Synthesis and in vitro cytotoxic evaluation of aminoquinones structurally related to marine isoquinolinequinones

Article abstract of DOI:10.3390/molecules17067042

The synthesis of 4-methoxycarbonyl-3-methylisoquinolinequinone (1) and a variety of its substitution products with amino-, alkylamino and halogen groups on the quinone nucleus is reported. The series of 6-, 7-and 6,7-subtituted isoquinolinequinones were evaluated in vitro for their cytotoxic activity using the MTT colorimetric method. All the newly synthesized compounds showed moderate to high potency against MRC-5 healthy lung fibroblasts and four human tumor cell lines: AGS gastric adenocarcinoma, SK-MES-1 lung, J82 bladder carcinoma, and HL-60 leukemia cells. Among the series, compounds 4b, 12 and 13 exhibited interesting antitumor activity against human gastric adenocarcinoma, human lung and human bladder carcinoma cancer cells. 7-Amino-6-bromoisoquinoline-5,8- quinone (13) was found to be the most promising active compound against the tested cancer cell lines, with IC₅₀ values in the 0.21-0.49 μM range, lower than the anti-cancer agent etoposide used as reference.

Full text of DOI:10.3390/molecules17067042

Molecules 2012, 17, 7042-7056; doi:10.3390/molecules17067042
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and in Vitro Cytotoxic Evaluation of Aminoquinones
Structurally Related to Marine Isoquinolinequinones
Virginia Delgado 1, Andrea Ibacache 1, Cristina Theoduloz 2 and Jaime A. Valderrama 1,3,
*
1
Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 6094411, Chile
Facultad de Ciencias de la Salud, Universidad de Talca, Talca 3460000, Chile
2
3
Instituto de Etno-Farmacología (IDE), Universidad Arturo Prat, Casilla 121, Iquique 1100000, Chile
* Author to whom correspondence should be addressed; E-Mail: jvalderr@uc.cl;
Tel.: +56-02-686-4432; Fax: +56-02-686-4744.
Received: 18 April 2012; in revised form: 23 May 2012 / Accepted: 25 May 2012 /
Published: 7 June 2012
Abstract: The synthesis of 4-methoxycarbonyl-3-methylisoquinolinequinone (1) and a
variety of its substitution products with amino-, alkylamino and halogen groups on the
quinone nucleus is reported. The series of 6-, 7- and 6,7-subtituted isoquinolinequinones
were evaluated in vitro for their cytotoxic activity using the MTT colorimetric method. All
the newly synthesized compounds showed moderate to high potency against MRC-5
healthy lung fibroblasts and four human tumor cell lines: AGS gastric adenocarcinoma,
SK-MES-1 lung, J82 bladder carcinoma, and HL-60 leukemia cells. Among the series,
compounds 4b, 12 and 13 exhibited interesting antitumor activity against human gastric
adenocarcinoma, human lung and human bladder carcinoma cancer cells. 7-Amino-6-
bromoisoquinoline-5,8-quinone (13) was found to be the most promising active compound
against the tested cancer cell lines, with IC50 values in the 0.21−0.49 M range, lower than
the anti-cancer agent etoposide used as reference.
Keywords: aminoisoquinoline-5,8-quinones; regioselectivity; anticancer; SAR analysis
1. Introduction
The search for biologically active natural products from marine sources continues to be an
important scientific field that offers promising opportunities for the development of new compounds
endowed with pharmacological properties [1–8]. Among the natural products with a quinoide system

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isoquinolinequinones form an important class characterized by their varied biological properties. For
instance, marine isoquinolinequinones such as caulibugulones A–D and mansouramycins A–C (Figure 1)
display a significant antitumor profile in many tumor cell lines [9,10]. Caulibugulones A–D, evaluated
for antitumor activity against the murine IC-2WT cell line [9] exhibit high potency. In these assays,
the A–C members displayed IC50 values ranging from 0.22 to 0.34 g/mL, whereas caulibugulone D
showed less cytotoxic activity (IC50 = 1.67 g/mL). According to these IC50 values against the
tested cell line it can be deduced that insertion of bromine or chlorine atoms at the 6-position in
caulibugulone A (0.34 g/mL), as in caulibugulone B (0.22 g/mL) and C (0.28 g/mL), is not an
important determinant of cytotoxicity. Furthermore, the replacement of the methylamino group in
caulibugulone A by the 2-hydroxyethylamino group, as in caulibugulone D, produces a decrease of the
cytotoxic activity. Evidences reported by Wipf and Lazo [11,12] indicate that caulibugulones are
selective in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases.
