxic and antitumor activities are summarized in Table 2.
The initial SAR analysis was focused on the effects of insertion of
the anilino group at the quinone nucleus of compounds 3aee. The
data of Table 2 indicate that, in all cases except 7b, the insertion of
the nitrogen substituent, as in 4a, 10, 16, 17 and 18, dramatically
increases the cytotoxic activity on all the evaluated cell lines,
compared to those of their precursors. It is noteworthy that the
presence of the anilino group is remarkable on the biological
activity in terms of the antitumor activity on gastric adeno-
etoposide drug (IC50: AGS: 0.36
included in the assays.
mM; SK-MES-1: 2.8 mM), which was
Comparison of the IC50 values for the mixture 4a þ 4b with that
for each isomer 4a and 4b indicates that 4a has a greater cytotoxic
potency than 4b, and no synergism occurs between the
regioisomers. Similarly, the screening of the couples of regioisom-
ers 6a/6b, 7a/7b, and 9a/b shows that compounds 6a, 7a and 9a are
more cytotoxic than their corresponding regioisomers 6b, 7b and
9b. It can be seen that this difference is remarkable for regioisomers
7a and 7b, in particular on gastric cancer cells where the former is
100 times more cytotoxic than the latter. In the case of regioisomers
8a and 8b, no significant differences were observed for their
cytotoxic activities on the cell lines. These results reveal that phe-
nylamino groups at the 8-position of the phenylamino-3,4-tetra-
hydro-phenanthridine-1,7,10(2H)-trione pharmacophore lead to
derivatives with enhanced cytotoxicity respect to the correspond-
ing regioisomers at the 9-position.
Considering that the cytotoxic activity of 1,4-quinones depends
largely on their hydrophobic, steric and electronic properties and
that hydrophobic and steric properties of the regioisomers of each
couple are closely similar, we attributed the difference of the bio-
logical activity between the regioisomers of the couples 4a/4b, 6a/
6b, 7a/7b and 9a/b to their redox capability. On the basis of this
assumption and on precedents on the relationships between redox
potentials and antitumor activity of quinones [8,18e21], we
decided to evaluate the redox potentials of the regioisomers 4a/4b,
6a/6b, 7a/7b and 9a/b by cyclic voltammetry.
The redox potentials of these compounds were measured by
cyclic voltammetry in acetonitrile as solvent, at room temperature,
using a platinum electrode and 0.1 M tetraethylammonium tetra-
fluoroborate as the supporting electrolyte [8]. Well-defined quasi-
reversible waves, the cathodic peak related to the reduction of
quinone, and the anodic one due to its reoxidation, were observed
for the compounds. The voltammograms were run in the potential
range 0.0e2.0 V versus non-aqueous Ag/Agþ. The first half-wave
potential values, EI1/2, evaluated from the voltammograms ob-
tained at a sweep rate of 100 mV sꢀ1, are summarized in Table 3.
The EI1/2 values for the first electron, which is related with the
formation of the semiquinone radical anion, are in the potential
range ꢀ492 to ꢀ399 mV [22]. The data of Table 3 indicate that the
insertion of phenylamino substituents at the quinone ring in
precursor 3a induces the displacement of the half wave potential
towards more negative values and the magnitude of this effect
carcinoma cells (IC50: 0.23e0.58
mM) and on lung cancer cells
(IC50: 1.3e3.1
mM), comparable to that exhibited by the anticancer
O
O
O
O
O
H
N
9
9
+
N
N
8
8
N
H
R
O
R
uncatalysed: 40
catalysed: 73
uncatalysed: 60
catalysed: 27
.
.
a
4
4b
R = H
2
1
O
O
O
3
4
O
R = Me
9
9
8
10
7
N
N
6
N
8
H
R
O
R
O
a.
10
3
R=H
.
3b
R= Me
Scheme 2. Reactions of 3a and 3b with aniline.

J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
3401
Table 1
Reaction of quinone 3aee with arylamines catalyzed with CeCl3$7H2O.
R1
R2
Aryl
Time (h)
Products
Yielda
a/bb
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
H
H
H
H
Me
Et
H
H
H
H
H
H
H
H
H
Ph
1:40
2:20
1:30
2
8
24
1:00
2:50
0:53
0:20
0:30
2:40
6:13
8:10
1:30
4a D 4b
5a D 5b
6a D 6b
7a D 7b
8a D 8b
9a D 9b
10
11
12
13
14
88
68
86
62
55
49
96
55
79
91
73
48
69
55
45
73/27
73/27
73/27
74/26
68/32
71/29
100
100
100
100
100
4-HO-Ph
4-MeO-Ph
2,5-(OMe)2-Ph
Ph
Ph
Ph
4-HO-Ph
4-MeO-Ph
4-F-Ph
2-MeO-Ph
2,5-(MeO)2-Ph
Ph
15
16
17
18
100
100
100
100
Ph
Furan-2-yl
Thiophen-3-yl
Ph
Ph
a
Isolated yields.
b
Determined by 1H NMR analysis.
depends on the nature and location of the nitrogen substituents.
Comparison of the first half wave potentials between the
regioisomers of each couple, reveals that the reduction for the
isomers substituted at the 8-position (4a, 6a, 7a and 9a) occurs at
more negative EI1/2 potentials than that of the corresponding
9-substituted isomer. According to reported precedents on the
electronic effect of substituents in 1,4-naphthoquinones on EI1/2
potentials [23], it can be deduced that, in the couples of
regioisomers, the nitrogen substituent located at the 8-position has
a greater electron-donor capacity than at the 9-position.
moderate activity (range 4.4e15.2 mM). Regarding the regioisomers
substituted at the 9-position, it was observed a similar decreasing
effect on the cytotoxic activity by substitution of the amino proton
in 4b by a methyl or ethyl group, as in 8b and 9b. According to
precedents reported by AguilareMartínez on the effect of substit-
uents in the aniline ring on the conjugation degree of the nitrogen
lone pair with the quinone system in anilino-1,4-naphthoquinones
[23], the effects of the N-alkylanilino substituents in 8a and 9a on
the biological activity could be related with a weak conjugation
degree between the donor and acceptor fragments due to molec-
ular planarity inhibition by steric hindrance induced by the N-alkyl
groups.
Compound 4a and its phenyl-substituted analogues 6a and 7a,
showed high cytotoxic activity (range 0.23e2.8 mM) on the cancer
cells, whereas compounds 8a and 9a, resulting from the replace-
ment of the amino proton in 4a by an alkyl group, showed
Comparison of IC50 values of compounds 4a, 6a and 7a with
those of their analogs 10, 12 and 15 reveals that insertion of
a methyl group at the 6-position of the phenylamino-3,4-tetrahy-
drophenanthridine-1,7,10(2H)-trione pharmacophore induces
a decrease of the cytotoxic activity on lung fibroblasts and bladder
cancer cell lines.
