Studies on quinones. Part 45: Novel 7-aminoisoquinoline-5,8-quinone derivatives with antitumor properties on cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2009.02.013

A variety of 7-aminoisoquinoline-5,8-quinone derivatives were prepared from 2,5-dihydroxyacetophenone, methyl aminocrotonate, and the corresponding amines, through a highly efficient three-step sequence. The members of this series were tested on normal hu

Full text of DOI:10.1016/j.bmc.2009.02.013

Bioorganic & Medicinal Chemistry 17 (2009) 2894–2901
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Studies on quinones. Part 45: Novel 7-aminoisoquinoline-5,8-quinone
derivatives with antitumor properties on cancer cell lines q
a
a
b
b
Jaime A. Valderrama a, , J. Andrea Ibacache , Verónica Arancibia , Jaime Rodriguez , Cristina Theoduloz
*
a Facultad de Quimica, Pontificia Universidad Católica de Chile, Casilla 306, Santiago, Chile
b Facultad de Ciencias de la Salud, Universidad de Talca, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of 7-aminoisoquinoline-5,8-quinone derivatives were prepared from 2,5-dihydroxyacetophe-
none, methyl aminocrotonate, and the corresponding amines, through a highly efficient three-step
sequence. The members of this series were tested on normal human fibroblasts and on a panel of three
human cancer cell lines and their redox properties were determined by cyclic voltammetry in acetoni-
trile. Both the cytotoxicity and antitumor activity of 7-phenylaminoisoquinoline-5,8-quinone derivatives
showed correlation with their half wave potentials and lipophilicities.
Received 19 December 2008
Revised 3 February 2009
Accepted 7 February 2009
Available online 14 February 2009
Keywords:
Isoquinolinquinones
Substitution
Ó 2009 Elsevier Ltd. All rights reserved.
Half-wave potential
Cytotoxicity
1. Introduction
3 3, Fig. 1).8,9 This structural array has stimulated the synthesis of
novel lead compounds that exhibited significant cytotoxicity on
Quinones are widespread in nature and are present in many
drugs such as anthracyclines, daunorubicin, doxorubicin, mitomy-
cin, mitoxantrones, and saintopin, which are used clinically in the
therapy of solid cancers. The cytotoxic effects of these quinones are
mainly due to the inhibition of DNA topoisomerase-II.2,3
human cancer cell lines.10–13
In a recent paper, we noticed that compounds 5 and 6, prepared
from substituted isoquinolinequinone 4 (Fig. 2), showed high anti-
tumor activity against representative cell tumor lines (0.8–5.9
compared to that of isoquinolinequinone precursor (2.1–
17.5
M).14
lM)
4
The quinoid anticancer agents undergo enzymatic reduction via
one or two electrons to give the corresponding semiquinone radi-
cal or hydroquinone. Under aerobic conditions the semiquinone
radical anion can yield its extra electron to molecular oxygen to
yield the parent quinone and superoxide radical anion. This reac-
tion 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 reac-
tion with the parent quinone to yield the semiquinone radical an-
ion. Both the semiquinone and the superoxide radical anion can
generate the hydroxyl radical, which is the cause of DNA strand
breaks.4–7
l
These 2,5-diaza-anthraquinones displayed antitumor potencies
comparable to that etoposide, an antineoplastic agent, evaluated
under the same conditions. Apparently, the nitrogen substituent
bonded to the quinone nucleus exerts influence on the redox prop-
erties of the quinone moiety improving the cytotoxic activity. Tak-
ing into account these observations and precedents on the
influence of the electronic and lipophilic characteristics of the sub-
stituents in substituted 1,4-naphthoquinones on the biological
activity,15–18 we were interested in the synthesis and antitumor
evaluation of a variety of 7-aminoisoquinoline-5,8-quinones hav-
ing a range of electronic and lipophilic characteristics. Since the
cytotoxic activity of quinoid compounds is related to the redox
properties of the electroactive quinone moiety, the effects of nitro-
gen substituents on the isoquinolinquinone system could provide
clues on the biological mechanism of their biological activity.
The molecular framework of several naturally occurring antitu-
moral agents contains an aminoquinonoid moiety as the key struc-
tural component, (e.g., streptonigrin 1, mitomycin C 2, cribrostratin
2. Chemistry
Isoquinolinequinone 4 was selected as a suitable precursor for
the preparation of aminoisoquinoline-5,8-quinone derivatives
due to its accessibility from commercially available starting
q
For Part 44, see Ref. 1.
* Corresponding author. Tel.: +56 02 6864432; fax: +56 02 6864744.