Figure 1. Structure of caulibugulones A–D and mansouramycins A–C.
The access to caulibugulones A–D via regioselective nucleophilic amination substitution at the
7-position in the reaction of isoquinoline-5,8-quinone with methylamine or 2-aminoethanol in
ethanol as solvent in the presence of CeCl3·7H2O has been simultaneously reported by Wipf [11] and
Tamagnan [13]. These amination reactions yield caulibugulones A and D accompanied by low amounts
of their corresponding 6-amino-substituted regioisomers. Further reaction of caulibugulone A with
N-chlorosuccinimide and N-bromosuccinimide provides access to caulibugulones B and C, respectively.
Regarding mansouramycins A–C, screening on a panel of up to 36 human tumor cell lines
displayed significant cytotoxicity, with pronounced selectivity for non-small cell lung cancer, breast
cancer, melanoma, and prostate cancer cells [10]. Mansouramycin C (4) proved to be the most
active compound, with an overall potency of 0.089 μM. It was followed by mansouramycin B
(mean IC50 = 2.7 μM) and then mansouramycin A, with a mean IC50 value of 13.44 μM. These data
indicate that insertion of a methoxycarbonyl group at C-1 or a chlorine atom at C-6 into the
7-methylaminoisoquinolinequinone pharmacophore induces an increase of antitumor activity.
As part of our research program aimed at the synthesis and structure-activity relationship (SAR) of
novel aminoquinones as potential antitumor agents [14–18] we report herein the synthesis and the
in vitro antitumor evaluation of a variety of amino-, alkylamino and alkylamino-haloisoquinolinequinone
derivatives, structurally related with the above mentioned marine isoquinolinequinones, as well the
SAR of this series.

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2. Results and Discussion
Isoquinolinequinone 1, unsubstituted at the 1-position and containing a methoxycarbonyl group on
the heterocyclic ring, as in mansouramycin C, was selected as a suitable precursor of the designed
aminoisoquinolinequinones. The synthesis of the target compound 1 was accomplished in 86% yield
from commercially available 2,5-dihydroxybenzaldehyde and methyl aminocrotonate, through a one-pot
procedure previously developed in our laboratory [19] (Scheme 1).
Scheme 1. Synthesis and reaction of isoquinolinequinone 1 with methylamine.
The amination reaction of quinone 1 was firstly examined with methylamine hydrochloride-sodium
acetate in ethanol at room temperature. The reaction went to completion in 3 h to give an 80:20
1
mixture of regioisomers 2a and 2b (as evaluated by H-NMR) in 74% yield. Further separation by
column chromatography on silica gel afforded pure compound 2a in 52% yield (Scheme 1). Our
efforts to isolate a pure sample of regioisomer 2b by chromatography were unsuccessful. Then we
examined the amination reaction of quinone 1 with 2-aminoethanol under the same conditions used
with methylamine. Analysis of the progress of the reaction by TLC reveals that the amination reaction
of 1 proceeds slowly (>7 h) and extensive decomposition of quinone 1 was detected. According to
precedents on the use of a Lewis acid to facilitate the reaction of quinones with amines [20–24],
compound 1 was allowed to react with 2-aminoethanol in ethanol in the presence of 5 mmol% of
CeCl3·7H2O. Under these conditions, the reaction went to completion in 3 h to give an 80:20 mixture
1
of regioisomers 3a and 3b (as evaluated by H-NMR) in 65% yield. Further separation by column
chromatography on silica gel afforded the major regioisomer 3a pure in 47% yield (Scheme 2).
Attempts to isolate a pure sample of the minor regioisomer 3b by chromatography were unsuccessful.

Molecules 2012, 17
Scheme 2. Reaction of isoquinolinequinone 1 with amines induced by acid-catalysis.