Comparison of the cytotoxic potency of 19 and 20 with their
corresponding precursors 7a and 7b demonstrate that the
replacement of the angular cyclohexenone ring by a fused phenolic
ring in phenylaminophenanthridinetriones 7a and 7b enhances the
cytotoxic potency against all of the tested lines. Evaluation of 19
and 20 also indicates that regioisomer 19, containing the phenyl-
aminosubstituent at the 8-position, exhibits a higher cytotoxic
HO
MeO
MeO
O
O
N
N
H
19
Pd(OAc)
AcOH
2
7a, 7b
potency (IC50 values: MRC-5 ¼ 0.53
1 ¼0.63 M; J82 ¼ 1.4 M) than that of its regioisomer 20 (IC50
values: MRC-5 ¼17.2 M; AGS ¼ 19.8 M; SK-MES-1 ¼13.5 M),
M). It is
mM; AGS ¼ 0.18
mM; SK-MES-
m
m
H
O
m
m
m
MeO
MeO
O
O
H
N
except on the bladder carcinoma cell line (J82 ¼ 0.61
m
worth to note that although compound 19 exhibits high cytotoxic
potency, compound 20 emerges as a promising lead compound as
antitumor agent on bladder carcinoma due to the IC50 and selective
N
index (SI ¼ IC50 fibroblasts/IC50 cancer cells) values (IC50 ¼ 0.61
mM;
SI ¼ 28).
20
Analysis of the data of Table 2 indicates that, in general, cyto-
toxicity was observed in all cancer cell lines, but that AGS and
Scheme 3. Preparation of phenenthridinequinones 19 and 20.

3402
J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
Table 2
Cytotoxic activity of 8- and 9- arylamino-3,4-tetrahydrophenanthridine-1,7,10(2H)-triones and their precursors.
Nꢁ
Structure
IC50 ꢂ SEMa
MRC-5b
(mM)
AGSc
SK-MES-1d
J82e
3a
20.2 ꢂ 1.1
19.4 ꢂ 1.0
25.7 ꢂ 1.7
15.8 ꢂ 0.7
3b
3c
3d
3e
20.5 ꢂ 0.9
22 ꢂ 1.3
13.9 ꢂ 1.1
68.0 ꢂ 3.4
37.1 ꢂ 2.2
3.5 ꢂ 0.2
73.0 ꢂ 4.4
96.9 ꢂ 7.7
76.7 ꢂ 3.8
5.6 ꢂ 0.3
57.6 ꢂ 4.0
80.4 ꢂ 6.4
>100
31.2 ꢂ 1.9
59.0 ꢂ 3.5
71 ꢂ 4.9
O
O
N
O
S
(4a D 4b)f
4.4 ꢂ 0.2
0.65 ꢂ 0.03
1.8 ꢂ 0.1
6.7 ꢂ 0.3
4a
1.4 ꢂ 0.1
0.23 ꢂ 0.01
1.3 ꢂ 0.1
2.1 ꢂ 0.1
(5a D 5b)f
2.3 ꢂ 0.1
0.49 ꢂ 0.02
2.7 ꢂ 0.1
1.8 ꢂ 0.1

J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
3403
Table 2 (continued )
Nꢁ
Structure
IC50 ꢂ SEMa
MRC-5b
(mM)
AGSc
SK-MES-1d
J82e
6a
7a
8a
9a
4b
6b
2.6 ꢂ 0.1
2.6 ꢂ 0.1
7.5 ꢂ 0.4
8.6 ꢂ 0.4
5.9 ꢂ 0.4
4.8 ꢂ 0.3
0.47 ꢂ 0.02
2.0 ꢂ 0.1
2.8 ꢂ 0.1
0.32 ꢂ 0.01
4.7 ꢂ 0.2
4.4 ꢂ 0.2
3.9 ꢂ 0.1
2.2 ꢂ 0.1
1.8 ꢂ 0.1
10.6 ꢂ 0.6
11.7 ꢂ 0.8
7.5 ꢂ 0.4
3.7 ꢂ 0.2
2.5 ꢂ 0.1
12.9 ꢂ 0.8
15.2 ꢂ 1.1
8.1 ꢂ 0.4
4.0 ꢂ 0.2
7b
46.4 ꢂ 2.9
32.9 ꢂ 2.0
21.3 ꢂ 1.1
56.7 ꢂ 2.8
8b
9b
7.3 ꢂ 0.4
8.3 ꢂ 0.4
8.7 ꢂ 0.3
8.4 ꢂ 0.4
19.6 ꢂ 1.0
17.1 ꢂ 0.9
17.7 ꢂ 0.9
19.5 ꢂ 1.2
(continued on next page)

3404
J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
Table 2 (continued )
Nꢁ
Structure
IC50 ꢂ SEMa
MRC-5b
(mM)
AGSc
SK-MES-1d
J82e
10
2.0 ꢂ 0.1
0.65 ꢂ 0.03
5.7 ꢂ 0.3
4.2 ꢂ 0.3
11
5.2 ꢂ 0.2
1.2 ꢂ 0.1
4.1 ꢂ 0.2
3.0 ꢂ 0.21
12
2.6 ꢂ 0.1
1.2 ꢂ 0.1
3.2 ꢂ 0.2
1.9 ꢂ 0.1
13
1.6 ꢂ 0.1
0.22 ꢂ 0.01
4.3 ꢂ 0.2
3.7 ꢂ 0.2
14
3.0 ꢂ 0.2
0.79 ꢂ 0.03
3.3 ꢂ 0.2
3.8 ꢂ 0.2
15
5.0 ꢂ 0.2
1.4 ꢂ 0.1
21.7 ꢂ 1.3
7.1 ꢂ 1.0
16
2.7 ꢂ 0.1
0.33 ꢂ 0.02
2.8 ꢂ 0.2
2.9 ꢂ 0.2
17
2.7 ꢂ 0.2
0.58 ꢂ 0.03
3.1 ꢂ 0.2
2.4 ꢂ 0.1

J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
3405
Table 2 (continued )
Nꢁ
Structure
IC50 ꢂ SEMa
MRC-5b
(mM)
AGSc
SK-MES-1d
J82e
18
2.8 ꢂ 0.2
0.35 ꢂ 0.02
0.36 ꢂ 0.15
2.9 ꢂ 0.2
2.5 ꢂ 0.1
Etoposide
e
3.9 ꢂ 0.21
2.8 ꢂ 0.18
0.80 ꢂ 0.04
a
Data represent average values for six independent determinations.
Normal human lung fibroblasts cells.
Human gastric adenocarcinoma cell line.
Human lung cancer cell line.
Human bladder carcinoma cell line.
