Studies on quinones. Part 46. Synthesis and in vitro antitumor evaluation of aminopyrimidoisoquinolinequinones

Article abstract of DOI:10.1016/j.ejmech.2010.08.040

In the search of structure-activity relationship studies and to explore the antitumor effect associated with the pyrimidoisoquinolinequinone scaffold, several diversily substituted 8-aminopyrimido[4,5-c]isoquinolinequinones were regioselectively synthesized. Variation in the structure of the nitrogen substituent bonded to the 8-position of the pyrimidoisoquinolinequinone system led to a set of alkylamino-, phenylamino- and alkyphenylamino derivatives. The cytotoxic activity of the aminoquinone derivatives was evaluated in vitro using the MTT colorimetric method against one normal cell line (MRC-5 lung fibroblasts) and four human cancer cell lines (AGS human gastric adenocarcinoma; SK-MES-1 human lung cancer cells, and J82 human bladder carcinoma; HL-60 human leukemia) in 72-h drug exposure assays. Among the series, five compounds exhibited interesting antitumor activity against AGS human gastric adenocarcinoma and human lung cancer cells. The SAR studies revealed 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.

Full text of DOI:10.1016/j.ejmech.2010.08.040

European Journal of Medicinal Chemistry 45 (2010) 5234e5242
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Studies on quinones. Part 46. Synthesis and in vitro antitumor evaluation of
aminopyrimidoisoquinolinequinonesq
,
,
,d
David Vásquez a d, Jaime A. Rodríguez b 1, Cristina Theoduloz b, Pedro Buc Calderon c
,
Jaime A. Valderrama a
,d,
*
a Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 6094411, Chile
b Facultad de Ciencias de la Salud, Universidad de Talca, Chile
c Université Catholique de Louvain, Louvain Drug Research Institute, Toxicology and Cancer Biology Research Group, PMNT Unit, 73, avenue E. Mounier, 1200 Bruxelles, Belgium
d Instituto de EtnoFarmacología (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 the search of structureeactivity relationship studies and to explore the antitumor effect associated
with the pyrimidoisoquinolinequinone scaffold, several diversily substituted 8-aminopyrimido[4,5-c]
isoquinolinequinones were regioselectively synthesized. Variation in the structure of the nitrogen
substituent bonded to the 8-position of the pyrimidoisoquinolinequinone system led to a set of alky-
lamino-, phenylamino- and alkyphenylamino derivatives. The cytotoxic activity of the aminoquinone
derivatives was evaluated in vitro using the MTT colorimetric method against one normal cell line (MRC-
5 lung fibroblasts) and four human cancer cell lines (AGS human gastric adenocarcinoma; SK-MES-1
human lung cancer cells, and J82 human bladder carcinoma; HL-60 human leukemia) in 72-h drug
exposure assays. Among the series, five compounds exhibited interesting antitumor activity against AGS
human gastric adenocarcinoma and human lung cancer cells. The SAR studies revealed 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.
Received 3 June 2010
Received in revised form
12 August 2010
Accepted 16 August 2010
Available online 21 August 2010
Keywords:
Aminopyrimido[4,5-c]isoquinolinequinones
Nucleophilic substitution
Antitumor activity
MTT assay
SAR analysis
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
are mainly due to the inhibition of DNA topoisomerase-II [3,4].
Because of the complex structure of most antitumor quinonoid
Cancer, second cause of mortality in the world, is characterized by
aderegulation of the cell cycle, which results ina progressive loss of the
cellular differentiation and a non-controlled cellular growth. Despite
the progress achieved in medicine during century, cancer is still
a leading life threatening pathology. Therefore, there is an increasing
need for new therapies, especially those that are based on current
knowledge of cancer biology as well as that taking advantage of the
cancer cells phenotype, described by Hanahan and Weinberg [2].
Quinones are widespread in nature and are present in many
drugs such as anthracyclines, daunorubicin, doxorubicin, mito-
mycin, mitoxantrones, and saintopin, which are used clinically in
the therapy of solid cancers. The cytotoxic effects of these quinones
compounds it is often difficult to separate the contributions of
chemical reactivity and the different pathways of metabolism to
overall biological activity. The quinoid anticancer agents undergo
enzymatic reduction via one or two electrons to give the corre-
sponding semiquinone radical or hydroquinone. Under aerobic
conditions the semiquinone radical anion can give its extra electron
to molecular oxygen to give the parent quinone and superoxide
radical anion. This reaction sequence, initiated by bioreduction of
the quinone followed by oxidation with dioxygen of the radical
anion intermediate, is known as redox-cycling, and it continues
until the system becomes anaerobic. The hydroquinone formed via
a two electron reduction, depending upon its stability, can be
excreted by the organism in a detoxification pathway or can
undergo a comproportianation reaction with the parent quinone to
yield the semiquinone radical anion. Both the semiquinone and the
superoxide radical anion can generate the hydroxyl radical, which
is the cause of DNA strand breaks [5e8].
q
For Part 45 of this series see Ref. [1].
* Corresponding author. Facultad de Química, Pontificia Universidad Católica de
Chile, Casilla 306, Santiago 6094411, Chile. Tel.: þ56
2
6864432; fax: þ56
2
6864744.
The molecular framework of several naturally occurring anti-
tumor agents contains an aminoquinonoid moiety as the key
E-mail address: jvalderr@uc.cl (J.A. Valderrama).
In memorial to Dr. Jaime A. Rodríguez who died on 13/10/09.
1
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.040

D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
5235
structural component (e.g. mitomycin C, cribrostatin 3, streptoni-
grin), [9e11]. This structural array has stimulated the synthesis of
novel compounds endowed with cytotoxic activity on human cancer
cell lines [1,12e14].
The structures of the new compounds 3e29 were established on
the basis of their nuclear magnetic resonance (1H NMR, 13C NMR,
2D NMR) spectra and elemental analyses. The location of the
nitrogen substituent at 8-position in compounds 3e15 was deter-
mined by means of HMBC experiments. For example, amino-
In a previous work we have reported that substituted 7-amino-
isoquinoline-5,8-quinones display antitumor activity against
several cell lines [1]. The QSAR analysis reveals that the half-wave
potential of 7-phenylaminoisoquinoline-5,8-quinones is an impor-
tant parameter determining the antitumoral activity on AGS gastric
adenocarcinoma and J82 bladder carcinoma cell lines. Recently, we
have reported preliminary results on the preparation and antitumor
evaluation of a number of aminopyrimido[4,5-c]isoquinoline-7,10-
quinones [15]. The results suggest that derivatives of this amino-
quinone scaffold might be goodcandidates as antitumorcompounds
and prompted us to explore the SAR of aminopyrimido[4,5-c]-
isoquinoline-7,10-quinones. Herein, we wish to report full details on
the regioselective synthesis of a broad variety of 8-aminopyrimido
[4,5-c]isoquinoline-7,10-quinone derivatives and the in vitro anti-
tumor evaluation on normal human lung fibroblasts MRC-5 and four
human tumor cells: AGS gastric adenocarcinoma, SK-MES-1 lung,
J82 bladder carcinoma and HL-60 leukemia human cancer cell lines.