7045
The amination reactions of quinone 1 with cyclohexylamine and morpholine under the above
acid-induced conditions were also explored. The experiments provided access to a mixture of the
corresponding alkylaminoisoquinolinequinones 4a,b and 5a,b (Scheme 2). In these cases, pure
samples of the regioisomers of each pair were isolated by column chromatography.
According to the amination reaction of quinone 1 with the studied alkyamines we can conclude that
the addition of nitrogen nucleophiles across the quinone double bond occurs with high regioselective
preference to give the 7-substituted regioisomer as the main product (Scheme 2).
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 nitrogen group at C-7 in compound 4a was deduced by the 3JC,H couplings
between the carbon at C-8 ( 180.83) with the protons at C-1 (9.17), at C-6 (5.77) and that of the
NH group ( 5.96). In the case of aminoquinone 4b, the location of the nitrogen substituent at C-6 was
established by 3JC,H coupling between the carbon at C-5 ( 181.71) with the proton at C-7 ( 5.79) and
3
the proton of the NH group (5.75). Also, the JC,H coupling between the carbon at C-8 (181.49)
with the proton at C-1 (9.29) can be observed (Figure 2).
Figure 2. HMBC correlations of regioisomers 4a and 4b.
Aminoisoquinolinequinones 2a, 3a, 4a and 5a were submitted to halogenations with
N-halosuccinimide to yield the corresponding alkylamino-haloisoquinolinequinones (Table 1).

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Table 1. Preparation of aminohaloisoquinolinequinones 6–11.
Yield
Yield
Substrate
2a
Product
N°
6
Substrate
3a
Product
N°
9
(%) *
(%) *
80
78
2a
3a
7
8
85
75
4a
5a
10
11
84
70
* Isolated yields.
We also explored the preparation of amino- and aminobromoisoquinolinequinones in order to
include them in the biological assays. Treatment of quinone 1 with sodium azide in acetic
acid, according to a previously reported procedure [15], afforded aminoisoquinolinequinone 12 as
the sole regioisomer in 51% yield (Scheme 3). The structure of compound 12 was established
by means of HMBC experiments. Further bromination of 12 with N-bromosuccinimide, provides
aminobromoisoquinolinequinone 13 in 86% yield.
Scheme 3. Preparation of amino- and aminobromoisoquinolinequinones 12 and 13.
Isoquinolinequinones 1, 2a, 3a, 4a, 4b, 5a, 6–13 were evaluated for their in vitro cytotoxic activity
against MRC-5 (healthy lung fibroblasts) and four human cancer cell lines: AGS (gastric), SK-MES-1
(lung), J82 (bladder), and HL-60 (leukemia), using the conventional microculture tetrazolium
reduction assay [25–27]. The broad variety of the synthesized compounds was designed in order to
gain insight into the influence of nitrogen and halogen groups on the quinone nucleus of the
isoquinolinequinone pharmacophore on the biological activity. Table 2 summarizes the data from these
evaluations. According to the IC50 values collected in Table 2, it is evident that insertion of the
nitrogen substituents in quinone 1, as in 2a, 3a, 4a, 4b, 5a and 5b, increases the cytotoxic activity in
all the evaluated cell lines, compared to those of precursor 1. The initial structure-activity relationship
(SAR) was focused on the nature and location of alkylamino at the quinone nucleus of the
isoquinolinequinone pharmacophore. The data of Table 2 for compounds 2a, 3a, 4a and 5a reveal that
the shape and polarity of the alkylamino group at the 7-position in the isoquinolinequinone moiety

Molecules 2012, 17
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does not significantly influence the biological activity. Concerning the effect of the position of the
alkylamino groups, the IC50 values for the regioisomers 4a/4b and 5a/5b indicate that substitution of
the nitrogen group at the 6-position of the quinone pharmacophore induces a greater effect on the
antitumor activity than at the 7-position. It can be seen that the effect of such substitution on the
antitumor potency on gastric adenocarcinoma and lung cancer cells reaches values nearly seven times
higher compared to regioisomer 4a.
Table 2. Cytotoxic activity of 1 and their aminoisoquinoline-5,8-quinone derivatives.