4a/4b [ 73/27; 5a/5b ¼ 73/27.
b
c
d
e
f
SK-MES-1 cell lines appear to be more sensitive to the compounds
overall. Among the compounds evaluated in the in vitro screen and
collected in Table 2, the members 4a, 7a, 16 and 18 (Fig. 2) were
selected for this study as the more significant antitumor members
on gastric adenocarcinoma and lung cancer cell lines, according to
their IC50 and SI values (w6.0e8.0). Compound 20 was also selected
and was considered as a promising lead compound due to its high
potency and selective index on bladder carcinoma cell line. Further
ways to prepare new analogs of compounds 4a, 7a,16, 18 and 20 are
under study.
and thienyl groups. The 8-arylamino-3,4-tetrahydrophenan-
thridine-1,7,10(2H)-triones 4a, 7a, 16, and 18 exhibit interesting
antitumor activity and selective index on gastric cancer cells,
comparable to that of etoposide. The replacement of the angular
cyclohexanone ring in compounds 7a and 7b by a fused phenolic
ring, as in compound 19 and 20, improves the cytotoxic potency on
some of the tested cell lines. This substitution is highly relevant on
regioisomer 7b because the resulting analog 20 is endowed with
high antitumor potency and selective index on bladder carcinoma
cells.
Given the high incidence of undesirable side-effects induced by
the majority of current anticancer drugs and by considering the
selective index of aminoquinones 4a, 7a, 16, 18 and, in particular,
that of phenylaminophenanthridinequinone 20, they appear as
promising and interesting leads, endowed of potential anticancer
activity. These results prompt us to design and synthesize more
new 6-aryl-substituted 8-phenylaminophenanthridinequinones in
order to discover more active and selective anticancer agents.
4. Conclusions
We have developed the regiospecific synthesis of a variety of
phenylamino-3,4-tetrahydrophenanthridine-1,7,10(2H)-trione
derivatives. The majority of the new aminoquinones expressed
in vitro cytotoxic activity against normal human lung fibro-
blasts (MRC-5) and on gastric adenocarcinoma (AGS), lung cancer
(SK-MES-1), and bladder carcinoma (J82) cell lines. The insertion of
a phenylamino group into the quinone nucleus of compounds 3aee
increases the cytotoxic potency, with respect to their precursors,
in almost all the evaluated cell lines. From the current investiga-
tion, structure activity relationships of the phenylaminoquinone
members demonstrate that phenylamino substituents at the
8-position of the quinone ring show increased antitumor activity
than those at the 9-position. The effect of such substitution is more
significant in enhancing the antitumor activity for those members
unsubstituted at the 6-position and substituted with phenyl, furyl
5. Experimental
5.1. Chemical synthesis
All reagents were commercially available reagent grade and
were used without further purification. Melting points were
determined on a Stuart Scientific SMP3 apparatus and are uncor-
rected. 1H NMR spectra were recorded on Bruker AM-200 and AM-
400 instruments in deuterochloroform (CDCl3). 13C NMR spectra
were obtained in CDCl3 at 50 and 100 MHz. 2D NMR techniques
(COSY, HMBC) and DEPT were used for signal assignment. Chemical
shifts are expressed in ppm downfield relative to tetramethylsilane
Table 3
First half-wave potentials and cytotoxic activity of 8- and 9- arylamino-3,4-tetra-
hydrophenanthridine-1,7,10(2H)-triones.
(TMS, d scale), and the coupling constants (J) are reported in Hertz.
Nꢁ
ꢀEI1/2 (mV)
IC50 ꢂ SEM (
m
M)
AGS
The elemental analyses were performed in a Fison SA, model EA-
1108 apparatus. HRMS were obtained on a Thermo Finnigan spec-
trometer, model MAT 95XP. Silica gel Merck 60 (70e230 mesh) was
used for preparative column chromatography, and TLC aluminum
foil 60F254 for analytical TLC.
Anhydrous acetonitrile (99.8%) for electrochemical evaluations
was obtained from Sigma-Aldrich. Quinones 3a and 3b were
prepared according to a previously reported procedure [11] and
acylhydroquinones were prepared by using a recently reported
solar photoacylation procedure [12].
MRC-5
SK-MES-1
J82
3a
4a
4b
6a
6b
7a
7b
9a
9b
367
452
430
492
460
440
420
451
399
20.2 ꢂ 1.1
1.4 ꢂ 0.1
5.9 ꢂ 0.4
2.6 ꢂ 0.1
4.8 ꢂ 0.3
2.6 ꢂ 0.1
46.4 ꢂ 2.9
8.6 ꢂ 0.4
19.6 ꢂ 1.0
19.4 ꢂ 1.0
0.23 ꢂ 0.01
3.9 ꢂ 0.1
25.7 ꢂ 1.7
1.3 ꢂ 0.1
7.5 ꢂ 0.4
2.0 ꢂ 0.1
3.7 ꢂ 0.2
1.8 ꢂ 0.1
21.3 ꢂ 1.1
11.7 ꢂ 0.8
17.7 ꢂ 0.9
15.8 ꢂ 0.7
2.1 ꢂ 0.1
8.1 ꢂ 0.4
2.8 ꢂ 0.1
4.0 ꢂ 0.2
2.5 ꢂ 0.1
56.7 ꢂ 2.8
15.2 ꢂ 1.1
19.5 ꢂ 1.2
0.47 ꢂ 0.02
2.2 ꢂ 0.1
0.32 ꢂ 0.01
32.9 ꢂ 2.0
4.4 ꢂ 0.2
17.1 ꢂ 0.9

3406
J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
O
O
O
MeO
MeO
O
O
O
O
N
N
N
N
H
N
H
N
H
O
O
4a
7a
16
HO
O
O
MeO
O
H
N
N
N
N
H
O
O
MeO
S
18
20
Fig. 2. Structures of selected quinones as the more significant antitumor members.
5.2. General procedure for the synthesis of acylhydroquinones 1cee
[12]
1581(C]O).1H NMR (200 MHz, CDCl3):
d 7.30 (s,1H, 6-H), 7.49 (d,1H,
J ¼ 3.0 Hz, 50- or 40-H), 7.71 (d, 1H, J ¼ 8.2 Hz, 4- or 3-H), 7.85 (d, 1H,
J ¼ 8.2 Hz, 3- or 4-H), 7.89 (m, 1H, 40- or 50-H), 8.32 (s, 1H, 20-H), 8.38
A 100 mL benzene solution of 1,4-benzoquinone (1 mmol) and
the required aldehyde (7.5 mmol), was placed into the outer jacket
of a Liebig condenser type. The solution was bubbled with nitrogen
(2 min), sealed with a septum and then irradiated for six days (total
illumination time of 30 h), under solar radiation conditions in the
range 800e1100 Watts/m2 (JanuaryeMarch) The solvent was
evaporated under reduced pressure and the residue was chroma-
tographed on silica gel (3:1 petroleum ether/ethyl acetate). The
starting aldehyde and the solvent were recovered and employed in
the next batches.
(s, 1H, 5-OH), 11.74 (s, 1H, 2-OH). 13C NMR (50 MHz, CDCl3):
d 116.9,
118.5, 122.1, 124.4, 125.9, 128.1, 132.1, 140.1, 148.7, 156.0, 193.6. Anal.
Calcd for C11H8O3S: C, 59.99; H, 3.66;S,14.56; found: C, 59.93; H, 3.65;
S, 14.17.