3
quinone 3 displayed JC,H couplings between the carbon at C-7
(d
179.5), the proton at C-6 (
d
9.30), the proton at C-9 (d6.52) and the
proton of the NeH group (
d
7.49). In the case of aminoquinones that
lack of the amino proton, such as compound 10, the location of the
nitrogen substituent was established by 3JC,H coupling between the
carbon at C-7 (
the proton at C-9 (
the carbon at C-9 (
nitrogen substituent. The observed NOE enhancement between
d
179.4) and the protons at C-6 (
6.17), together with the 4JC,H coupling between
111.9) and the methyl protons ( 3.43) of the
d9.07), and
d
d
d
the methyl protons (d 3.43) and the proton at C-9 (d 6.52) supported
the assignment.
Regarding the position of the nitrogen substituent in
3
compounds 16e29, this was deduced by the JC,H coupling of C-7
with the proton at C-9, the proton of the amino group, together
with the 4JC,H coupling of C-7 with the protons of the methyl group
at C-6. The location of the nitrogen substituent in compounds 24,
25 and 27 was deduced in a similar manner to that mentioned for
compound 10.
2. Results and discussion
On the basis of results reported by Pratt [16] on the catalytic
action of a Lewis acid, such as cerium chloride, to promote regio-
selective 6-amination reactions of quinoline-5,8-quinone and its
further applications on the regioselective synthesis of amino-N-
heterocyclic quinones [1,17e21], we assume that the effect of the
catalyst CeCl3$7H2O to induce the attack of the amines at the 8-
position in quinones 1 and 2 may be ascribed to coordination of the
cerium ion to the heterocyclic nitrogen atom and/or the carbonyl
group at the C-10. The coordination 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 substitu-
tion via nucleophilic attack by the amines.
2.1. Chemistry
The synthesis of the aminopyrimido[4,5-c]isoquinolinequinones
was achieved by amination reaction of quinones 1 and 2 with a variety
of primary and secondary amines in ethanol in the presence of 5% mol
of CeCl3$7H2O and under aerobic conditions (Scheme 1). The nucleo-
philic substitution reaction takes place at room temperature to give,
under regiospecific manner, the corresponding aminoquinones in
moderate to good yields. The low yield formation of aminoquinones 15
and 29 probably is due to the fact that 1-adamantylamine has a low
nucleophilicity due to steric hindrance around the nitrogen atom. Since
the nucleophilic substitution on quinone 2 with p-nitroaniline was
unsuccessful, the nitrophenyl derivative 20 was prepared, albeit in low
yields, by microwave irradiation (MWI) of the precursors loaded on the
acid clay montmorillonite KSF (Scheme 1). Table 1 summarizes the
results of the preparation of the 8-aminopyrimidoisoquinolinequinone
derivatives 3e29.
2.2. Cytotoxic activities
The newly synthesized 8-aminopyrimidoisoquinolinequinone
derivatives 3e13, 15e29 and their precursors 1 and 2 were evalu-
ated for in vitro anticancer activity against normal human lung
Scheme 1. Synthesis of 8-aminopyrimido[4,5-c]isoquinolinequinone derivatives.

5236
D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
Table 1 (continued)
Table 1
Substituent types and yield of the synthesized pyrimidoisoquinolinquinones 3e29.
R3R2N
Compound R1 Yield (%) Compound R1
Yield (%)
14
15
H
H
49
6
28
29
Me 65
Me 16
fibroblasts MRC-5 and four human tumor cells: AGS gastric
adenocarcinoma, SK-MES-1 lung, J82 bladder carcinoma and HL-60
leukemia in 72-h drug exposure assays. The cytotoxicity of the
compounds was measured using a conventional microculture
R3R2N
Compound R1 Yield (%) Compound R1
Yield (%)
3
4
H
H
99
40
16
17
Me 99
tetrazolium reduction assay [22e24]. The average IC50 values (mM)
are collected in Table 2. The broad variety of the synthesized
compounds was designed in order to gain insight upon the effects
of the substituted amino group at C-8 and the methyl group at C-6
of the pyrimidoisoquinolinequinones on the biological activity. The
cytotoxic and antitumor activities are summarized in Table 2.
The initial structureeactivity relationship (SAR) focused on the
effects of insertion of phenyl-, phenylalkyl- and alkylamino groups
at the 8-position in precursors 1 and 2. The data of Table 2 indicate
that insertion on quinone 1 leads to a variety of aminoquinones (3,
5e15) with enhanced cytotoxic and antitumor activity compared to
that of their precursor 1, except for compound 4. A similar effect is
observed in quinone 2 where the insertion of substituted amino
groups at the 8-position increases the biological activity respect to
their precursor, except for compounds 21, 23 and 25. It is note-
worthy that the presence of nitrogen substituents on the biological
activity is remarkable in terms of the antitumor activity on gastric
adenocarcinoma cells, particularly for compounds 16, 19 and 20
which are 82-fold more active than precursor 2.
Next, theSARanalysis wasfocused ontheeffectsof themethyl and
nitrogen substituent into the pyrimidoisoquinolinequinone chro-
mophore. The data of Table 2 indicate that insertion of a methyl group
at the 6-position of the phenylaminoquinones 3, 4 and 6, as in 16, 17
and19, induces anincreaseofthecytotoxic and antitumoractivity. On
the contrary, introduction of a methyl group at the 6-position in
phenylaminoquinones 5, as in 18, induces a decrease of the biological
activity. These effects produced by the methyl group in such position,
could be attributed to its electronic and/or lipophilic properties,
which modify the ability of the pharmacophore to interact with the
biological targets involved in the cancer cell death. Comparison of the
biological activity of the phenylaminoquinone 16 to that of its 6-
methyl substituted analogues 17e25 indicates that insertion in 16, of
the electron-donor substituents OH and OMe at para-position,
induces a decrease of the cytotoxic and antitumor activity. In the case
of the electron-withdrawing substituents located at para-position,
such as F and NO2, no significant effect on the biological activity is
detected. These facts clearly demonstrate the influence on the bio-
logical activity of the donoreacceptor properties of the nitrogen
substituent into the pyrimidoisoquinolinquinone pharmacophore.
As can be seen in Table 2, insertion in 16 of a methoxy group at
the ortho-position, as in 21 and 23, or replacement of the amino
proton by an ethyl group, as in 25, produces a decrease of cytotoxic
and antitumor activity. Taking into account precedents reported by
Aguilar-Martínez [25] concerning the effect of substituents in the
aniline ring on the conjugation degree of the nitrogen lone pair
with the quinone system in anilino-1,4-naphthoquinones, the
effects of substituents in 21, 23 and 25 on the biological activity
could be related with a low conjugation degree between the donor
Me 46
Me 99
Me 72
5
6
H
H
e
99
91
e
18
19
20
e
Me 39
Me 95
Me 65
7
8
H
H
95
74
21
22
9
H
81
23
Me 79
10
11
H
H
61
55
24
25
Me 63
Me 58
nC4H9NHe
12
13
H
H
75
74
26
27
Me 75
Me 78

D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
5237
Table 2
Cytotoxic activity of aminopyrimidoisoquinolinquinone derivatives and their precursors.