IC50 ± SEM a (M)
N°
1
Structure
MRC-5 b
AGS c
SK-MES1 d
J82 e
HL-60 f
4.5 ± 0.3
25.9 ± 1.6
14.81 ± 0.74 14.81 ± 0.74
17.34 ± 1.64
2a
3a
4a
4b
8.1 ± 0.3
7.9 ± 0.3
10.1 ± 0.6
3.5 ± 0.2
2.3 ± 0.1
4.0 ± 0.2
5.22 ± 0.38
5.99 ± 0.36
5.33 ± 0.26
5.1 ± 0.4
7.2 ± 0.4
12.0 ± 1.0
9.74 ± 0.76
6.16 ± 0.36
10.67 ± 0.64
2.2 ± 0.1
6.7 ± 0.4
0.59 ± 0.03
1.75 ± 0.09
1.69 ± 0.09
4.00 ± 0.22
3.0 ± 0.2
1.57 ± 0.13
2.99 ± 0.15
5a
5b
8.56 ± 0.27
3.77 ± 0.21 1.20 ± 0.06
1.76 ± 0.11
2.99 ± 0.12
2.11 ± 0.13

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Table 2. Cont.
IC50 ± SEM a (M)
SK-MES1 d
N°
6
Structure
MRC-5 b
AGS c
J82 e
HL-60 f
2.6 ± 0.2
1.8 ± 0.1
1.66 ± 0.08
2.0 ± 0.1
2.64 ± 0.21
7
8
1.9 ± 0.2
2.7 ± 0.2
7.1 ± 0.5
2.2 ± 0.2
1.9 ± 0.0
1.1 ± 0.0
2.6 ± 0.2
1.15 ± 0.05
2.13 ± 0.13
1.29 ± 0.08
1.2 ± 0.1
2.2 ± 0.2
1.2 ± 0.1
2.3 ± 0.1
2.63 ± 0.13
3.57 ± 0.16
2.34 ± 0.14
2.15 ± 0.12
9
10
0.56 ± 0.03 2.14 ± 0.13
11
12
13
9.53 ± 0.38
1.8 ± 0.1
8.15 ± 0.49 1.46 ± 0.06 6.97 ± 0.35
2.61 ± 0.18
2.16 ± 0.09
0.49 ± 0.02
2.23 ± 0.09
1.2 ± 0.1
0.93 ± 0.03
1.1 ± 0.1
0.28 ± 0.01
0.26 ± 0.01 0.21 ± 0.01 0.29 ± 0.01
Etoposide
0.33 ± 0.02
0.58 ± 0.02 1.83 ± 0.09 3.49 ± 0.16
a
b
Data represent mean average values for six independent determinations; Normal human lung fibroblasts
c
d
e
cells; Human gastric adenocarcinoma cell line; Human lung cancer cell line; Human bladder carcinoma
cell line; f Leukemia cell line.
Next, the SAR analysis was focused on the effects of insertion of chlorine and bromine atoms at the
6-position of the 7-substituted alkylaminoisoquinolinequinones 2a, 3a, 4a and 5a. Comparison of the
IC50 values of halo derivatives 6–11 with those of their corresponding precursors indicates that the
substitution of halogen atom at the 6-position enhances the antitumor potency in all the evaluated cell
lines, except for compound 11 on lung fibroblasts and gastric cancer cells.

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According to the data of Table 2, aminoisoquinolinequinone 12 showed more cytotoxic activity
(0.93–2.16 µM) on the cancer cell lines than the N-alkyl 7-substituted analogs 2a, 3a, 4a and 5a. It is
noteworthy that compound 12 exhibits higher antitumor potency on lung and bladder cell lines than the
anticancer drug etoposide.
Comparison of the cytotoxic potency of compound 12 (0.93−2.16 µM) with that of its
bromoderivative 13 (IC50: 0.21−0.49 µM) reveals that insertion of the bromine atom at the 6-position
of 12 induces a high increase of the cytotoxic activity in all the evaluated cell lines. It is noteworthy
that compounds 12 and 13 exhibit higher antitumor potency and selectivity index (SI = IC50
fibroblasts/IC50 cancer cells. 12: 0.8–1.9; 13: 0.5–1.3) in almost all cell lines than the anticancer drug
etoposide (SI: 0.1–0.6). Among the tested analogs of the series, compounds 4b, 12 and 13 are the most
significant antitumor members. Compound 13 was selected as a promising lead compound due to its
high potency on the tested tumor cell lines and selectivity index.