5.3. General procedure for the preparation of 8- and 9-
phenylamino-3,4-dihydrophenanthridine-1,7,10(2H)-triones
A suspension of quinone 3a (1 mmol), the required amine
(2 mmol), CeCl3$7H2O (0.05 mmol), and ethanol (20 mL) was left
with stirring at rt after completion of the reaction as indicated by
TLC. The reaction mixture was partitioned between chloroform and
water and the organic layer was washed with water (3 ꢃ 15 mL).
The dried extract was evaporated under reduced pressure and the
residue was column chromatographed (10:90 AcOEt/petroleum
ether) to yield the mixture of regioisomers. These were analysed by
1H NMR to evaluate the proportion between the 8- and 9-phenyl-
aminophenanthridinequinone derivatives. Column chromatog-
raphy of the mixture, using CH2Cl2 as eluent, provided pure
samples of the regioisomers.
The solar photolysis were performed at the Canchones Experi-
mental Center in Iquique/Chile (latitude 20ꢁ26043.8000 S, 990 m
above sea level), which is located in the Atacama desert.
5.2.1. 2,5-Dihydroxybenzophenone (1c)
Prepared from 1,4-benzoquinone and benzaldehyde (91%),
orange solid, mp 121e123 ꢁC (lit. [24]: 125e126 ꢁC), IR (KBr): cmꢀ1
3456 (OH), 1687 (C]O), 1225 (CeO). 1H NMR (200 MHz, CDCl3):
d
6.91 (m, 2H, 40- þ 60-H), 6.96 (s, 1H, 6-H), 7.04 (m, 2H, 50- or 30-H
and 20-H), 8.18 (d, 1H, J ¼ 6.9 Hz, 3- or 4-H), 8.06 (d, 1H, J ¼ 6.9 Hz,
4- or 3-H), 7.12 (m, 2H, 30- or 50-H þ 5-OH), 11.34 (bs, 1H, 2-OH). 13C
NMR (50 MHz, CDCl3):
d
119.6,119.9,123.4, 125.5, 129.1, 129.5, 129.7,
5.3.1. 8- and 9-Phenylamino-3,4-dihydrophenanthridine-
1,7,10(2H)-trione (4a, 4b) [10]
130.4, 130.6, 132.7, 138.9, 149.9, 206.1.
The mixture of regioisomers was prepared from 3a and aniline
(1:40 h, 88%); red solid; isomers proportion: 73:27 ¼4a:4b.
Compound 4a (less polar): red solid, mp 179e182 ꢁC; IR (KBr): cmꢀ1
3441 (NeH), 1668 (C]O), 1610, 1566 (C]O quinone). 1H NMR
5.2.2. (2,5-dihydroxyphenyl)(furan-2-yl)methanone (1d)
Prepared from 1,4-benzoquinone and furan-2-carbaldehyde
(88%) orange solid, mp, 125.5e126.5 ꢁC. IR (KBr): cmꢀ1 3330
(OeH), 1562 (C]O). 1H NMR (200 MHz, acetone-d6):
d
6.66 (s, 1H,
(400 MHz, CDCl3):
d
2.21 (q, J ¼ 6.5 Hz, 2H, 3-H), 2.89 (t, J ¼ 6.5 Hz,
6-H), 6.80 (dd, 1H, J ¼ 3.6, 2.7 Hz, 50- or 40-H), 7.12 (d, 1H, J ¼ 8.3 Hz,
4- or 3-H), 7.79 (d, 1H, J ¼ 8.3 Hz, 3- or 4-H), 7.49 (m, 1H, 30-H), 8.01
(dd, 1H, J ¼ 3.6, 2.7 Hz, 40- or 50-H), 8.21 (bs, 1H, 5-OH), 11.45 (s, 1H,
2H, 2-H), 3.13 (t, J ¼ 6.5 Hz, 2H, 4-H), 6.43 (s, 1H, 9-H), 7.23 (m, 3H,
arom.), 7.25 (m, 2H, arom.), 7.41 (s, 1H, NH), 9.24 (s, 1H, 6-H). 13C
NMR (100 MHz, CDCl3):
d 21.37, 33.40, 39.13, 104.99, 122.67, 124.25
2-OH). 13C NMR (50 MHz, acetone-d6):
d
113.3, 116.5, 116.7, 119.4,
(2C), 126.09, 128.90 (2C), 129.84, 136.92, 140.45, 143.74, 149.66,
169.79, 180.80, 181.42, 198.00; HRMS (Mþ): m/z calcd for
C19H14N2O3: 318.10044; found: 318.10012.
121.8, 125.3, 148.8, 150.2, 152.7, 157.2, 185.1. Anal. Calcd for C11H8O4:
C, 64.71; H, 3.95; found: C, 64.80; H, 3.85.
Compound 4b: red solid, mp 182e184 ꢁC; IR (KBr): cmꢀ1 3447
5.2.3. (2,5-Dihydroxyphenyl)(thiophen-3-yl)methanone (1e)
Prepared from 1,4-benzoquinone and thiophene-3-carbaldehyde
(87%), orange solid, mp 135e136 ꢁC. IR (KBr): cmꢀ1 3334 (OeH),
(NeH), 1703 (C]O), 1607, 1590 (C]O quinone). 1H NMR (400 MHz,
CDCl3):
d
2.22 (q, J ¼ 6.5 Hz, 2H, 3-H), 2.89 (t, J ¼ 6.5 Hz, 2H, 2-H),
3.18 (t, J ¼ 6.5 Hz, 2H, 4-H), 6.31 (s, 1H, 8-H), 7.23 (m, 3H, arom.),

J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
3407
7.25 (m, 2H, arom.), 7.41 (s, 1H, NH), 9.33 (s, 1H, 6-H). 13C NMR
(100 MHz, CDCl3): 21.50, 33.39, 39.26, 102.24, 122.67, 124.25 (2C),
126.25, 128.89 (2C), 129.83, 136.93, 140.44, 143.75, 150.75, 167.65,
180.79, 181.97, 197.62. HRMS (Mþ): m/z calcd for C19H14N2O3:
318.10044; found: 318.10005.
68:32 ¼ 8a:8b. Compound 8a (less polar): red solid, mp
152e153 ꢁC; IR (KBr): cmꢀ1 3447 (NeH), 1686 (C]O), 1561, 1546
d
(C]O quinone). 1H NMR (400 MHz, CDCl3)
d: 2.23 (q, J ¼ 6.5 Hz,
2H, 3-H), 2.90 (t, J ¼ 6.5 Hz, 2H, 2-H), 3.13 (t, J ¼ 6.5 Hz, 2H, 4-H),
3.42 (s, 3H, NMe), 6.11 (s, 1H, 9-H), 7.13 (m, 2H, arom), 7.31 (m, 1H,
arom), 7.40 (m, 2H, arom), 9.04 (s, 1H, 6-H). 13C NMR (100 MHz,
5.3.2. 8- and 9-(4-Hydroxyphenylamino)-3,4-
CDCl3) d: 21.24, 33.27, 39.20, 43.16, 60.40, 111.70, 125.41 (2C),
dihydrophenanthridine-1,7,10(2H)-trione (5a,b)
126.93, 127.92, 129.79 (2C), 139.73, 147.17, 150.07, 150.60, 168.75,
180.68, 180.96, 198.01. HRMS (Mþ): m/z calculated for C20H16N2O3:
332.11609; found: 332.11569.