IC50 ꢀ SEMa
(mM)
Nꢁ
MRC-5b
AGSc
SK-MES-1d
J82e
HL-60f
1
3
4
5
6
7
8
9
10
11
12
13
15
2
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Etoposide
39.9 ꢀ 2.2
9.2 ꢀ 0.6
47.4 ꢀ 3.8
20.5 ꢀ 1.2
6.4 ꢀ 0.3
8.2 ꢀ 0.6
16.6 ꢀ 1.2
30.9 ꢀ 1.5
4.3 ꢀ 0.2
7.8 ꢀ 0.5
8.4 ꢀ 0.4
7.9 ꢀ 0.4
6.1 ꢀ 0.4
72.8 ꢀ 3.4
4.6 ꢀ 0.3
17.4 ꢀ 1.0
49.6 ꢀ 2.5
4.0 ꢀ 0.2
5.9 ꢀ 0.3
>100
75.2 ꢀ 3.9
2.5 ꢀ 0.2
15.5 ꢀ 0.9
2.8 ꢀ 0.2
3.0 ꢀ 0.2
4.0 ꢀ 0.3
5.2 ꢀ 0.4
9.1 ꢀ 0.5
2.1 ꢀ 0.1
3.9 ꢀ 0.3
2.5 ꢀ 0.2
4.6 ꢀ 0.3
2.0 ꢀ 0.1
82.1 ꢀ 4.4
1.0 ꢀ 0.1
3.3 ꢀ 0.3
5.5 ꢀ 0.3
1.0 ꢀ 0.1
1.0 ꢀ 0.1
>100
1.9 ꢀ 0.1
31.7 ꢀ 1.9
4.7 ꢀ 0.2
19.2 ꢀ 1.3
5.2 ꢀ 0.4
3.9 ꢀ 0.3
2.2 ꢀ 0.1
4.2 ꢀ 0.2
0.36 ꢀ 0.02
>100
95.3 ꢀ 4.4
10.2 ꢀ 0.6
41.3 ꢀ 2.5
15.6 ꢀ 0.9
9.4 ꢀ 0.5
13.4 ꢀ 0.7
21.3 ꢀ 1.7
43.7 ꢀ 2.2
14.0 ꢀ 0.7
8.9 ꢀ 0.9
11.0 ꢀ 0.7
8.8 ꢀ 0.6
9.3 ꢀ 0.6
78.6 ꢀ 3.9
3.8 ꢀ 0.2
7.0 ꢀ 0.4
24.2 ꢀ 1.2
7.7 ꢀ 0.5
5.0 ꢀ 0.4
>100
43.6 ꢀ 2.5
9.3 ꢀ 0.6
>100
15.8 ꢀ 1.1
7.6 ꢀ 0.6
47.5 ꢀ 2.9
7.6 ꢀ 0.5
>100
20.8 ꢀ 1.7
12.9 ꢀ 0.9
9.7 ꢀ 0.8
4.6 ꢀ 0.2
9.5 ꢀ 0.8
31.5 ꢀ 1.7
5.7 ꢀ 0.4
11.6 ꢀ 0.9
>100
7.2 ꢀ 0.6
55.9 ꢀ 2.8
6.5 ꢀ 0.5
5.6 ꢀ 0.4
5.7 ꢀ 0.3
17.3 ꢀ 0.9
30.9 ꢀ 1.5
9.3 ꢀ 0.7
8.5 ꢀ 0.7
6.3 ꢀ 0.3
3.3 ꢀ 0.3
4.5 ꢀ 0.4
67.5 ꢀ 3.6
2.9 ꢀ 0.2
3.9 ꢀ 0.2
16.0 ꢀ 0.8
2.6 ꢀ 0.2
3.2 ꢀ 0.3
>100
5.6 ꢀ 0.5
2.9 ꢀ 0.2
>100
6.2 ꢀ 0.4
2.3 ꢀ 0.1
6.9 ꢀ 0.5
20.9 ꢀ 1.3
>100
>100
>100
>100
12.1 ꢀ 0.7
87.7 ꢀ 4.4
19.9 ꢀ 1.4
4.8 ꢀ 0.2
11.8 ꢀ 5.9
25.6 ꢀ 2.0
3.9 ꢀ 0.21
27.3 ꢀ 1.6
83.9 ꢀ 4.2
15.2 ꢀ 1.0
6.4 ꢀ 0.3
15.6 ꢀ 1.1
16.1 ꢀ 0.8
2.5 ꢀ 0.15
29.2
>100
>100
>100
17.9 ꢀ 0.9
17.3 ꢀ 1.2
14.1 ꢀ 0.7
26.8 ꢀ 1.3
2.8 ꢀ 0.18
19.7 ꢀ 1.6
33.8 ꢀ 1.7
29.9 ꢀ 2.4
20.8 ꢀ 1.5
0.80 ꢀ 0.04
a
Data represent mean average values for six independent determinations.
Normal human lung fibroblasts cells.
Human gastric adenocarcinoma cell line.
Human lung cancer cell line.
b
c
d
e
f
Human bladder carcinoma cell line.
Human leukemia cell line.
and acceptor fragments due to the molecular planarity inhibition
by steric hindrance.
the assays. Among the compounds evaluated in the in vitro screen,
derivatives 5, 16, 19, 20 and 22 (Fig. 3) were selected for this study
as the more significant antitumor members. Although compound 5
exhibited less antitumor activity than compounds 16, 19, 20 and 22,
it was included into the selected group since it showed the highest
selectivity index for AGS and SK-MES-1 cell lines.
No linear correlation could be found between the IC50 of the
evaluated phenylaminoquinones with any single variable such as
electronic, lipophilic or steric parameters (data not shown).
Therefore, a combination of these molecular descriptors could
explain the differences on the antitumor activity.
It is noteworthy that insertion of a fluorine atom at the ortho-
position in compounds 3 and 16, as in 8 and 22, induces a decrease of
the cytotoxic and antitumor activity respect to their precursors.
However, no significant influence on these biological properties was
observed when a fluorine atom is substituted at the para-position in
compounds 3 and 16, as in 6 and 19. These results demonstrate that
the ortho-fluorophenylamino groupexerts a greater influence on the
antitumor activity than that of its para-isomers.
Regarding the effect of the structure of the alkylamino group at
8-position in the pyrimidoisoquinolinquinone chromophore, in
particular n-butyl- and adamantyl- substituents (compounds 12/26
and 15/29), no significant differences on the biological activity were
observed. These facts suggest that the structure of the alkylamino
group, in these compounds, does not significantly influence on the
cytotoxic activity. It should be noted that the introduction of
a methyl group in compounds 12, 13 and 15 leads to the analogues
26, 27 and 29 with lower activity.
Analysis of the data in Table 2 indicates that, in general, cyto-
toxity was observed in all the cancer cell lines but that AGS and SK-
MES-1 cell lines appeared to be more sensitive to the compounds
overall. Aminoquinones 3, 5, 6, 12, 15, 16, 19, 20 and 22 exhibit
interesting antitumor properties on gastric adenocarcinoma and
lung cancer cells, expressed by their IC50 values and selectivity
index (IC50 fibroblasts/IC50 cancer cells) (Figs. 1 and 2).
3. Conclusion
We have developed the regiospecific synthesis of a variety of 8-
aminopyrimido[4,5-c]isoquinolinequinone
derivatives.