3. Experimental
3.1. General
All reagents were commercially available reagent grade and were used without further purification.
1
Melting points were determined on a Stuart Scientific SMP3 apparatus and are uncorrected. H-NMR
spectra were recorded on Bruker AM-200 and Avance-400 instruments in deuterochloroform (CDCl3).
13C-NMR spectra were obtained in CDCl3 at 50 and 100 MHz. Bidimensional NMR techniques and
DEPT were used for signal assignment. Chemical shifts are expressed in ppm downfield relative to
tetramethylsilane and the coupling constants (J) are reported in Hertz. The HRMS spectra 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 aluminum foil 60F254 for analytical TLC.
3.2. Chemistry
4-Methoxycarbonyl-3-methylisoquinoline-5,8-quinone (1). A suspension of 2,5-dihydroxybenzaldehyde
(1 mmol), methyl 3-aminocrotonate (1 mmol), Ag2O (2 mmol) and MgSO4 (0.5 g) in CH2Cl2 (25 mL)
was stirred at rt for 3 h. The mixture was filtered, the solids were washed with CH2Cl2 and the solvent
removed under reduced pressure. The residue was column cromatographed over silica gel (90:10
CH2Cl2/AcOEt) to yield pure quinone 1 (86%) as yellow solid, mp 109–111.5 °C; IR max 1731
1
(C=O ester), 1668 (C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.65 (s, 3H, Me), 4.03 (s, 3H,
13
CO2Me), 7.04 (s, 2H, 6- and 7-H), 9.22 (s, 1H, 1-H); C-NMR (100 MHz): δ 22.69, 53.11, 122.18,
125.15, 133.39, 138.38, 138.58, 148.57, 161.83, 167.68, 183.38, 183.60; HRMS (M+): m/z calcd for
C12H9NO4: 231.05315; found: 231.05229.
7-Methylamino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (2a). A suspension of quinone 1
(1 mmol), methylamine hydrochloride (2 mmol) and AcONa (2 mmol) in ethanol (20 mL) was stirred
at rt for 1.5 h. The solvent was removed under reduced pressure to give an orange solid containing
regioisomers 2a and 2b (74%) in 80:20 proportion. The major regioisomer 2a was isolated by column
chromatography over silica gel (90:10 CH2Cl2/AcOEt): red solid (52 %), mp 209.5–211 °C; IR max

Molecules 2012, 17
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1
3279 (N-H), 1740 (C=O ester), 1681 (C=O quinone); H-NMR: (400 MHz, DMSO): δ 2.66 (s, 3H,
Me), 2.95 (d , 3H, J = 5.4 Hz, NHMe), 4.04 (s, 3H, CO2Me), 5.75 (s, 1H, 6-H), 6.09 (br s, 1H, NH),
9.18 (s, 1H, 1-H); 13C-NMR (100 MHz) δ: 23.02, 29.36, 53.21, 101.16, 122.08, 126.38, 136.38,
148.10, 148.82, 163.02, 168.94, 180.24, 180.55; HRMS (M+): m/z calcd for C13H12N2O4: 260.0797;
found: 260.0731.
7-(2-Hydroxyethylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (3a). A suspension of
quinone 1 (1 mmol), 2-aminoethanol (2 mmol), CeCl3.7H2O (0.05 mmol) and ethanol (25 mL) was left
with stirring at rt for 3 h. The solvent was removed under reduced pressure to give an orange solid
containing regioisomers 3a and 3b (65%) in 80:20 proportion. The major regioisomer 3a was isolated
by column chromatography over silica gel (90:10 CH2Cl2/AcOEt); red solid (47%), mp 195.5–197 °C;
1
IR max 3399 (O-H), 3186(N-H), 1700 (C=O ester), 1600 (C=O quinone); H-NMR (400 MHz,
DMSO): δ 2.52 (s, 3H, Me), 3.27 (dt, 2H, J = 5.8, 11.7 Hz, NHCH2), 3.59 (dt, 2H, J = 5.6, 11.2 Hz,
CH2OH), 3.88 (s, 3H, CO2Me), 4.89 (t, 1H, J = 5.7 Hz, OH), 5.79 (s, 1H, 6-H), 7.75 (t, 1H, J = 5.9 Hz,
13
NH), 9.06 (s, 1H, 1-H); C-NMR (100 MHz): δ 22.35, 44.77, 52.63, 58.47, 99.87, 122.43, 125.18,
135.92, 147.36, 149.04, 161.13, 168.25, 178.91, 180.24; HRMS (M+): m/z calcd for C14H14N2O5:
290.0903; found: 290.0891.