E-mail address: jvalderr@uc.cl (J.A. Valderrama).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.013

J. A. Valderrama et al. / Bioorg. Med. Chem. 17 (2009) 2894–2901
2895
O
O
O
O
O
OCONH2
H2N
H2N
Me
MeO
H2N
OMe
N
O
N
CO2H
N
NH
Me
N
O
H2N
Me
Me
OH
O
Me
OMe
1
2
3
Figure 1. Structure of streptonigrin (1), mitomycin C (2) and cribrostatin 3 (3).
Me
O
Me
Me
N
O
Me
O
Me
N
N
N
N
H
Me
CO2Me
Me
CO2Me
Me
CO2Me
O
O
O
5
6
4
Figure 2. Structure of anti-tumoral diaza-anthraquinones 5 and 6 prepared from 4.
products and to its high regioselectivity in substitution reactions
with nucleophiles according to LUMO energies predicted by fron-
tier molecular orbital (FMO) theory.14 This compound was pre-
pared in 74% yield from 2,5-dihydroxyacetophenone and methyl
3-aminocrotonate using our previously reported one pot proce-
dure.14 First we explored the reaction of isoquinolinquinone 4 with
aniline. After considerable experimentation in different solvents
(ethanol, dichloromethane and acetonitrile) and Lewis acid cata-
lysts (CeCl3ꢀ7H2O, InBr3, FeCl3), the reaction was performed by
treating 1 equiv of 4 with 2 equiv of aniline, and 0.5 equiv of
CeCl3ꢀ7H2O in ethanol for 4 h at room temperature. Under these
conditions, anilinoquinone 7 was isolated in 93% yield (Scheme 1).
The structure of 7 was assigned on the basis of the HMQC/
responding substitution products 8–17 in good to excellent yields.
Aminoisoquinolinequinone 18 was prepared in 45% yield by reac-
tion of 4 with sodium azide in acetic acid. Table 1 summarizes
the results of the preparation of the amino isoquinolinequinone
derivatives.
All new compounds were fully characterized on the basis of
their 1H, 13C NMR spectra and high resolution mass spectra.
Regarding the location of the amino group in products 8–18, they
were established by analysis of their corresponding HMBC spectra.
In all cases long-range couplings between the carbon at C-8, the vi-
nyl proton at C-6, and the protons of the methyl group at C-1 were
observed.
3
3. Biological results and discussion
HMBC experiments. The HMBC spectrum of 7 shows JC,H and
4JC,H coupling of the C-8 carbon (d 181.6 ppm) with the proton at
C-6 (d 6.36 ppm) and the protons of the methyl group at C-1 (d
3.00 ppm), respectively.
The newly synthesized 7-aminoisoquinolinequinone deriva-
tives 7–18 were evaluated for in vitro anticancer activity against
human normal cells: MRC-5 human lung fibroblasts and three hu-
man tumor cells: AGS gastric adenocarcinoma, SK-MES-1 lung can-
cer, and J82 bladder carcinoma, in 72 h drug exposure MTT assays.
The cytotoxicity of the compounds was measured using a conven-
tional microculture tetrazolium assay.21 As indicated in Table 2,
the tested compounds showed moderate to good activity in the
in vitro antitumor screening expressed by the IC50 values.
The data of Table 2 indicate that substitution of one of the ami-
no-protons in the parent compound 18 by phenyl- or 4-R-phenyl
groups, as in 7–10, increases the cytotoxic activity on the normal
and almost all the cancer cell lines. On the other hand, the substi-
It is noteworthy that no regioisomers were detected in the reac-
tion assays of quinone 4 with aniline either in presence or absence
of the CeCl3ꢀ7H2O, thus indicating that the arylamination reaction
is totally regiocontrolled. The control of the regioselectivity could
be ascribed to the large difference between the LUMO coefficients
of the C-6 and C-7 carbon atoms of quinone 4 (C-6 = 0.3142 and C-
7 = 0.3949 eV),14 which is probably enhanced by coordination of
the catalyst with the nitrogen atom and/or the carbonyl oxygen
at the 5-position of the isoquinoline system.19,20
Isoquinolinequinone 4 was reacted with a variety of aryl- and
alkylamines under the aforementioned conditions to yield the cor-
NH2
MeO2C
H2N
H
OH
OH
O
Me
1
O
Me
H
7
8
N
Me
7
6
N
N
Me
CeCl3.7H2O, CH2Cl2
Ag2O, CH2Cl2
6
Me
CO2Me
Me
CO2Me
O
O
O
7
4
Scheme 1. Preparation of 7-anilinoisoquinolinequinone 7.

2896
J. A. Valderrama et al. / Bioorg. Med. Chem. 17 (2009) 2894–2901
Table 1 (continued)
Table 1
Preparation of 7-aminoisoquinolinequinone derivatives 7–18
No.
Structure
Yielda (%)
No.