The
majority of the new aminoquinones expressed in vitro cytotoxic
activity against normal human lung fibroblasts (MRC-5) and on
gastric adenocarcinoma (AGS), lung cancer (SK-MES-1), and bladder
carcinoma (J82) cell lines. The substitution of phenylamino- and
alkylamino groups into the 8-position of quinones 1 and 2 increases
the cytotoxic potency, with respect to their precursors, in almost all
the evaluated cell lines. This substitution effect on the biological
activity is remarkable in the antitumor activity on gastric adeno-
carcinoma cells, for some of the aminoquinones prepared from 2,
which were 82-fold more active than the precursor.
Compounds 16, 19, 20 and 22 have antitumor activity and
selectivity index on lung cancer cells at levels comparable to those
obtained for the antitumor drug etoposide, which was included in
From the current investigation, structureeactivity relationships
of the phenylaminoquinone members suggest substituents at the
ortho or para-position of the phenyl ring will determine the

5238
IC50(µM)
D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
interesting lead, endowed of potential anticancer activity. These
results prompt us to design and synthesize more new amino-
pyrimido[4,5-c]isoquinolinequinone derivatives, structurally
25
20
15
10
5
MRC-5
AGS
related to 5, in order to discover new, more active and selective
anticancer agents.
4. Experimental
4.1. Chemistry
All reagents were commerciallyavailable reagent grade and were
used without further purification. Melting points were determined
on a Stuart Scientific SMP3 apparatus and are uncorrected. 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, NOESY)
and DEPT were used for signal assignment. Chemical shifts are
expressed in ppm downfield relative to tetramethylsilane (TMS,
0
Selectivity
index
3.7
3
7.3
5
2.1
6
3.4
12
3.1
15
4.6
16
4.0
19
5.9
20
3.3
22
10.8
Compound numbers
Fig. 1. Comparative MTT assay for MRC-5 and AGS cell lines.
IC50(µM)
d
scale), and the coupling constants (J) are reported in Hertz. The
25
20
15
10
5
elemental analyses were performed in a Fison SA, model EA-1108
apparatus. HRMS were obtained on a Thermo Finnigan spectrom-
eter, model MAT 95XP. The reaction induced by microwave irradia-
tion was performed in a 10 mL sealed tube with a single mode cavity
Discover Microwave apparatus. Silica gel Merck 60 (70e230 mesh)
was used for preparative column chromatography, and TLC
aluminum foil 60F254 for analytical TLC. Quinones 1 and 2 were
prepared according to a previously reported procedure [26].
MRC-5
SK-MES-1
0
4.2. General procedure for the synthesis of 8-aminopyrimidoisoquin
olinequinone derivatives
Selectivity
1.3
3.2
1.1
1.3
1.4
1.6
1.5
1.8
2.7
1.6
index
3
5
6
12
15
16
19
20
22
Compound numbers
Fig. 2. Comparative MTT assay for MRC-5 and SK-MES-1.
A suspension of quinone 1 (40 mg, 0.1476 mmol) or 2 (50 mg,
0.1753 mmol), the required amine (2 equiv.), CeCl3$7H2O (5% mmol
respect to 1 or 2), and ethanol (5 mL) was left with stirring at room
temperature until completion of the reaction as indicated by TLC.
The reaction mixture was evaporated under reduced pressure and
the residue was column chromatographed (1:1:2 AcOEt/CH2Cl2/
light petroleum) toyield the corresponding aminoquinone (Table 1).
antitumor activity. The effect of such substitutions were more
significant in enhancing the antitumor activity for the 6-methyl
substituted 8-phenylaminopyrimido[4,5-c]isoquinolinequinones
than those of the 6-unsubstituted analogs.
Pyrimido[4,5-c]isoquinolinequinone derivatives 5, 16, 19, 20 and
22 exhibited interesting antitumor activity and selective index
comparable to that of reference drug etoposide. Given the high
incidence of undesirable side-effects induced by the majority of
currently anticancer drugs and by considering the selective index of
aminoquinone 5, this compound appeared as promising and
4.2.1. 2,4-Dimethyl-2H, 4H-8-(phenylamino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (3)
Prepared from 1 and aniline (3 h, 99%): red solid, mp 236e237 ꢁC;
1H NMR (400 MHz, CDCl3):
d 3.50 (s, 3H, 2-NMe), 3.77 (s, 3H, 4-NMe),
6.52 (s,1H, 9-H), 7.25 (m, 3H, 20-, 40- and 60-H), 7.44 (t, 2H, J ¼ 7.9 Hz,
30- and 50-H), 7.49 (s,1H, NeH), 9.30 (s,1H, 6-H); 13C NMR (100 MHz,
Fig. 3. Structure of the selected as the more significant antitumor members.

D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
5239
CDCl3):
d
29.2, 30.6, 105.7, 107.1, 121.4, 122.4, 125.9, 129.8, 136.9,
7.49 (s, 1H, NeH), 9.31 (s, 1H, 6-H); 13C NMR (100 MHz, CDCl3):
d
124.9 (d, 1C, J ¼ 3.5 Hz, 60-C,), 125.5 (d, 1C, J ¼ 11.6 Hz, 10-C), 126.6 (d,
1C, J ¼ 7.5 Hz, 40-C), 146.8, 151.4, 152.6, 155.1 (d, 1C, J ¼ 248.3 Hz, 20-
C), 155.6, 155.6, 158.5, 179.7, 180.6. Anal. Calcd. for C19H13N4O4: C,
60.00; H, 3.45; N, 14.73. Found: C, 59.12; H, 3.19; N, 14.27.
143.4, 145.5, 150.9, 152.3, 155.2, 158.3, 179.5, 180.9. Anal. Cald. for
C19H14N4O4: C, 62.98; H, 3.89; N, 15.46. Found: C, 62.88; H, 3.52; N,
15.32.
29.2, 30.8, 106.1, 107.1, 116.5 (d, 1C, J ¼ 19.7 Hz, 30-C), 121.2, 123.3,
4.2.2. 2,4-Dimethyl-2H, 4H-8-(40-hydroxyphenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (4)
Prepared from 1 and p-hydroxyaniline (6 h, 40%); violet solid, mp
4.2.7. 2,4-Dimethyl-2H, 4H-8-(20,50-dimethoxyphenylamino)
pyrimido[4,5-c]isoquinoline-1,3,7,10-tetraone (9)
305e306 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d 3.27 (s, 3H, 2-NMe),
3.59(s, 3H, 4-NMe), 5.93 (s,1H, 9-H), 6.83 (d, 2H, J ¼8.8 Hz, 20- and60-
Prepared from 1 to 2,5-dimethoxyaniline (6 h, 81%): violet
H), 7.18 (d, 2H, J ¼ 8.8 Hz, 30- and 50-H), 9.18 (s, 1H, 6-H), 9.20 (s, 1H,
solid mp 260e261 ꢁC; 1H NMR (400 MHz, CDCl3):
d 3.49 (s, 3H, 4-
NeH), 9.63 (s,1H, OeH); 13C NMR (100 MHz, DMSO-d6):
d
29.1, 30.6,
NMe), 3.76 (s, 3H, 2-NMe), 3.80 (s, 3H, OMe), 3.88 (s, 3H, OMe),
6.62 (s, 1H, 9-H), 6.67 (dd, 1H, J ¼ 8.9, 2.7 Hz, 40-H), 6.88 (d, 1H
J ¼ 8.9 Hz, 30-H), 6.97 (d, 1H, J ¼ 2.7 Hz, 60-H), 7.96 (s, 1H, NeH),
103.1, 107.4, 116.4, 122.1(2C), 125.9, 129.3(2C), 137.4, 145.4, 146.4,
151.2, 155.3, 155.9, 158.5, 179.8, 180.1. Anal. Calcd. for C19H14N4O5: C,
60.32; H, 3.73; N, 14.81. Found: C, 60.11; H, 3.83; N, 15.01.