6- and 7-(Cyclohexylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (4a,4b). A suspension
of quinone 1 (1 mmol), cyclohexylamine (2 mmol), CeCl3·7H2O (0.05 mmol) and ethanol (25 mL) was
left with stirring at rt for 2 h. The solvent was removed under reduced pressure to give an orange
solid containing regioisomers 4a and 4b in 86:14 ratio. These compounds were isolated by column
chromatography over silica gel (90:10 CH2Cl2/AcOEt).
Compound 4a (less polar, 80%): orange solid, mp 161.5–163.5 °C; IR max 3,246 (N-H), 2,927 and
1
2,856 (CH), 1741 (C=O ester), 1685 (C=O quinone); H-NMR (400 MHz, CDCl3): δ 1.35 (m, 5H,
CH2), 1.68 (m, 1H, CH2), 1.81 (m, 2H, CH2), 2.03 (m, 2H, CH2), 2.65 (s, 3H, Me), 3.30 (m,1H, CH),
13
4.03 (s, 3H, CO2Me), 5.77 (s, 1H, 6-H), 5.96 (d, 1H, J = 7.5 Hz, NH), 9.17 (s, 1H, 1-H); C-NMR
(100 MHz): δ 22.99, 24.54 (2C), 25.42, 31.83 (2C), 51.44, 53.16, 101.25, 122.11, 126.23, 136.34,
146.52, 148.03, 162.89, 168.93, 180.13, 180.83; HRMS (M+): m/z calcd for C18H20N2O4: 328.14227;
found: 328.14083.
Compound 4b (9.5%): red solid, mp 158.5–160 °C; IR max 3246 (N-H); 2927 and 2856 (CH); 1741
1
(C=O ester); 1685 (C=O quinone); H-NMR (400 MHz, CDCl3): δ 1.35 (m, 5H, CH2), 1.68 (m, 1H,
CH2), 1.81 (m, 2H, CH2), 2.03 (m, 2H, CH2), 2.65 (s, 3H, Me), 3.30 (m, 1H, CH), 4.05 (s, 3H,
13
CO2Me), 5.75 (d, 1H, J = 7.8 Hz, NH), 5.79 (s, 1H, 6-H), 9.28 (s, 1H, 1-H), C-NMR (100 MHz): δ
22.64, 24.54 (2C), 25.47, 31.86 (2C), 51.39, 53.26, 101.23, 123.05, 124.92, 132.44, 146.31, 148.73,
159.91, 168.51, 181.49, 181.71;HRMS (M+): m/z calcd for C18H20N2O4: 328.14227; found: 328.14070.
6- and 7-Morpholino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (5a,5b). A suspension of
quinone 1 (1 mmol), morpholine (2 mmol), CeCl3·7H2O (0.05 mmol) and ethanol (25 mL) was left
with stirring at rt for 2 h. The solvent was removed under reduced pressure to give an orange solid

Molecules 2012, 17
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containing regioisomers 5a and 5b in 87:13 proportion. These compounds were isolated by column
chromatography over silica gel (90:10 CH2Cl2/AcOEt).
Compound 5a (less polar, 72%): dark orange solid, mp 169.5–171.5 °C; IR max 1722 (C=O ester),
1,696 and 1,638 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.65 (s, 3H, Me), 3.59 (t, 4H, J = 4.7 Hz,
N-CH2), 3.87 (t, 4H, J = 4.7 Hz, O-CH2), 4.03 (s, 3H, CO2Me), 6.01 (s, 1H, 6-H), 9.14 (s, 1H, 1-H);
13C-NMR (100 MHz): δ 22.90, 49.21 (2C), 53.28, 66.49 (2C), 110.92, 123.98, 125.36, 134.86, 148.72,
152.78, 161.98, 168.72, 181.16, 181.75; HRMS (M+): m/z calcd for C16H16N2O5: 316.10593;
found: 316.10521.