Structure
Yielda (%)
O
O
Me
H
O
Me
N
H
N
N
16
68
N
7
93
Me
CO2Me
Me
CO2Me
O
O
O
Me
O
Me
H
N
N
N
17
97
N
8
91
Me
CO2Me
HO
Me
O
O
CO2Me
O
Me
O
Me
N
H2N
H
N
N
18
45
9
89
Me
CO2Me
MeO
Me
CO2Me
O
O
a
Isolated by column chromatography.
O
O
Me
H
N
N
tution of one of the amino-protons in 18 by alkyl groups, as in 14–
17, decreases the antitumor activity against all of the tested cancer
cell lines.
Comparison of the IC50 values for phenylaminoquinone 7 and its
N-alkyl derivatives 10 and 11 shows that substitution of the ami-
no-proton in 7 by an alkyl group decreases the cytotoxic activity.
On the other hand, comparison of the IC50 values of the aryl-
aminoisoquinolinequinones 7–10 indicates that the substituents
located on the 4-position of the arylamino group have a relatively
significant influence on their antitumor activities.
10
11
12
13
84
90
92
30
F
Me
CO2Me
MeO
O
O
Me
H
N
N
Me
CO2Me
MeO
It is noteworthy that the introduction of methoxy groups into
the 2,5-positions of 7, as in arylaminoisoquinolinequinone 11, in-
duces a remarkable decreasing cytotoxic effect on the MRC-5 and
AGS cell lines and a suppression of the antitumor activity on the
SK-MES-1 and J82 cell lines. These findings, along with the ob-
served antitumor activity of compound 9, suggest that the cyto-
toxic activity of the arylaminoisoquinolinequinone chromophore
depends on the location of methoxy substituents on the phenyl
ring.
Replacement of the amino-proton in phenylaminoquinone 7 by
an alkyl group, as in 12 and 13, induces a slight modification of the
antitumor activity in the case of a methyl group, but a significant
decrease of the biological activity for the ethyl group. Apparently,
the size of the alkyl group bonded to the nitrogen atom of the phe-
nylamino group has an influence on the biological activity.
Comparison of the IC50 values for the aminoisoquinolinequi-
none derivatives 14–17 shows that the presence of an alkylamino
group, as in 14–16, is more relevant on the antitumor activity of
the pharmacophore than that of an dialkylamino group as in 17.
It is well-known that the cytostatic and antimicrobial activity of
numerous quinones emerges from their ability to act as potent
inhibitors of electron transport, as uncouplers of oxidative phos-
phorylation, as intercalating agents in the DNA double helix, as
bioreductive alkylating agents of biomolecules, and as producers
of reactive oxygen radicals, by redox cycling under aerobic condi-
tions. In all these cases the mechanism of action in vivo requires
bioreduction of the quinones as the first activating step.22–24
Therefore, those molecular properties that provide proof of the
reduction capability of quinonoid compounds could provide valu-
able information on the mechanism of biological activity and aid
Me
N
O
Me
N
Me
O
O
CO2Me
Et
N
Me
N
Me
CO2Me
O
O
Me
H
N
nBut
N
14
15
83
Me
CO2Me
O
O
Me
H
N
Adamantyl
N
6
Me
CO2Me
O

J. A. Valderrama et al. / Bioorg. Med. Chem. 17 (2009) 2894–2901
2897
Table 2
Cytotoxic activity, half wave potential (E1/2), and lipophilicity (logP) of aminoisoquinolinequinone derivatives 7–18
O
Me
R
N
Me
O
CO2Me
No.
7
R
MRC-5b
5.6 0.3
8.3 0.4
9.0 0.4
AGSc
IC50 SEMa
(lM)
II
SK-MES-1d
4.2 0.3
J82e
EI1=2 (mV)
E
(mV)
LogP
1=2
H
N
2.1 0.2
1.3 0.1
2.3 0.2
5.8 0.2
592
1172
953
1.443
1.054
1.317
H
8
4.2 0.2
7.2 0.4
4.4 0.2
7.4 0.5
603
622
HO
N
H
9
1220
MeO
N
H
N
10
11
3.7 0.3
2.2 0.1
3.9 0.2
>100
4.2 0.3
>100
646
573
1234
1164
1.602
1.190
F
H
N
MeO
31.3 1.9
19.9 1.2
OMe
Me
N
12
13
14
8.8 0.5
3.3 0.2
8.1 0.5
6.8 0.3
8.7 0.5
11.3 0.7
7.8 0.4
5.2 0.2
588
418
687
1034
1074
1339
2.232
2.570
1.020
Et
N
17.9 0.8
13.3 0.7
14.8 0.8
10.7 0.6
H
N
HN
15
7.7 0.5
4.6 0.3
9.5 0.5
12.1 0.7
374
1225
1.607
H
N
16
17
16.6 0.9
24.1 1.1
10.5 0.6
9.6 0.5
18.1 1.0
28.7 1.6
14.9 0.9
30.8 1.7
722
647
1221
1245
1.330
ꢁ0.243
ꢁ741
O
N
18
10.1 0.7
3.9 0.2
4.2 0.3
5.9 0.4
2.5 0.2
5.9 0.3
2.8 0.2
982
—
1278
—
H2N
—
Etoposide
0.36 0.02
a
b
c
Data represent mean average values for six independent determinations.