9.27 (s, 1H, 6-H); 13C NMR (100 MHz, CDCl3):
d 29.4. 30.8. 56.1.
56.5. 106.4. 107.3. 107.5. 110.1. 112.2. 121.7. 127.4. 142.5. 145.4. 145.7.
151.1. 152.6. 154.1. 155.4. 158.5. 179.7. 181.1. Anal. Calcd. for
C21H18N4O6: C, 59.71; H, 4.30; N, 13.26. Found: C, 59.42; H, 4.08;
N, 13.33.
4.2.3. 2,4-Dimethyl-2H, 4H-8-(40-methoxyphenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (5)
Prepared from 1 and p-anisidine (3 h, 99%): red solid mp
236.5e237.5 ꢁC; 1H NMR (400 MHz, CDCl3):
d 3.49 (s, 3H, 2-NMe),
3.79 (s, 3H, 4-NMe), 3.85 (s, 3H, 40-OMe), 6.33 (s, 1H, 9-H), 6.96 (d,
2H, J ¼ 8.8 Hz, 30- and 50-H), 7.19 (d, 2H, J ¼ 8.8 Hz, 20- and 60-H), 7.36
4.2.8. 2,4-Dimethyl-2H, 4H-8-(N-methylphenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (10)
(s, 1H, NeH), 9.28 (s, 1H, 6-H); 13C NMR (100 MHz, CDCl3):
d
29.2,
Prepared from 1 and N-methylaniline (16 h, 61%): red solid mp
30.5, 55.5,104.7,107.1,115.0(2C),121.5,124.6(2C),129.5,145.7,150.9,
152.2,155.2, 157.9,158.3,179.6,180.7. Anal. Calcd. for C20H16N4O5: C,
61.22; H, 4.11; N, 14.28. Found: C, 60.74; H, 3.84; N, 14.10.
185e185.5 ꢁC 1H NMR (400 MHz, CDCl3):
d 3.43 (s, 3H, Me), 3.50 (s,
3H, 4-NMe), 3.74 (s, 3H, 2-NMe), 6.17 (s,1H, 9-H), 7.12 (d, 2H, 20- and
60-H), 7.32 (t, 1H, 40-H), 7.42 (t, 2H, 30- and 50-H), 9.07 (s, 1H, 6-H);
13C NMR (100 MHz, CDCl3):
d 29.2, 30.6, 43.1, 106.3, 111.9, 123.0,
4.2.4. 2,4-Dimethyl-2H, 4H-8-(40-fluorophenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (6)
125.4(2C), 126.9, 129.8(2C), 144.6, 147.2, 150.4, 151.0, 152.5, 154.7,
158.7, 179.4180.5. Anal. Calcd. for C20H16N4O4: C, 63.82; H, 4.28; N,
14.89. Found: C, 63.45; H, 3.98; N, 14.67.
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 50e60 ꢁC. The mixture was
neutralized with NaHCO3 and then extracted with ethyl acetate
(2 ꢂ 15 mL). The organic extract was dried over Na2SO4, filtered and
evaporated under reduced pressure to yield crude p-fluoroaniline.
Quinone 1 (40 mg, 0.1476 mmol) was reacted with p-fluoroaniline
under the standard conditions (3 h, rt) to give compound 6 (91%), as
4.2.9. 2,4-Dimethyl-2H, 4H-8-(N-ethylphenylamino)pyrimido[4,5-
c]isoquinoline-1,3,7,10-tetraone (11)
Prepared from 1 and N-ethylaniline (16 h, 55%): red solid mp
212e213 ꢁC 1H NMR (400 MHz, CDCl3):
CH3CH2), 3.49 (s, 3H, 4-NMe), 3.74 (s, 3H, 2-NMe), 3.87 (q, 2H,
J ¼ 7.09 Hz, CH3CH2), 6.09 (s, 1H, 9-H), 7.11 (m, 2H, 20- and 60-H),
7.34 (m, 1H, 40-H), 7.43 (m, 2H, 30- and 50-H), 9.07 (s, 1H, 6-H); 13C
d
1.28 (t, 3H, J ¼ 7.09 Hz,
red solid, mp 260e265 ꢁC 1H NMR (400 MHz, CDCl3):
d 3.49 (s, 3H,
4-NMe), 3.77 (s, 3H, 2-NMe), 6.35 (s, 1H, 9-H), 7.15 (m, 2H, 20- and
60-H), 7.25 (m, 2H, 30- and 50-H), 7.42 (s, 1H, NeH), 9.29 (s, 1H, 6-H).
NMR (100 MHz, CDCl3): d 11.7, 29.3, 30.6, 41.0, 106.4, 112.1, 113.1
(2C), 117.5, 122.8, 129.3(2C), 147.2, 149.3, 150.5, 151.1, 152.6, 155.5,
158.5, 179.5, 180.4. Anal. Calcd. for C21H18N4O4: C, 64.61; H, 4.65; N,
14.35. Found: C, 64.87, H, 4.72, N, 14.66.
13C NMR (100 MHz, CDCl3): 29.3, 30.7, 106.4, 107.4, 116.9 (d, 2C, 30-
d
and 50-C, J ¼ 22.7 Hz), 124.8 (d, 2C, 20- and 60-C, J ¼ 8.2 Hz), 133.4 (d,
1C, 40-C, J ¼ 2.7 Hz), 147.4, 150.7, 152.6, 155.8, 156.5, 158.6, 160.6,
179.4, 180.4. Anal. Calcd. for C19H13FN4O4: C, 60.00; H, 3.45; N,
14.73. Found: C, 60.04; H, 3.34; N, 14.60.
4.2.10. 2,4-Dimethyl-2H, 4H-8-(n-butylamino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (12)
Prepared from 1 and n-butylamine (3 h, 75%): red solid mp
4.2.5. 2,4-Dimethyl-2H, 4H-8-(20-methoxyphenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (7)
188e189 ꢁC 1H NMR (400 MHz, CDCl3):
d
0.99 (t, 3H, J ¼ 7.4 Hz,
CH3C3H6),1.45 (m, 2H, CH3CH2C2H4),1.70 (m, 2H, C2H5CH2CH2), 3.20
(m, 2H, CH2C3H7), 3.50 (s, 3H, 4-NMe), 3.77 (s, 3H, 2-NMe), 5.69 (s,
1H, 9-H), 5.82 (s, 1H, NeH), 9.39 (s, 1H, 6-H); 13C NMR (100 MHz,
Prepared from 1 and o-anisidine (5 h, 95%): red solid mp >300
(d); 1H NMR (400 MHz, CDCl3):
d 3.47 (s, 3H, 4-NMe), 3.74 (s, 3H, 2-
NMe), 3.90 (s, 3H, 20-OMe), 6.55 (s, 1H, 9-H), 6.98 (m, 2H, 40- and 50-
H), 7.14 (m, 1H, 60-H), 7.38 (m, 1H, 30-H), 7.89 (m, 1H, NeH), 9.27 (s,
CDCl3): d 13.7, 20.2, 29.3, 30.4, 30.6, 42.5, 103.4, 107.4, 116.4, 144.2,
150.7, 151.3, 151.5, 154.5, 158.4, 180.2, 180.4. Anal. Calcd. for
C17H18N4O4: C, 59.64; H, 5.30; N, 16.37. Found: C, 59.78; H, 5.45; N,
15.98.