The mixture of regioisomers was prepared from 3a and
p-hydroxyaniline (2:20 h, 68%), purple solid, proportion of
regioisomers: 73:27 ¼ 5a:5b. 1H NMR (400 MHz, acetone-d6):
Compound 8b: red solid mp 133e135 ꢁC; IR (KBr): cmꢀ1 3448
d
2.22 (q, J ¼ 6.4 Hz, 2H, 3-H), 2.90 (t, J ¼ 6.4 Hz, 2H, 2-H), 3.12
(NeH), 1691 (C]O), 1627, 1577 (C]O quinone); 1H NMR (200 MHz,
(t, J ¼ 6.4 Hz, 2H, 4-H), 5.96 (s, 0.27H, 8-H), 6,04 (s, 0.73H, 9-H), 6,95
(d, J ¼ 8,4 Hz, 2H, arom), 7.25 (d, J ¼ 8.4 Hz, 2H, arom), 8.06 (s, 1H,
OH), 8.51 (s, 1H, NH), 9.15 (s, 0.27H, 6-H); 9,17 (s, 0.73H, 6-H).
CDCl3)
d
: 2.26 (q, J ¼ 6.4 Hz, 2H, 3-H), 2.78 (t, J ¼ 6.4 Hz, 2H, 2-H),
3.20 (t, J ¼ 6.4 Hz, 2H, 4-H), 3.50 (s, 3H, NMe), 5.85 (s, 1H, 8-H), 7.26
(m, 3H, arom), 7.48 (m, 2H, arom), 9.27 (s, 1H, 6-H); HRMS (Mþ):
m/z calcd for C20H16N2O3: 332.11609; found: 332.11572.
5.3.3. 8- and 9-(4-Methoxyphenylamino)-3,4-
dihydrophenanthridine-1,7,10(2H)-trione (6a, 6b)
5.3.6. 8- and 9-(N-Ethylphenylamino)-3,4-dihydrophenanthridine-
1,7,10(2H)-trione (9a, 9b)
The mixture of isomers was prepared from 3a and N-ethylani-
line, (24 h, 49%), isomers proportion: 71:29 ¼ 9a:9b. Compound 9a
(less polar) was isolated as orange oil. IR (KBr): cmꢀ1 3430 (NeH);
1683 (C]O); 1561, 1545 (C]O quinone). 1H NMR (400 MHz,
The mixture of regioisomers was prepared from 3a and p-ani-
sidine, (1:30 h, 86%); purple solid, isomers proportion:
73:27 ¼6a:6b. Compound 6a (less polar): purple solid mp
162.5e164.5 ꢁC; IR (KBr): cmꢀ1 3448 (NeH), 1674 (C]O), 1611, 1598
(C]O quinone). 1H NMR (400 MHz, CDCl3):
d
2.21 (q, J ¼ 6.6 Hz, 2H,
3-H), 2.87 (t, J ¼ 6.6 Hz, 2H, 2-H), 3.11 (t, J ¼ 6.6 Hz, 2H, 4-H), 3.80
(s, 3H, OMe), 6.22 (s, 1H, 9-H), 6.92 (d, J ¼ 8.8 Hz, 2H, 20- and 60-H),
7.16 (d, J ¼ 8.8 Hz, 2H, 30- and 50-H), 7.40 (s, 1H, NH), 9.20 (s, 1H,
CDCl3):
d
: 1.28 (t, J ¼ 6.1 Hz, 3H, CH2CH3), 2.23 (q, J ¼ 6.5 Hz, 2H,
3-H), 2.90 (t, J ¼ 6.5 Hz, 2H, 2-H), 3.12 (t, J ¼ 6.5 Hz, 2H, 4-H), 3.86
(t, J ¼ 6.1 Hz, 3H, Me), 4.11 (q, J ¼ 6.1 Hz, 2H, CH2CH3), 6.04 (s, 1H,
9-H), 7.11 (m, 2H, arom), 7.33 (m,1H, arom), 7.42 (m, 2H, arom), 9.05
6-H). 13C NMR (100 MHz, CDCl3):
d 21.35, 33.35, 39.13, 55.59, 104.13,
115.04 (2C), 124.33, 124.81 (2C), 128.96, 129.48, 140.63, 144.60,
149.53, 157.97, 169.69, 180.87, 181.16, 198.15. HRMS (Mþ): m/z calcd
for C20H16N2O4: 348.1110; found: 348. 1107.
(s, 1H, 6-H). 13C NMR (400 MHz, CDCl3):
d 12.21, 14.22, 21.45, 29.71,
33.26, 39.22, 49.79, 60.40, 110.93, 126.36 (2C), 127.19, 129.93 (2C),
145.14, 150.01, 150.18, 168.66, 180.88, 181.11, 198.06. HRMS (Mþ):
m/z calcd for C21H18N2O3: 346.13174; found: 346.13153.
Compound 6b: purple solid, mp 168e169 ꢁC; IR (KBr): cmꢀ1
3442 (NeH), 1703 (C]O), 1599, 1510 (C]O quinone). 1H NMR
Compound 9b was isolated as orange oil. IR (KBr): cmꢀ1 3448
(200 MHz, CDCl3):
d
2.24 (q, J ¼ 6.8 Hz, 2H, 3-H), 2.91 (t, J ¼ 6.8 Hz,
(NeH), 1695 (C]O), 1600, 1567 (C]O quinone). 1H NMR (400 MHz,
2H, 2-H), 3.14 (t, J ¼ 6.8 Hz, 2H, 4-H), 3.83 (s, 3H, OMe), 6.15 (s, 1H,
8-H), 6.97 (d, J ¼ 8.9 Hz, 2H, 20- and 60-H), 7.16 (d, J ¼ 8.9 Hz, 2H,
30- y 50-H), 7.42 (s, 1H, NH), 9.35 (s, 1H, 6-H). HRMS (Mþ): m/z calcd
for C20H16N2O4: 348.1110; found: 348.1102.
CDCl3):
d
1.25 (t, J ¼ 6.7 Hz, 3H, CH2CH3), 2.24 (q, J ¼ 6.5 Hz, 2H,
3-H); 2.80 (t, J ¼ 6.5 Hz, 2H, 2-H); 3.20 (t, J ¼ 6.5 Hz, 2H, 4-H); 3.64
(t, J ¼ 6.7 Hz, 3H, Me); 3.92 (q, J ¼ 6.7 Hz, 2H, CH2CH3); 5.70 (s, 1H,
8-H); 7.15 (m, 2H, arom); 7.36 (m, 1H, arom); 7.49 (m, 2H, arom);
9.25 (s, 1H, 6-H). HRMS (Mþ): m/z calcd for C21H18N2O3: 346.13174;
found: 346.13114.