Human lung fibroblasts cells.
Human gastric adenocarcinoma cell line.
Human lung cancer cell line.
d
e
Human bladder carcinoma cell line.

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J. A. Valderrama et al. / Bioorg. Med. Chem. 17 (2009) 2894–2901
in designing new molecules with greater biological activity. Based
on this general precedent and those reported on the effects of sub-
stituents on the redox properties of 2(4-R-phenylamino)-1,4-naph-
thoquinones,14 a voltammetric study of compounds 7–18 was
made in order to explain their biological activity through the elec-
tronic effect of the substituents on the aminoquinone
chromophore.
atom by alkyl substituents, as in 12 and 13, produced a less nega-
tive EI1=2 for the corresponding AQ with respect to the parent com-
pound 7.
According to the results of the electrochemical study, the
electron capability of the quinone nucleus in compounds 7–13,
evaluated as a function of their half-wave potentials for wave I
(EI1=2), the electron donor effect of the substituents decreases in
Electrochemical results. The redox potentials of the compounds
were measured by cyclic voltammetry in acetonitrile as a solvent
at room temperature, using a platinum electrode and 0.1 M tetra-
ethylamonium tetrafluoroborate as the supporting electrolyte.
The voltammograms were run in the potential range 0.0–2.0 V ver-
sus non-aqueous Ag/Ag+.
the
following
order:
4-F > 4-OH > 4-OMe > H > N-Me > 2,5-
(OMe) > N-Et.
Linear regression analysis provided satisfactory relationships
between the log IC50 values and EI for the AGS gastric adenocar-
1=2
cinoma and J82 bladder carcinoma cell lines (Eqs. 1 and 2). In these
regression equations n is the number of the test compounds and r
is the correlation coefficient. Compound 11 was not included in
this analysis because its IC50 values in two of the tested cell lines
Figure 3 shows the typical redox behaviour of the aminoisoqu-
inolinequinone derivatives, such as 11, that proceeded in two one-
electron diffusion stages: AQ + e = AQÅꢁ and AQÅꢁ + e = AQ2ꢁ
.
lie at concentrations greater than 100 lM.
The first wave is due to the addition of an electron to the qui-
none (AQ) to give a semiquinone radical anion (AQÅꢁ) which by a
subsequent addition of a second electron produces a hydroquinone
dianion (AQ2ꢁ). It is interesting to note that formation of the sem-
iquinone radical anion and its reaction with dioxygen to yield the
superoxide anion are crucial features required in the antitumoral
mechanism of drugs by redox cycling.
2 ¼ 0:826logIC50 ¼ 0:0029ðE1=2Þþ2:1307 n ¼ 6 r2 ¼ 0:826
2 ¼ 0:833logIC50 ¼ 0:0022ðE1=2Þþ2:0900 n ¼ 6 r2 ¼ 0:833
ð1Þ
ð2Þ
The relationship between log IC50 and EI1=2, which corresponds to a
regression line, indicates that for the studied aminoquinones, the
more negative the EI1=2 the stronger the anti-tumor promoting effect
on the AGS and J82 cell lines. This behaviour agrees with reported
precedents on the relationship between redox potentials and inhib-
itory effects of 2-aza-anthraquinones on Epstein-Barr virus early
antigen (EBV-EA) activation.25,26
The half-wave potential values, E1/2, for both waves were eval-
uated from the voltammograms obtained at a sweep rate of
100 mV sꢁ1, as E1/2 = (Epa + Epc)/2, where Epa and Epc correspond
to anodic and cathodic peak potentials, respectively. Table 2 shows
the half-wave potential values E1/2 for both waves. The EI values
1=2
As lipophilicity is an important parameter affecting the biolog-
ical activity of quinonoid compounds,15,27 the logP descriptors of
arylaminoisoquinolinquinone derivatives 7–13 were calculated
using the AM1 semiempirical method. Apparent relationships be-
tween the values of log IC50 and logP were obtained for the AGS
gastric adenocarcinoma and J82 bladder carcinoma cell lines
according to regression Eqs. 3 and 4, respectively.
for the first electron, corresponding to the formation of the semi-
quinone radical anion, are in the potential range ꢁ374 to
ꢁ982 mV. For the second one-electron transfer that produces the
II
1=2
corresponding dianion, the E
ꢁ1339 mV.