Compound 5b (17%): dark red solid, mp 159.5–162 °C; IR max 1724 (C=O ester), 1,678 and 1,634
(C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.66 (s, 3H, Me), 3.47 (t, 4H, J = 4.8, N-CH2), 3.85 (t,
4H, J = 4.8 Hz, O-CH2), 4.03 (s, 3H, CO2Me), 6.01 (s, 1H, 6-H), 9.23 (s, 1H, 1-H); 13C-NMR (100 MHz):
δ 22.89, 49.10 (2C), 53.25, 66.44 (2C), 111.30, 122.43, 125.41, 135.43, 148.35, 153.21, 160.76,
168.54, 182.10, 182.30; HRMS (M+): m/z calcd for C16H16N2O5: 316.10593; found: 316.10445.
3.3. General Procedure for the Synthesis of 6-Bromo (or 6-chloro)-7-aminoisoquinolinequinone Derivatives
A solution of the 7-aminoisoquinolinequinone derivative (1 mmol), the corresponding
N-bromosuccinimide (NBS) or N-chlorosuccinimide (NCS) (1 mmol) and methanol (15 mL) was left
with stirring at rt after completion of the reaction as indicated by TLC. The solvent was removed under
reduced pressure and the residue was column cromatographed over silica gel (90:10 CH2Cl2/AcOEt) to
yield the corresponding 7-amino-6-haloisoquinolinequinone derivative.
6-Bromo-4-methoxycarbonyl-7-methylamino-3-methylisoquinoline-5,8-quinone (6). Prepared from 2a
and NBS (30 min, 80%): dark red solid, mp 170–172 °C; IR max 3331 (N-H); 1730 (C=O ester), 1687
1
(C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.65 (s, 3H, Me), 3.48 (d , 3H, J = 5.8 Hz, NHMe),
13
4.05 (s, 3H, CO2Me), 6.39 (br s, 1H, NH), 9.16 (s, 1H, 1-H); C-NMR (100 MHz): δ 23.09, 33.34,
53.39, 121.29, 126.56, 134.72, 146.86, 148.62, 163.23, 168.42, 174.40, 178.84 (the signal of the C-6
was not observed in this experiment); HRMS (M+): m/z calcd for C13H11N2O4Br: 337.99023;
found: 337.98951.
6-Chloro-4-methoxycarbonyl-7-methylamino-3-methylisoquinoline-5,8-quinone (7). Prepared from 2a
and NCS (3 h, 85%): dark red solid, mp 158–160 °C; IR max 3331 (N-H); 1773 (C=O ester), 1693
1
(C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.65 (s, 3H, Me), 3.48 (d , 3H, J = 6.0 Hz, NHMe),
13
4.05 (s, 3H, CO2Me), 6.30 (br s, 1H, NH), 9.15 (s, 1H, 1-H); C-NMR (100 MHz): δ 23.09, 32.82,
53.37, 121.25, 126.52, 135.11, 144.83, 148.50, 163.41, 168.36, 174.83, 179.27 (the signal of the
C-6 was not observed in this experiment); HRMS (M+): m/z calcd for C13H11N2O4Cl: 294.0407;
found: 294.04002
6-Bromo-2-(hydroxyethylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (8). Prepared
from 3a and NBS (30 min, 75%): dark red solid, mp 134–136 °C; IR max 3463 (O-H), 3320 (N-H),
1
1720 (C=O ester), 1685 (C=O quinone); H-NMR (400 MHz, DMSO): δ 2.63 (s, 3H, Me), 3.92 (dt,
2H, J = 5.1, 11.2 Hz, CH2OH), 4.06 (s, 3H, CO2Me), 4.11 (dt, 2H, J = 5.3, 10.8 Hz, NHCH2), 6.65 (br s,

Molecules 2012, 17
7052
1H, NH), 9.13 (s, 1H, 1-H); 13C-NMR (100 MHz): δ 22.98, 47.25, 53.40, 61.36, 121.49, 126.41,
134.60, 146.84, 148.61, 163.02, 168.52, 174.19, 178.66, (the signal of the C-6 was not observed in this
experiment); HRMS (M+): m/z calcd for C14H13N2O5Br: 368.00079; found: 368.00005.