5.3.4. 8- and 9-(2,5-Dimethoxyphenylamino)-3,4-
dihydrophenanthridine-1,7,10(2H)-trione (7a,b)
The mixture of regioisomers was prepared from 3a and 2,5-
dimethoxyaniline, (2 h, 62%), purple solid, isomers proportion:
74:26 ¼ 7a:b. Compound 7a (less polar): purple solid, mp
179e181 ꢁC; IR (KBr): cmꢀ13449 (NeH),1705 (C]O),1625,1593 (C]
5.4. General procedure for the preparation of the 6-substituted
8-amino-6-methyl-3,4-dihydrophenanthridine-1,7,10(2H)-triones
O quinone). 1H NMR (400 MHz, CDCl3):
d
2.27 (q, J ¼ 6.6 Hz, 2H, 3-H),
A suspension of quinone 3bee (1 mmol), the required amine
(2 mmol), CeCl3$7H2O (0.05 mmol), and ethanol (20 mL) was left
with stirring at rt after completion of the reaction as indicated
by TLC. The reaction mixture was partitioned between chloro-
form/water and the organic layer was washed with water
(3 ꢃ15 mL). The dried extract was evaporated under reduced
pressure and the residue was column chromatographed (10:90
AcOEt/petroleum ether) to yield the corresponding substituted
aminophenanthridinequinone.
2.90 (t, J ¼ 6.5 Hz, 2H, 2-H), 3.16 (t, J ¼ 6.5 Hz, 2H, 4-H), 3.80 (s, 3H,
OMe), 3.88 (s, 3H, OMe), 6.56 (s,1H, 9-H), 6.68 (dd, J ¼ 8.5; 3.4 Hz,1H,
40-H), 6.88(d, J ¼ 8.5 Hz,1H, 30-H), 6.98(d, J ¼ 3.4 Hz,1H, 60-H), 7.96 (s,
1H, NH), 9.27 (s, 1H, 6-H). 13C NMR (100 MHz, CDCl3):
d 21.38, 33.40,
39.16, 55.97, 56.29, 105.50, 107.68, 110.01, 111.97, 127.06, 140.49 (2C),
142.63, 145.42 (2C), 149.74, 153.86, 169.66, 180.75, 181.42, 198.05.
HRMS (Mþ): m/z calcd for C21H18N2O5: 378.12155; found: 378.12067.
Compound 7b: purple solid; mp 175e177 ꢁC; IR (KBr): cmꢀ1
3329 (NeH), 1687 (C]O), 1638, 1598 (C]O quinone). 1H NMR
(200 MHz, CDCl3):
d
2.28 (q, J ¼ 6.6 Hz, 2H, 3-H), 2.93 (t, J ¼ 6.6 Hz,
5.4.1. 8-(Phenylamino)-6-methyl-3,4-dihydrophenanthridine-
1,7,10(2H)-trione (10) [10]
2H, 2-H), 3.21 (t, J ¼ 6.6 Hz, 2H, 4-H), 3.80 (s, 3H, OMe); 3.86 (s, 3H,
OMe), 6.49 (s, 1H, 8-H), 6.70 (dd, J ¼ 8.9, 3.0 Hz, 1H, 40-H), 6.87 (d,
J ¼ 8.9 Hz, 1H, 30-H), 6.98 (d, J ¼ 3.0 Hz, 1H, 60-H), 7.89 (s, 1H, NH),
9.37 (s, 1H, 6-H). HRMS (Mþ): m/z calcd for C21H18N2O5: 378.1215;
found: 378.1206.
Prepared from 3b and aniline, (1 h, 96%), red solid, mp 180-
183.5 ꢁC; IR (KBr): cmꢀ1 3442 (NeH), 1696 (C]O), 1591, 1564 (C]O
quinone). 1H NMR (400 MHz, CDCl3):
d
2.23 (q, J ¼ 6.4 Hz, 2H, 3-H),
2.89 (t, J ¼ 6.4 Hz, 2H, 2-H), 2.94 (s, 3H, Me), 3.07 (t, J ¼ 6.4 Hz, 2H,
4-H), 6.38 (s, 1H, 9-H), 7.23 (m, 3H, arom), 7.41 (m, 2H, arom), 7.69
5.3.5. 8- y 9-(N-Methylphenylamino)-3,4-dihydrophenanthridine-
1,7,10(2H)-trione (8a, 8b) [10]
The mixture of regioisomers was prepared from 3a and
N-methylaniline, (8 h, 55%), red solid, isomers proportion:
(s, 1H, NH). 13C NMR (400 MHz, CDCl3):
d 21.49, 26.46, 33.30, 39.21,
103.45, 122.56, 122.67, 125.95, 128.20, 129.83 (2C), 137.23, 143.53
(2C), 144.78, 162.38, 167.75, 181.58, 182.07, 198.65. HRMS (Mþ): m/z
calcd for C20H16N2O3: 332.11609; found: 332.11539.

3408
J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
5.4.2. 8-(4-Hydroxyphenylamino)-6-methyl-3,4-
dihydrophenanthridine-1,7,10(2H)-trione (11)
d
2.25 (q, J ¼ 6.8 Hz, 2H, 3-H), 2.90 (t, J ¼ 6.8 Hz, 2H, 2-H), 2.99
(s, 3H, Me), 3.07 (t, J ¼ 6.8 Hz, 2H, 4-H), 3.79 (s, 3H, OMe), 3.87
(s, 3H, OMe), 6.53 (s, 1H, 9-H), 6.65 (dd, J ¼ 8.8, 2.8 Hz, 1H, 40-H),
6.87 (d, J ¼ 2.8 Hz,1H, 60-H), 6.97 (d, J ¼ 8.8 Hz,1H, 30-H), 8.09 (s,1H,
Prepared from 6 and p-hydroxyaniline, (2:30 h, 55%), purple
solid, mp 215.5e217.5 ꢁC; IR (KBr): cmꢀ1 3331 (NeH), 1681 (C]O),
1598, 1517 (C]O quinone). 1H NMR (400 MHz, DMSO):
d
2.22
NH). 13C NMR (400 MHz, CDCl3):
d 14.39, 21.57, 26.60, 33.40, 39.32,
(q, J ¼ 6.4 Hz, 2H, 3-H), 2.88 (t, J ¼ 6.4 Hz, 2H, 2-H), 2.90 (s, 3H, Me),
3.07 (t, J ¼ 6.4 Hz, 2H, 4-H), 6.16 (s, 1H, 9-H), 6.89 (d, J ¼ 8.4 Hz, 2H,
30- and 50-H), 7.08 (d, J ¼ 8.4 Hz, 2H, 20- and 60-H), 7.74 (s, 1H, NH),
56.13, 56.45, 104.11, 107.64, 109.94, 112.06, 122.77, 127.43, 128.24,
143.68, 145.47, 154.01, 162.59, 167.75, 181.72, 182.23, 198.75. HRMS
(Mþ): m/z calcd for C22H20N2O5: 392.13721; found: 392.13642.