values fall in the range ꢁ603 to
Since arylaminoisoquinolinequinone derivatives 7–13 showed
higher antitumor activity than the alkyl derivatives 13–17, we fo-
cused our attention on the electrochemical behavior of these com-
pounds in order to explain their biological activity through the
electronic effect of the substituents on the electroactive quinone
moiety. The data for these compounds indicate that the introduc-
tion of electron-donor groups (F, OH and OMe) into the phenyl sub-
stituent of compound 7, as in 8–10, displaced the EI1=2 potentials for
the first reduction wave towards a more negative region with re-
spect to 7. On the contrary, the insertion of two methoxy groups
in 6 as well as in 9, and the replacement of the amino hydrogen
n ¼ 6 log IC50 ¼ 0:5421 log P ꢁ 0:3706 n ¼ 6 r2 ¼ 0:711
ð3Þ
2
n ¼ 6 log IC50 ¼ 0:4382ðlog PÞ ꢁ 1:0739 log P þ 1:3651
n ¼ 6 r2 ¼ 0:738
ð4Þ
4. Conclusion
The aim of this work was to test 7-aminoisoquinoline-5,8-qui-
none derivatives with substituents having a range of electronic
and lipophilic properties, in order to help elucidate the mechanism
of their antitumor action. We have developed a high yield synthe-
sis of 7-arylamino- and 7-alkylaminoisoquinoline-5,8-quinones 7–
17 using acid-induced substitution reactions of isoquinolinequi-
none 4 with alkyl- and arylamines. Compounds 7–17 and 7-amin-
oisoquinolinquinone 18, prepared by addition of hydrazoic acid to
quinone 4, expressed in vitro cytotoxic activity against human lung
fibroblasts (MRC-5), gastric adenocarcinoma (AGS), lung cancer
(SK-MES-1), and bladder carcinoma (J82) cell lines. The analysis
of the screening shows that replacement of the amino-proton of
the parent compound 7-aminoisoquinoline-5,8-quinone 18 by
aryl- or alkyl groups induces significant changes in the cytotoxic
activity due to the nature of the substituents. The 7-phen-
ylaminoisoquinolinquinones 7–10, 12, and 13 exhibited higher
antitumoral activity than that of the 7-alkylaminoisoquinolinqui-
none 14–17.
EII
0.000004
0.000002
0.000000
-0.000002
-0.000004
-0.000006
-0.000008
pc
EIpc
EII
pa
EIpa
-1500
-1000
-500
0
500
Potential mV vs Ag/Ag+
The electrochemical study of the electron capacity of the qui-
none nucleus in compounds 7–13, evaluated as a function of their
half wave potentials E1/2, shows that the electron donor effect of
Figure 3. Cyclic voltammogram of compound 11 in 0.1 M Et4NBF4/acetonitrile
obtained in Pt electrode, scan rate 100 mV/s. The cathodic (EIpc, EIIpc) and anodic
(EIpa, EIIpa) peak potentials are indicated in the figure.

J. A. Valderrama et al. / Bioorg. Med. Chem. 17 (2009) 2894–2901
2899
the substituents decrease in the following order: 4-F > 4-OH > 4-
OMe > H > N-Me > 2,5-(OMe) > N-Et.
HRMS (M+): m/z calcd for C19H16N2O5: 352.10592; found:
352.10593.
The QSAR analysis of the data revealed that for the evaluated
phenylaminoisoquinolinquinones 7–10, 12 and 13, the first reduc-
tion potential E1/2 is an important parameter determining the anti-
tumoral activity on AGS gastric adenocarcinoma and J82 bladder
carcinoma cell lines.
5.2.3. Methyl 7-(4-methoxyphenyl)amino-1,3-dimethyl-5,8-
dioxo-5,8-dihydroisoquinoline-4-carboxylate (9)
Prepared from 4 and p-anisidine (4 h, 89% yield): violet solid,
mp 166–167 °C; IR: mmax 3436 NH, 1733 (C@O ester), 1676
and1604 (C@O quinone); 1H NMR (400 MHz): d 2.64 (s, 3H,
3-Me), 3.01 (s, 3H, 1-Me), 3.86 (s, 3H, OMe), 4.03 (s, 3H, CO2Me),
6.19 (s, 1H, 6-H), 6.99 (d, 2H, 20- and 60-H), 7.18 (d, 2H, 30- and
50-H), 7.64 (s, 1H, NH) 13C NMR (100 MHz): d 22.93, 26.08,
52.97, 55.58, 101.62, 115.04 (2C), 119.97, 124.9 (2C), 125.17,
129.44, 137.99, 146.29, 158.09, 160.86, 161.21, 169.20, 181.17,
181.72.
5. Experimental
5.1. Chemical synthesis
All reagents were commercially available reagent grade and
were used without further purification. Melting points were deter-
mined on a Stuart Scientific SMP3 apparatus and are uncorrected.
The IR spectra were recorded on an FT Bruker spectrophotometer
HRMS (M+): m/z calcd for C20H18N2O5: 366.12157;
found:366.12149.
using KBr disks, and the wave numbers are given in cmꢁ1 1H
.