1H, 6-H); 13C NMR (100 MHz, CDCl3):
d 29.2, 30.7, 55.6, 104.4, 107.0,
110.6, 114.9, 118.7, 121.3, 121.3, 136.5, 146.5, 147.5, 150.6, 152.4, 155.1,
156.4, 158.4, 179.8, 180.8. Anal. Calcd. for C20H16N4O5: C, 61.22; H,
4.11; N. 14.28. Found: C, 60.20; H, 3.88; N, 14.22.
4.2.11. 2,4-Dimethyl-2H, 4H-8-(4-morpholino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (13)
4.2.6. 2,4-Dimethyl-2H, 4H-8-(20-fluorophenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (8)
Quinone 1 (40 mg; 0.1476 mmol) was reacted with o-fluo-
roaniline [prepared from o-fluoronitrobenzene (141.1 mg, 1 mmol)
and iron powder (1 g, 17.9 mmol)] under the standard conditions
(6 h, rt) to give compound 8 (74%), red solid mp 207e208 ꢁC; 1H
Prepared from 1 and morpholine (4 h, 74%): red solid mp
198e199 ꢁC 1H NMR (400 MHz, CDCl3):
d 3.49 (s, 3H, 4-NMe),
3.56 (m, 4H, 30- and 50-H), 3.75 (s, 3H, 2-NMe), 3.86 (m, 4H, 20-
and 60-H,), 6.10 (s, 1H, 9-H), 9.20 (s, 1H, 6-H); 13C NMR (100 MHz,
CDCl3):
d 29.1, 30.5, 48.8(2C), 66.3(2C), 106.2, 112.0, 123.1, 144.1,
150.9, 151.5, 152.6, 154.6, 158.3, 180.3, 180.4. Anal. Calcd. for
C17H16N4O5: C, 57.30; H, 4.53; N, 15.72. Found: C, 57.02; H, 4.32;
N, 15.55.
NMR (400 MHz, CDCl3):
d 3.49 (s, 3H, 4-NMe), 3.77 (s, 3H, 2-NMe),
6.42 (s, 1H, 9-H), 7.23 (m, 3H, 40-, 50- and 60-H), 7.44 (m, 1H, 30-H),

5240
D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
4.2.12. 2,4-Dimethyl-2H, 4H-8-(cyclohexylamino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (14)
J ¼ 22.9 Hz, 30- and 50-C), 119.8, 124.8 (d, 1C, J ¼ 8.3 Hz, 20- and 60-C),
133.3 (d,1C, 40-C, J ¼ 2.7 Hz),145.2,147.2,150.9,152.7,158.6,160.6 (d,
1C, J ¼ 247 Hz, 40-C),165.5,180.0,181.8. Anal. Calcd. for C20H15FN4O4:
C, 60.91; H, 3.83; N, 14.21. Found: C, 60.72; H, 3.95; N, 13.37.
Prepared from 1 and cyclohexylamine (4 h, 49%): red solid mp
178e179 ꢁC 1H NMR (400 MHz, CDCl3):
d 1.33e2.05 (m, 10H,
5 ꢂ CH2), 3,30 (m, 1H, 10-H), 3.46 (s, 3H, 4-NMe), 3.72 (s, 3H, 2-
NMe), 5.80 (s, 1H, 9-H), 5.88 (d, 1H, J ¼ 5.9 Hz, NeH), 9.15 (s, 1H, 6-
4.2.18. 2,4,6-Trimethyl-2H, 4H-8-(40-nitrophenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (20)
H); 13C NMR (100 MHz, CDCl3):
d 24.4, 25.3, 29.4, 30.8, 32.0, 51.7,
104.6, 107.3, 121.3, 143.4, 145.5, 150.9, 153.4, 155.3, 158.4, 180.2,
180.4. Anal. Calcd. for C19H20N4O4: C, 61.95; H, 5.47; N,15.21. Found:
C, 61.77; H, 5.31; N, 15.03.
A mixture of p-nitroaniline (40.7 mg, 0.3506 mmol), quinone 2
(50 mg, 0.1753 mmol) and montmorillonite KSF (0.5 g) into a 10 mL
microwave vial was irradiated at 100 ꢁC and 100 W for 5 min. The
mixture was thoroughly washed with CH2Cl2, the extract dried over
Na2SO4 and evaporated under reduced pressure. The residue was
column chromatographed (1:1:7 CH2Cl2/ethyl acetate/light petro-
leum) to give 20 (22%): orange solid mp 269e270 ꢁC; 1H NMR
4.2.13. 2,4-Dimethyl-2H, 4H-8-(1-adamantylamino)pyrimido[4,5-
c]isoquinoline-1,3,7,10-tetraone (15)
Prepared from 1 and 1-adamantylamine (24 h, 6%): red solid mp
110e111 ꢁC 1H NMR (400 MHz, CDCl3):
d
1.73 (m, 6H, adamantyl),
(400 MHz, DMSO-d6): d 2.93 (s, 3H, 6-Me), 3.27 (s, 3H, 4-NMe), 3.58
2.03 (m, 6H, adamantyl), 2.19 (m, 3H, adamantyl), 3.49 (s, 3H, 4-
(s, 3H, 2-NMe), 6.57 (s, 1H, 9-H), 7.66 (d, 2H, J ¼ 9.1 Hz, 20- and 60-H),
NMe), 3.74 (s, 3H, 2-NMe), 5.81 (s, 1H, NeH), 6.14 (s, 1H, 9-H), 9.19
8.24 (d, 2H, J ¼ 9.1 Hz, 30- and 50-H), 9.70 (s, 1H, NeH); 13C NMR
(s, 1H, 6-H); 13C NMR (100 MHz, CDCl3):
d
29.3, 29.3, 30.7, 36.2, 41.2,
(100 MHz, DMSO-d6): d 26.5, 28.5, 29.8. 105.4. 106.4, 119.7, 121.4
52.8, 104.9, 107.5, 121.5, 144.3, 145.7, 150.7, 152.9, 155.4, 158.4, 180.3,
180.4. Anal. Calcd. for C23H24N4O4: C, 65.70; H, 5.75; N, 13.33.
Found: C, 65.54, H, 5.77, N, 13.27.
(2C), 125.1(2C), 142.4, 144.3, 145.0, 146.9, 150.8, 152.5, 158.0, 164.3,
179.5, 181.5. Anal. Calcd. for C20H15N5O6: C, 57.01; H, 3.59; N, 16.62.
Found: C, 56.76; H, 3.21; N, 17.01. HRMS (Mþ): m/z calcd for
C20H15N5O6 [Mþ]: 421.10223; found: 421.09972.