9.03 (s, 1H, OH). 13C NMR (400 MHz, DMSO):
d 21.35, 26.35, 29.66,
33.13, 102.13, 116.51 (2C), 122.69, 125.07 (2C), 128.18, 128.36, 143.78,
146.07, 155.89, 162.12, 167.42, 181.57, 181.76, 198.84. HRMS (Mþ):
m/z calcd for C20H16N2O4: 348.11102; found: 348.11028.
5.4.7. 6-Phenyl-8-phenylamino-3,4-dihydrophenanthridine-
1,7,10(2H)-trione (16)
Prepared from quinone 3c and aniline (6:15 h, 69%), red solid,
mp 193.5e195 ꢁC. IR (KBr): cmꢀ1 3442 (NeH), 1682 (C]O), 1592,
5.4.3. 8-(4-Methoxyphenylamino)-6-methyl-3,4-
1559 (C]O quinone). 1H NMR (400 MHz, CDCl3)
d: 2.26
dihydrophenanthridine-1,7,10(2H)-trione (12) [10]
(q, J ¼ 6.4 Hz, 3-H), 2.94 (t, J ¼ 6.4 Hz, 2H, 2-H), 3.14 (t, J ¼ 6.4 Hz, 2H,
Prepared from 3b and p-aniꢀsi1dine, (53 min, 79%); purple solid,
4-H), 6.46 (s, 1H, 9-H), 7.21 (m, 3H, arom), 7.40 (m, 2H, arom), 7.48
mp 130e131.5 ꢁC; IR (KBr): cm 3442 (NeH), 1674 (C]O), 1560,
(m, 6H, NH y arom). 13C NMR (400 MHz, CDCl3)
d: 21.57, 33.48,
1519 (C]O quinone). 1H NMR (400 MHz, CDCl3):
d
2.22
39.32, 103.80, 122.59 (2C), 126.06, 128.36 (2C), 128.59 (2C), 128.73,
129.34, 129.95 (2C), 137.21, 140.11, 144.23, 144.79, 161.98, 167.82,
171.28, 180.49, 181.83, 198.60.HRMS (Mþ): m/z: calcd for
C25H18N2O3: 394.13174; found: 394.13074.
(q, J ¼ 6.8 Hz, 2H, 3-H), 2.89 (t, J ¼ 6.8 Hz, 2H, 2-H), 2.94 (s, 3H,
Me), 3.06 (t, J ¼ 6.8 Hz, 2H, 4-H), 3.82 (s, 3H, OMe), 6.19 (s, 1H,
9-H), 6.92 (d, J ¼ 9.0 Hz, 2H, 20-and 60-H), 7,16 (d, J ¼ 9.0 Hz, 2H,
30- and 50-H), 7.56 (s, 1H, NH). 13C NMR (400 MHz, CDCl3):
d 21.49,
26.44, 33.29, 39.23, 55.67, 102.62, 115.04 (2C), 122.66, 124.82 (2C),
128.31, 129.82, 143.75, 145.63, 157.90, 162.29, 167.67, 181.70, 181.79,
198.80. HRMS (Mþ): m/z calcd for C21H18N2O4: 362.12665; found:
362.12587.
5.4.8. 8-Phenylamino-6-(2-furyl)-3,4-dihydrophenanthridine-
1,7,10(2H)-trione (17)
Prepared from quinone 3dꢀ(18:10 h, 55%) and aniline, red solid,
mp 166e167 ꢁC. IR (KBr): cm 3448 (NeH), 1683 (C]O), 1590,
1557 (C]O quinone). 1H NMR (400 MHz, CDCl3):
d 2.18
5.4.4. 8-(2-Methoxyphenylamino)-6-methyl-3.4-
dihydrophenanthridine-1.7.10(2H)-trione (13)
(q, J ¼ 6.8 Hz, 2H, 3-H), 2.85 (t, J ¼ 6.8 Hz, 2H, 2-H), 3.05 (t,
J ¼ 6.8 Hz, 2H, 4-H), 6.38 (s, 1H, 9-H), 6.57 (m, 1H, eCH furyl), 7.15
(m, 3H, arom), 7.19 (s, 1H, eCH furyl), 7.35 (m, 2H, arom), 7.50 (s, 1H,
Prepared from 3b and o-anisidine (30 min, 73%), purple solid,
mp 170e172 ꢁC; IR (KBr): cmꢀ1 3448 (NeH). 1672 (C]O) 1619, 1591
NH), 7.57 (s, 1H, CH furyl). 13C NMR (400 MHz, CDCl3)
d: 21.49,
(C]O quinone). 1H NMR (400 MHz. CDCl3):
d
2.24 (q. J ¼ 6.4 Hz. 2H,
33.48, 39.32, 103.59, 112.31, 115.06, 115.28, 122.20, 122.71 (2C),
126.15, 128.32, 129.97 (2C), 137.21, 144.66, 145.17, 149.41, 151.90,
167.92, 179.89, 181.71, 198.39. HRMS (Mþ): m/z calcd for
C23H16N2O4: 384.11101; found: 384.11051.
3-H), 2.89 (t, J ¼ 6.4 Hz, 2H, 2-H), 2.98 (s, 3H, Me), 3.06 (t, J ¼ 6.4 Hz,
2H, 4-H), 3.92 (s, 3H, OMe), 6.47 (s, 1H, 9-H), 6.95 (d, J ¼ 8.4 Hz, 1H,
60-H), 7.00 (m. 1H, 50-H), 7.14 (dd, J ¼ 7.6; 8.4 Hz, 1H, 40-H), 7.37
(d. J ¼ 7.6 Hz, 1H, 30-H), 8.04 (s, 1H, NH). 13C NMR (400 MHz, CDCl3):
d
21.48, 26.47, 33.27, 39.21, 55.91, 103.64, 111.29, 120.95, 121.00,
5.4.9. 8-Phenylamino-6-(3-thiophen-3-yl)-3,4-
122.66, 125.80, 126.64, 128.12, 143.59, 143.84, 151.15, 162.38, 167.59,
181.64, 182.05, 198.59. HRMS (Mþ): m/z calcd for C21H18N2O4:
362.12665; found: 362.12555.
dihydrophenanthridine-1,7,10(2H)-trione (18)
Prepared from quinone 3c aꢀn1d aniline 39 (1:30 h, 45%), red solid
mp 168e170 ꢁC. IR (KBr): cm 3443 (NeH), 1682 (C]O), 1614,
1591 (C]O quinone). 1H NMR (400 MHz, CDCl3)
d: 2.24
5.4.5. 8-(4-Fluorophenylamino)-6-methyl-3,4-
dihydrophenanthridine-1,7,10(2H)-trione (14)
(q, J ¼ 6.4 Hz, 2H, 3-H), 2.92 (t, J ¼ 6.4 Hz, 2H, 2-H), 3.11 (t, J ¼ 6.4 Hz,
2H, 4-H), 6.45 (s, 1H, 9-H), 7.22 (m, 3H, phenyl), 7.25 (s, 1H, thienyl),
7.39 (m, 3H, phenyl and thienyl), 7.52 (s, 1H, NH), 7.72 (m, 1H,
A suspension of p-fluoronitrobenzene (141.1 mg, 1 mmol), iron
powder (1 g, 17.9 mmol) and a 1:1:1 mixture of water/ethanol/
acetic acid (45 mL) was stirred for 1 h at 50-60 ꢁC. The mixture was
neutralized with NaHCO3 and then extracted with ethyl acetate
(2 ꢃ15 mL). The organic extract was dried over NaSO4, filtered and
evaporated under reduced pressure to yield crude p-fluoroaniline.