NMR spectra were run on Bruker AM-200 and AM-400 instruments
in deuterochloroform (CDCl3). Chemical shifts are expressed in
ppm downfield relative to tetramethylsilane (TMS, d scale), and
the coupling constants (J) are reported in Hertz. 13C NMR spectra
were obtained in CDCl3 at 50 and 100 MHz. 2D NMR techniques
(COSY, HMBC) and DEPT were used for signal assignment. Chemi-
cal shifts are reported in d ppm downfield fromTMS, and J-values
are given in Hertz. HRMS were obtained on a Thermo Finnigan
spectrometer, model MAT 95XP. Silica gel Merck 60 (70–230 mesh)
was used for preparative column chromatography, and TLC alumi-
num foil 60F254 for analytical TLC. Acetonitrile anhydrous (99.8%)
for electrochemical evaluations was obtained from Sigma–Aldrich.
5.2.4. Methyl 7-(4-fluorophenyl)amino-1,3-dimethyl-5,8-dioxo-
5,8-dihydroisoquinoline-4-carboxylate (10)
Prepared from 4 and 4-fluoroaniline (24 h, 84% yield): orange
solid, mp 161–163 °C; IR: mmax 1720 (C@O ester), 1672 and 1614
(C@O quinone); 1H NMR (400 MHz): d 2.61 (s, 3H, 3-Me), d 2.97
(s, 3H, 1-Me), 4.00 (s, 3H, CO2Me), 6.17 (s, 1H, CH), 7.12 (m, 2H,
arom.), 7.24 (m, 2H, arom.), 7.65 (s, 1H, NH); 13C NMR
(100 MHz): d 23.11, 26.24, 53.16, 102.26, 116.84, 117.06, 120.00,
125.31, 125.51, 132.93, 132.96, 137.83, 146.17, 159.61, 161.13,
162.07, 169.22, 181.56, 181.62.
HRMS (M+): m/z calcd for C19H15FN2O4: 354.10156; found:
354.10281.
5.2. General procedure for the synthesis of 7-
aminoisoquinolinequinone derivatives 7–17
5.2.5. Methyl 7-(2,5-dimethoxyphenyl)amino-1,3-dimethyl-5,8-
dioxo-5,8-dihydroisoquinoline-4-carboxylate (11)
Prepared from 4 and 2,5-dimethoxyaniline (19 h, 90% yield):
purple solid, mp 205.5–207 °C; IR: mmax 3454 (NH), 1725 (C@O es-
A suspension of quinone 4 (1 mmol), the required amine
(2 mmol), CeCl3ꢀ7H2O (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, the organic extract was washed with water
(2 ꢂ 15 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was column chromatographed over silica
gel (petroleum ether/ethyl acetate 90:10) to yield the correspond-
ing aminoisoquinolinequinone derivative (Table 1).
ter), 1678 and 1633 (C@O quinone); 1H NMR (400 MHz): d 2.58 (s,
3H, 3-Me), 2.96 (s, 3H, 1-Me), 3.74 (s, 3H, OCH3), 3.84 (s, 3H, OCH3),
3.98 (s, 3H, CO2Me), 6.43 (s, 1H, 6-H), 6.66 (dd, 1H, J = 8.5; 3.0 Hz,
40-H), d 6.84 (d, 1H, J = 3.0 Hz, 60-H), d 6.91 (d, 1H, Jorto = 8,5 Hz, 30-
H), 8.12 (s, 1H, NH); 13C NMR (100 MHz): d 22.67, 26.11, 53.00,
55.89, 56.25, 102.91, 107.95, 110.07, 111.94, 119.99, 125.07,
126.96, 137.77, 144.21, 145.58, 153.78, 160.97, 161.10, 169.18,
181.48, 181.59.
HRMS (M+): m/z calcd for C21H20N2O6: 396.13214; found:
396.13377.
5.2.1. Methyl 7-phenylamino-1,3-dimethyl-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylate (7)
Prepared from 4 and aniline (3 h, 93% yield): red solid, mp 202–
203.5 °C; IR: mmax 1736 (C@O ester), 1627 and 1625 (C@O qui-
none); 1H NMR (400 MHz): d 2.60 (s, 3H, 3-Me), 3.20 (s, 3H, 1-
Me), 4.00 (s, 3H, CO2Me), 6.41 (s, 1H, 6-H), 7.30 (m, 3H, arom.),
7.50 (m, 2H, arom.), 7.70 (s, 1H, NH); 13C NMR (100 MHz): d 23.0,
26.2, 53.1, 102.4, 119.9, 122.9 (2C), 125.2, 126.3 (2C), 129.8,
136.9, 137.8, 145.5, 160.2, 161.5, 169.1, 181.5, 181.6.