4.2.14. 2,4,6-Trimethyl-2H, 4H-8-(phenylamino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (16)
4.2.19. 2,4,6-Trimethyl-2H, 4H-8-(20-methoxyphenylamino)
pyrimido[4,5-c]isoquinoline-1,3,7,10-tetraone (21)
Prepared from
2 and aniline (2 h, 99%): red solid mp
222e223 ꢁC; 1H NMR (400 MHz, CDCl3):
d
3.05 (s, 3H, 6-Me), 3.51
Prepared from 2 and o-anisidine (6 h, 95%): red solid mp
(s, 3H, 2-NMe), 3.78 (s, 3H, 4-NMe), 6.51 (s, 1H, 9-H), 7.28 (m, 3H, 20-
266e267 ꢁC; 1H NMR (400 MHz, CDCl3):
d 3.03 (s, 3H, 6-Me), 3.48
, 40- and 60-H), 7.47 (t, 2H, J ¼ 8.0 Hz, 30- and 50-H), 7.65 (s, 1H, NeH).
(s, 3H, 4-NMe), 3.75 (s, 3H, 2-NMe), 3.92 (s, 3H, OMe), 6.55 (s, 1H, 9-
13C NMR (100 MHz, CDCl3):
d
27.0, 29.1. 30.2, 103.9, 106.2, 119.9,
H), 7.01 (m, 2H, 40- and 50-H), 7.16 (m, 1H, 60-H), 7.41 (m, 1H, 30-H),
122.3(2C), 125.7, 129.8(2C), 137.2, 144.5, 148.9, 151.2, 153.1, 158.6,
165.6, 180.2, 182.0. Anal. Calcd. for C20H16N4O4: C, 63.82; H, 4.28; N,
14.89. Found: C, 63.77; H, 3.99; N, 14.93.
8.06 (s, 1H, NeH). 13C NMR (100 MHz, CDCl3):
d 27.0, 29.1, 30.1, 55.7,
102.9, 106.7, 110.5, 114.9, 118.6, 119.9, 121.4, 136.3, 145.3, 147.5, 149.3,
151.4,153.2,158.3,165.2,180.4,180.8. Anal. Calcd. for C21H18N4O5: C,
62.06; H, 4.46; N, 13.79. Found: C, 61.46; H, 4.11; N, 13.48.
4.2.15. 2,4,6-Trimethyl-2H, 4H-8-(40-hydroxyphenylamino)
pyrimido[4,5-c]isoquinoline-1,3,7,10-tetraone (17)
4.2.20. 2,4,6-Trimethyl-2H, 4H-8-(20-fluorophenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (22)
Prepared from 2 and p-hydroxyaniline (6 h, 46%): red solid mp
281e281.5 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d
2.91 (s, 3H, 6-Me),
Quinone 2 (50 mg, 0.1753 mmol) was reacted with o-fluo-
roaniline [prepared from o-fluoronitrobenzene (141.1 mg, 1 mmol)
and iron powder (1 g,17.9 mmol)] to give 22 (3 h, 65%): red solid mp
3.25 (s, 3H, 2-NMe), 3.57 (s, 3H, 4-NMe), 5.87 (s, 1H, 9-H), 6.96 (d,
2H, J ¼ 8.9 Hz, 20- and 60-H), 7.19 (d, 2H, J ¼ 8.9, 30- and 50-H), 9.13 (s,
1H, NeH), 9.57 (s, 1H, OeH); 13C NMR (100 MHz, DMSO-d6):
d
27.0,
211e212 ꢁC; 1H NMR (400 MHz, CDCl3):
d 3.03 (s, 3H, 6-Me), 3.48 (s,
29.2, 30.2, 103.0, 106.6, 120.1, 122.1(2C), 125.9, 129.3(2C), 137.4,
145.9, 148.5, 151.2, 153.0, 158.6, 165.6, 180.2, 181.6. Anal. Calcd. for
C20H16N4O5: C, 61.22; H, 4.11; N, 14.28. Found: C, 61.58; H, 4.60; N,
14.61.
3H, 4-NMe), 3.75 (s, 3H, 2-NMe), 6.40 (s, 1H, 9-H), 7.21 (m, 3H, 40-,
50- and 60-H), 7.44 (m, 1H, 30-H), 7.61 (s, 1H, NeH); 13C NMR
(100 MHz, CDCl3):
d 26.9, 29.2, 30.1, 103.9, 107.2, 116.5 (d, 1C,
J ¼ 19.2 Hz, 30-C), 120.1, 123.3, 124.9 (d, 1C, J ¼ 3.7 Hz, 60-C), 125.5 (d,
1C, J ¼ 11.7 Hz,10-C),126.6 (d,1C, J ¼ 7.6 Hz, 40-C); 145.4,149.5,151.5,
153.4, 155.1 (d, 1C, J ¼ 248.1 Hz, 20-C), 158.4, 165.4, 180.1, 180.4. Anal.
Calcd. for C20H15FN4O4: C, 60.91; H, 3.83; N, 14.21. Found: C, 61.23;
H, 4.01; N, 14.33.
4.2.16. 2,4,6-Trimethyl-2H, 4H-8-(40-methoxyphenylamino)
pyrimido[4,5-c]isoquinoline-1,3,7,10-tetraone (18)
Prepared from 2 and p-anisidine (3 h, 99%): red solid mp
242e243 ꢁC; 1H NMR (400 MHz, CDCl3):
d 3.00 (s, 3H, 6-Me), 3.47 (s,
3H, 2-NMe), 3.74 (s, 3H, 4-NMe), 3.84 (s, 3H, OMe), 6.27 (s, 1H, 9-H),
4.2.21. 2,4,6-Trimethyl-2H, 4H-8-(20,50-dimethoxyphenylamino)
pyrimido[4,5-c]isoquinoline-1,3,7,10-tetraone (23)
6.94(d, 2H, J¼ 8.3Hz, 20-and60-H),7.18(d, 2H,J¼ 8.3Hz, 30-and50-H),
7.47 (s, 1H, NeH); 13C NMR (100 MHz, CDCl3):
d
26.9, 29.1, 30.2, 55.6,
Prepared from 2 to 2,5-dimethoxyaniline (6 h, 79%): red solid
103.0, 106.3, 115.0(2C), 120.0, 124.5(2C), 129.8, 145.4, 149.2, 151.2,
153.0, 157.8, 158.6. 165.6, 180.3, 181.7. Anal. Calcd. for C21H18N4O5: C,
62.06; H, 4.46; N, 13.79. Found: C, 62.17; H, 4.53; N, 13.87.
mp 255e256 ꢁC; 1H NMR (400 MHz, CDCl3):
d 3.02 (s, 3H, 6-Me),
3.48 (s, 3H, 4-NMe), 3.75 (s, 3H, 2-NMe), 3.80 (s, 3H, OMe), 3.89 (s,
3H, OMe), 6.59 (s, 1H, 9-H), 6.66 (dd, 1H, J ¼ 8.9, 2.6 Hz, 60-H), 6.88
(d, 1H, J ¼ 8.9 Hz, 30-H), 6.99 (d, 1H, J ¼ 2.6 Hz, 40-H), 8.1 (s, 1H,
4.2.17. 2,4,6-Trimethyl-2H, 4H-8-(40-fluorophenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (19)
NeH). 13C NMR (100 MHz, CDCl3):
d 27.0, 29.1, 30.2, 56.0, 56.3,104.4,
106.2,107.1,109.8,112.0,127.4,143.4,145.2,148.9,151.2,153.0,153.9,
158.6, 166.0. 180.2. 182.0. Anal. Calcd. for C22H20N4O6: C, 60.55; H,
4.62; N, 12.84. Found: C, 60.19; H, 4.49; N, 12.56.