Quinone 3b was reacted with p-fluoraniline under the standard
conditions (20 min, 40%) to give compound 14 (91%), red solid, mp
121.5e123 ꢁC; IR (KBr): cmꢀ1 3341 (NeH), 1678 (C]O), 1614, 1594
thiophenyl). 13C NMR (400 MHz, CDCl3)
d: 21.56, 33.51, 39.34,
103.73, 122.47, 122.61(2C), 125.13 (2C), 126.14, 127.59, 128.59,
128.71, 129.99,137.22 , 140.54, 144.54, 144.90, 156.51, 168.02, 180.52,
181.93, 198.60. HRMS (Mþ): m/z calcd for C23H16N2O3S: 400.08816;
found: 400.08753.
5.5. 8-(2,5-Dimethoxyphenylamino)-1-hydroxyphenanthridine-
7,10-diona (19)
(C]O quinone). 1H NMR (400 MHz, CDCl3):
d
2.24 (q, J ¼ 6.4 Hz, 2H,
3-H), 2.89 (t, J ¼ 6.4 Hz, 2H, 2-H), 2.97 (s, 3H, Me), 3.07 (t, J ¼ 6.4 Hz,
A suspension of aminoquinone 7a (46.7 mg), Pd(OAc)2 (52 mg)
and glacial acetic acid (5 mL) was heated to reflux for 17 h. The
reaction mixture was cooled, neutralized with solid sodium
hydrogencarbonate and filtered. The filtrate was diluted with water
(23 mL) and then extracted with ethyl acetate (2 ꢃ 15 mL). The
organic extract was washed with water (2 ꢃ 15 mL), dried over
sodium sulfate, filtered and evaporated under reduced pressure.
The residue was chromatographed on silicagel (CH2Cl2) to give
quinone 19 (32 mg, 69%), as violet solid, mp 200e202 ꢁC. IR (KBr):
cmꢀ1 3443 (NeH), 1650, 1580 (C]O quinone). 1H RMN (400 MHz,
2H, 4-H), 6.23 (s, 1H, 9-H), 7.12 (m, 2H, arom), 7.22 (m, 2H, arom),
7.54 (s, 1H, NH). 13C NMR (400 MHz, CDCl3):
d 21.55, 26.55, 29.88,
33.38, 39.29, 103.25, 116.28, 117.02, 122.65, 125.10, 125.18, 128.33,
133.15, 143.61, 145.39, 162.57, 167.93, 181.59, 182.15, 198.79. HRMS
(Mþ): m/z calcd for C20H15FN2O3: 350.10666; found: 350.10692.
5.4.6. 8-(2,5-Dimethoxyphenylamino)-6-methyl-3,4-
dihydrophenanthridine-1,7,10(2H)-trione (15)
Prepared from 3b and 2,5-dimethoxyaniline, (2:40 h, 48%),
purple solid, mp 165e167.5 ꢁC; IR (KBr): cmꢀ1 3401 (NeH), 1674
(C]O), 1626, 1556 (C]O quinone). 1H NMR (400 MHz, CDCl3):
CDCl3): d 3.83 (s, 3H, OMe), 3.91 (s, 3H, OMe), 6.69 (s, 1H, 9-H), 6.74
(dd, J ¼ 8.8, 2.8 Hz, 1H, 40-H), 6.92 (d, J ¼ 8.8 Hz, 1H, 30-H), 7.02 (d,

J.A. Valderrama et al. / European Journal of Medicinal Chemistry 46 (2011) 3398e3409
3409
J ¼ 2.8 Hz,1H, 60-H), 7.23 (m,1H, 3-H), 7.72 (m,1H, 2-H), 7.81 (m,1H,
5.8. Electrochemical measurements [8]
4-H); 8.30 (s, 1H, NH); 9.57 (s, 1H, 6-H), 13.66 (s, 1H, eOH). 13C RMN
(400 MHz, CDCl3):
d
55.97, 56.32, 104.80, 108.30, 110.71, 112.08,
Cyclic voltammograms of compounds were obtained on a Bio-
analytical Sytem BAS CV-50W electrochemical analyzer. A small
capacity measuring cell was equipped with a platinum disc as
working electrode, a Ag/10 nM Ag (MeCN) reference electrode for
non aqueous solvent with a platinum wire auxiliary electrode,
a mechanical mini-stirrer and a capillary to supply an inert argon
114.18, 116.39, 121.50, 121.75, 126.35 (2C), 134.69, 142.50, 145.75,
146.97, 153.86, 153.91, 156.33, 181.17, 188.45. HRMS (Mþ): m/z calcd
for C21H16N2O5: 380.13722; found: 380.13719.
5.6. 9-(2,5-Dimethoxyphenylamino)-1-hydroxyphenanthridine-
atmosphere. A 0.1 M solution of tetrabutylammonium tetra-
7,10-dione (20)
fluoroborate in acetonitrile was used as supporting electrolyte.
According to the procedure for the preparation of compound 19,
quinone 20 was synthesized in 63% from 7b (61.4 mg) and Pd(OAc)2
(68 mg) after 19 h reflux. Compound 20 was isolated by column
chromatography as a pure violet solid, mp 206e207 ꢁC. IR (KBr):
cmꢀ1 3399 (NeH), 1620, 1566 (C]O quinone). 1H RMN (200 MHz,
Acknowledgement
We thank the Fondo Nacional de Ciencia y Tecnología (Grants Nꢁ
1060591 and 1100376) for financial support to this study.
CDCl3): d 3.83 (s, 3H, OMe), 3.91 (s, 3H, OMe), 6.66 (s, 1H, 8-H), 6.73
References
(dd, J ¼ 9.0, 3.4 Hz, 1H, 40-H), 6.90 (d, J ¼ 9.0 Hz, 1H, 30-H), 7.02 (d,
J ¼ 3.5 Hz,1H, 60-H), 7.26 (m,1H, 3-H), 7.77 (m,1H, 2-H), 7.81 (m,1H,
4-H), 8.29 (s, 1H, NH), 9.56 (s, 1H, 6-H), 13.61 (s, 1H, OH). HRMS
(Mþ): m/z calcd for C21H16N2O5: 380.13722; found: 380.13710.
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