HRMS (M+): m/z calcd for C19H16N2O4: 336.11101; found:
336.11093.
5.2.6. Methyl 7-(methylphenylamino)-1,3-dimethyl-5,8-dioxo-
5,8-dihydroisoquinoline-4-carboxylate (12)
Preparedfrom4andN-methylaniline(9:25 h, 92%yield):darkor-
ange oil; IR (neat) mmax: 1735 (C@O ester), 1634 and 1633 (C@O qui-
none); 1H NMR (400 MHz): d 2.55 (s, 3H, 3-Me), 2.65 (s, 3H, 1-Me),
3.39 (s, 3H, NMe), 3.97 (s, 3H, CO2Me), 5.97 (s, 1H, 6-H), 7.00–7.35
(m, 5H, arom.); 13C NMR (100 MHz): d 22.67, 23.90, 24.89, 42.76,
53.00, 109.03, 122.60, 124.31, 125.19 (2C), 126.86, 129.77 (2C),
137.13, 147.10, 154.07, 159.76, 169.16, 181.01, 182.92.
HRMS (M+): m/z calcd for C20H18N2O4: 350.12666; found:
350.12607.
5.2.2. Methyl 7-(4-hydroxyphenyl)amino-1,3-dimethyl-5,8-
dioxo-5,8-dihydroisoquinoline-4-carboxylate (8)
Prepared from 4 and 4-aminophenol (5 h, 91% yield): purple so-
lid, mp 233–235 °C; IR: mmax 3339 (NH), 3300 OH), 1732 (C@O es-
ter), 1678 and1600, (C@O quinone); 1H NMR (400 MHz): d 2.57 (s,
3H, 3-Me), 3.01 (s, 3H, 1-Me), 3.95 (s, 3H, CO2Me), 6.03 (s, 1H, CH),
6.88 (d, 2H, J = 8.5 Hz, 30- and 50-H), 7.09 (d, 2H, J = 8.5 Hz, 20- and
60-H), 8.56 (s, 1H, NH); 9.22 (s, 1H, OH); 13C NMR (100 MHz): d
22.50, 25.73, 52.46, 100.55, 116.09 (2C), 119.99, 120.1, 124.66,
125.33 (C2), 128.12, 137.90, 147.21, 155.90, 160.18, 168.88,
180.34, 181.58.
5.2.7. Methyl 7-(ethylphenylamino)-1,3-dimethyl-5,8-dioxo-
5,8-dihydroisoquinoline-4-carboxylate (13)
Prepared from 4 and N-ethylaniline (47 h, 30% yield): dark or-
ange solid, mp 124–125.5; IR: mmax 1731 (CO2Me), 1681 and
1627 (C@O); 1H NMR (400 MHz): d 1.21 (t, 3H, J = 5.0 Hz, Me),
2.58 (s, 3H, 3-Me), 2.69 (s, 3H, 1-Me), 3.87 (q, 2H, J = 5.0 Hz,
CH2), 4.00 (s, 3H, CO2Me), 5.94 (s, 1H, 6-H), 7.10–7.41 (m, 5H,
arom.).13C NMR (100 MHz):
d 12.20, 14.22, 22.68, 24.83,

2900
J. A. Valderrama et al. / Bioorg. Med. Chem. 17 (2009) 2894–2901
29.71, 49.56, 52.97, 108.32, 122.81, 124.23, 126.08, 127.11, 129.80,
137.16, 145.12, 153.75, 159.56, 159.64, 169.20, 180.93, 180.43.
HRMS (M+): m/z calcd for C21H20N2O4: 364.14227; found:
364.14217.
HRMS (M+): m/z calcd for C13H12N2O4: 260.07971; found:
260.07978.
5.3. Anticancer assay21
5.2.8. Methyl 7-(n-butylamino)-1,3-dimethyl-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylate (14)
The cell lines used in this work were obtained from the Amer-
ican Type Culture Collection (ATCC, Manasas, VA, USA). They in-
cluded MRC-5 normal human lung fibroblasts (CCL-171), AGS
human gastric adenocarcinoma cells (CRL-1739), SK-MES-1 hu-
man lung cancer cells (HTB-58), and J82 human bladder carci-
noma cells (HTB-1). After the arrival of the cells, they were
proliferated in the corresponding 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% 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, and AGS cells in Ham F-12. The MEM
Prepared from 4 and n-butylamine (2:20 h, 83% yield): orange
solid, mp 97–99 °C; IR: mmax 3378 (NH), 1735 (CO2Me), 1677 and
1610 (C@O); 1H NMR (400 MHz): d 0.94 (t, 3H, J = 7.0 Hz, Me),
1.40 (m, 2H, CH2), 1.62 (m, 2H, CH2), 2.55 (s, 3H, 3-Me), 2.88 (s,
3H, 1-Me), 3.14 (t, 2H, J = 7.0 Hz, CH2), 3.97 (s, 3H, CO2Me), 5.66
(s, 1H, NH), 6.09 (s, 1H, 6-H); 13C NMR (100 MHz): d 14.11,
20.14, 22.83, 25.91, 30.15, 42.38, 52.87, 100.00, 120.05, 125.28,
138.35, 148.44, 160.63, 160.94, 169.35, 180.09, 181.41.