Quinone 2 (50 mg, 0.1753 mmol) was reacted with p-fluoraniline
[prepared from p-fluoronitrobenzene (141.1 mg, 1 mmol) and iron
powder (1 g, 17.9 mmol)] to give 22 (3 h, 72%), red solid mp
216e217 ꢁC; 1H NMR (400 MHz, CDCl3):
d
3.00 (s, 3H, 6-Me), 3.47 (s,
4.2.22. 2,4,6-Trimethyl-2H, 4H-8-(N-methylphenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (24)
3H, 4-NMe), 3.74 (s, 3H, 2-NMe), 6.30 (s,1H, 9-H), 7.13 (m, 2H, 20- and
60-H), 7.24 (m, 2H, 30- and 50-H), 7.50 (s, 1H, NeH). 13C NMR
Prepared from 2 and N-methylaniline (16 h, 63%): red solid mp
(100 MHz, CDCl3):
d
26.9, 29.1, 30.2, 103.7, 106.3, 116.9 (d, 1C,
182.5e183.5 ꢁC (d). 1H NMR (400 MHz, CDCl3):
d 2.70 (s, 3H, 6-Me),

D. Vásquez et al. / European Journal of Medicinal Chemistry 45 (2010) 5234e5242
5241
3.41 (s, 3H, NMe), 3.48 (s, 3H, 4-NMe), 3.72 (s, 3H, 2-NMe), 6.10 (s,
4.3. Anticancer assay
1H, 9-H), 7.11 (d, 2H, J ¼ 7.6 Hz, 20- and 60-H), 7.27 (m, 1H, 40-H), 7.42
(t, 2H, J ¼ 7.8 Hz, 30- and 50-H); 13C NMR (100 MHz, CDCl3):
d
25.9,
The cell lines used in this work were obtained from the American
Type Culture Collection (ATCC, Manasas, VA, USA). They included
MRC-5 normal human lung fibroblasts (CCL-171), AGS human
gastric adenocarcinoma cells (CRL-1739), SK-MES-1 human lung
cancer cells (HTB-58), and J82 human bladder carcinoma cells (HTB-
1). After the arrival of the cells, they were proliferated in the corre-
sponding culture medium as suggested by the ATCC. The cells were
stored in medium containing 10% glycerol in liquid nitrogen. The
viability of the cells after thawing was higher than 90%, as assessed
by trypan blue exclusion test. Cells were sub-cultured once a week
and medium was changed every two days. Cells were grown in the
following media: MRC-5, SK-MES-1, and J82 in MEM, AGS cells in
Ham F-12; and HL-60 in RPM1. The MEM medium contained 2 mM
glutamine, 1 mM sodium pyruvate, and 1.5 g/L sodium hydro-
gencarbonate. Ham F-12 was supplemented with 2 mM -glutamine
and 1.5 g/L sodium hydrogencarbonate. RPM1 contained 1 mM
sodium pyruvate and 2 g/L sodium hydrogencarbonate. All media
were supplemented with 10% heat-inactivated FBS, 100 IU/mL
29.0, 30.1, 42.2, 105.0, 108.6, 121.8, 125.1(2C), 126.5, 129.7(2C), 147.1,
147.4, 151.3. 152.3, 154.0, 158.6, 164.4, 181.2, 181.4. Anal. Calcd. for
C21H18N4O4: C, 64.61; H, 4.65; N, 14.35. Found: C, 64.31; H, 4.64; N,
14.03.
4.2.23. 2,4,6-Trimethyl-2H, 4H-8-(N-ethylphenylamino)pyrimido
[4,5-c]isoquinoline-1,3,7,10-tetraone (25)
Prepared from 3 and N-ethylaniline (16 h, 58%): red solid mp
205e206 ꢁC 1H NMR (400 MHz, CDCl3):
d
1.28 (t, 3H, J ¼ 7.10 Hz,
CH3CH2), 2.70 (s, 3H, 6-Me), 3.48 (s, 3H, 4-NMe), 3.72 (s, 3H, 2-
NMe), 3.83 (q, 2H, J ¼ 7.10 Hz, CH3CH2), 6.04 (s, 1H, 9-H), 7.07 (d,
2H, J ¼ 7.5 Hz, 20- and 6-H), 7.29 (m, 1H, 40-H), 7.39 (t, 2H,
L-
J ¼ 7.7 Hz, 30- and 50-H). 13C NMR (100 MHz, CDCl3):
d 11.8, 26.1,
29.0, 30.2, 41.2, 105.2, 108.3, 112.9(2C), 117.3, 121.5, 129.5(2ꢂ),
147.3, 148.5, 148.8, 151.3, 152.5, 158.7, 165.4, 180.8, 181.5. Anal.
Calcd. for C22H20N4O4: C, 65.34; H, 4.98; N, 13.85. Found: C, 65.48;
H, 4.68; N, 13.92.
L
penicillin, and 100 mg/mL streptomycin in a humidified incubator
4.2.24. 2,4,6-Trimethyl-2H, 4H-8-(n-butylamino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (26)
with 5% CO2 in air at 37 C. For the experiments, cells were plated at
a density of 50,000 cells/mL in 96-well plates. One day after seeding,
the cells were treated with the medium containing the compounds
Prepared from 2 and n-butylamine (2 h, 75%): red solid mp
146e147 ꢁC 1H NMR (400 MHz, CDCl3):
CH3C3H6), 1.44 (m, 2H, CH3CH2C2H4), 1.68 (m, 2H, C2H5CH2CH2),
2.95 (s, 3H, 6-Me), 3.18 (m, 2H, CH2C3H7), 3.47 (s, 3H, 4-NMe), 3.73
(s, 3H, 2-NMe), 5.78 (s, 1H, 9-H), 5.91 (s, 1H, NeH); 13C NMR
d
0.98 (t, 3H, J ¼ 7.4 Hz,
at concentrations ranging from 0 up to 100 mM during 3 days. Finally
the MTT reduction assay was carried out. The final concentration of
MTT was 1 mg/mL. The compounds were dissolved in DMSO (1%
final concentration) and complete medium. Untreated cells
(medium containing 1% DMSO) were used as controls. Each exper-
iment was carried out in sextuplicate.
(100 MHz, CDCl3):
d 13.7, 20.2. 26.8. 29.1, 30.2, 30.3. 42.4. 100.7,
106.4,120.2, 147.9, 149.6,151.3,152.9,158.8,185.3, 180.2, 180.8. Anal.
Calcd. for C18H20N4O4: C, 60.66; H, 5.66; N,15.72. Found: C, 60.74; H,
5.54; N, 15.34.
Acknowledgement
4.2.25. 2,4,6-Trimethyl-2H, 4H-8-(4-morpholino)pyrimido[4,5-c]
isoquinoline-1,3,7,10-tetraone (27)
We thank Fondo Nacional de Ciencia y Tecnología (Grant Nꢁ
1060591) for financial support to this study.
Prepared from 2 and morpholine (3 h, 78%): red solid mp
196e197 ꢁC; 1H NMR (400 MHz, CDCl3):
d 2.90 (s, 3H, 6-Me), 3.46
(s, 3H, 4-NMe), 3.49 (m, 4H, 30- and 50-H), 3.73 (s, 3H, 2-NMe), 3.86
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