HRMS (M+): m/z calcd for C17H20N2O4: 316.14231; found:
316.14207.
5.2.9. Methyl 7-(1-adamantylamino)-1,3-dimethyl-5,8-dioxo-
5,8-dihydroisoquinoline-4-carboxylate (15)
medium contained 2 mM
and 1.5 g/L sodium hydogencarbonate. Ham F-12 was supple-
mented with 2 mM -glutamine and 1.5 g/L sodium hydrogencar-
bonate. All media were supplemented with 10% heat-inactivated
FBS, 100 IU/mL penicillin, and 100 g/mL streptomycin in
humidified incubator with 5% CO2 in air at 37 °C. For the experi-
ments, 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 med-
ium containing the compounds at concentrations ranging from 0
L-glutamine, 1 mM sodium pyruvate,
Prepared from 4 and 1-adamantylamine (7 d, 6% yield): orange
L
oil; IR (neat): mmax: 3388 (NH), 1735 (CO2Me), 1634 and 1676 (C@O
quinone); 1H NMR (400 MHz): d 2.14–1.23 (m, 15H, adamantyl),
2.60 (s, 3H, 3-Me), 2.95 (s, 3H, 1-Me), 4.00 (s, 3H, CO2Me), 6.01
(s, H, 6-H), 6.93 (s, H, NH).
l
a
HRMS (M+): m/z calcd for C17H20N2O4: 394.18927; found:
394.18903.
up to 100 lM during 3 days, and finally the MTT reduction assay
5.2.10. Methyl 7-cyclohexylamino-1,3-dimethyl-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylate (16)
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 experiment was carried out in
sextuplicate.
Prepared from 4 and cyclohexylamine (3 h, 68% yield): orange
oil; IR: mmax 3388 (NH), 1735 (CO2Me), 1634 and 1676 (C@O); 1H
NMR (400 MHz): d 1.40 (m, 6H, 3 ꢂ CH2), 1.66 (m, 1H, CH), 1.78
(m, 2H, CH2), 2.01 (m, 2H, CH2), 2.59 (s, 3H, 3-Me), 2.93 (s, 3H, 1-
Me), 4.00 (s, 3H, CO2Me), 5.73 (s, 1H, 6-H), 6.05 (s, 1H, NH); 13C
NMR (100 MHz): d 23.03, 24.60 (2C), 25.55 (2C), 26.15, 31.94,
51.45, 53.08, 100.07, 120.32, 125.46, 138.57, 147.35, 160.80,
161.16, 169.54, 180.29, 181.85.
5.4. Electrochemical measurements28
Cyclic voltammograms of compounds were obtained on a
Bioanalytical Sytem BAS CV-50 W electrochemical analyzer. A
small capacity measuring cell was equipped with a platinum
disc as working electrode, a Ag/10 nM Ag (MeCN) reference elec-
trode for non aqueous solvent, with a platinum wire auxiliary
electrode, a mechanical mini-stirrer, and a capillary to supply
an inert argon atmosphere. A 0.1 M solution of tetrabutylammo-
nium tetrafluoroborate in acetonitrile was used as supporting
electrolyte.
HRMS (M+): m/z calcd for C19H22N2O4: 342.15796; found:
342.15808.
5.2.11. Methyl 7-[4-morpholino)-1,3-dimethyl-5,8-dioxo-5,8-
dihydroisoquinoline-4-carboxylate (17)
Prepared from 4 and morpholine (2:50 h, 97% yield): solid, mp
191–193 °C; IR: mmax 1735 (CO2Me),1633 and 1681 (C@O quinone),
1H NMR (400 MHz): d 2.58 (s, 3H, 3-Me), 2.87 (s, 3H, 1-Me), 3.52 (t,
4H, J = 5 Hz, 2- and 6-H0), 3.87 (t, 4H, J = 5 Hz, 3- and 5-H0), 4.00 (s,
3H, CO2Me), 5.93 (s, 1H, 6-H). 13C NMR (100 MHz): d 22.87, 25.49,
49.19 (2C), 53.15, 66.55 (2C), 108.69, 122.77, 124.46, 136.92,
155.01, 159.99, 160.20, 169.20, 181.26, 183.41.
Acknowledgement
We thank the Fondo Nacional de Ciencia y Tecnología (Grant
No. 1060591) for financial support of this study.
HRMS (M+): m/z calcd for C17H18N2O5: 330.12158; found:
330.12228.
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