6-Chloro-2-(hydroxyethylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (9). Prepared
from 3a and NCS (4 h, 78%): dark red solid, mp 123–125 °C; IR max 3461 (O-H), 3325 (N-H), 1716
(C=O ester), 1687 (C=O quinone); 1H-NMR (400 MHz, CDCl3): 1H-NMR (400 MHz, CDCl3): δ 2.65
(s, 3H, Me), 3.92 (m, 2H, NHCH2), 4.05 (s, 3H, CO2Me), 4.07 (m, 2H, CH2OH), 6.58 (br s, 1H, NH),
13
9.16 (s, 1H, 1-H); C-NMR (100 MHz): δ 23.09, 4.82, 53.42, 61.71, 121.38, 126.48, 134.99, 144.53,
148.59, 163.35, 168.41, 174.67, 179.22, (the signal of the C-6 was not observed in this experiment);
HRMS (M+): m/z calcd for C14H13N2O5Cl: 324.0513; found: 324.05073.
6-Bromo-7-(cyclohexylamino)-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (10). Prepared
from 4a and NBS (15 min, 84% yield): dark red solid, mp 132.5–134.5 °C; IR max 3312 (N-H), 3008
and 2858 (C-H), 1735 (C=O ester), 1687 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 1.34 (m, 5H,
CH2), 1.68 (m, 1H, CH2), 1.81 (m, 2H, CH2), 2.03 (m, 2H, CH2), 2.64 (s, 3H, Me), 4.05 (s, 3H,
13
CO2Me), 4.58 (m,1H, CH), 6.24 (br s, 1H, NH), 9.15 (s, 1H, 1-H); C-NMR (100 MHz): δ 23.05,
24.52 (2C), 25.29, 34.50 (2C), 53.32, 61.58, 121.36, 126.59, 134.80, 148.65, 149.99, 163.16, 168.38,
174.32, 178.90, (the signal of the C-6 was not observed in this experiment); HRMS (M+): m/z calcd for
C18H19N2O4Br: 406.05280; found: 406.05196.
6-Bromo-7-morpholino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (11). Prepared from 5a
and NBS (30 min, 70%): violet solid, mp 127.5–129 °C; IR max 2955 and 2856 (C-H), 1722
(C=O ester), 1696 and 1638 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.66 (s, 3H, Me), 3.70 (t,
4H, J = 4.5 Hz, N-CH2), 3.90 (t, 4H, J = 4.5 Hz, O-CH2), 4.05 (s, 3H, CO2Me), 9.19 (s, 1H, 1-H);
13C-NMR (100 MHz): δ 22.87, 52.59 (2C), 53.42, 67.51 (2C), 115.51, 122.43, 126.05, 133.51, 149.00,
152.08, 162.25, 168.21, 176.25, 180.24; HRMS (M+): m/z calcd for C16H15N2O5Br: 394.01643;
found: 394.01457.
7-Amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (12). A solution of sodium azide
(1.2 mmol) in water (5 mL) was added, with stirring, to a previously heated solution (40 °C) of
quinone 1 (1 mmol) in glacial acetic acid (10 mL). The mixture was stirred at rt for 3.5 h, diluted with
water and then extracted with AcOEt (3 × 15 mL). The organic phase was washed with saturated
aqueous solution of NaHCO3, dried (MgSO4) and the solvent was removed under reduced pressure.
The residue was column cromatographed over silica gel (80:20 CH2Cl2/AcOEt) to yield aminoquinone
12 as an orange solid (51%), mp 189–191 °C; IR max 3376 and 3140 (N-H), 1732 (C=O ester), 1691
1
(C=O quinone); H-NMR (400 MHz, DMSO): δ 2.66 (s, 3H, Me), 4.05 (s, 3H, CO2Me), 5.38 (br s,
2H, NH2), 6.03 (s, 1H, 6-H), 9.20 (s, 1H, 1-H); 13C-NMR (100 MHz): δ 23.37, 52.68, 102.44, 122.47,
125.17, 135.92, 147.19, 150.88, 161.00, 168.34, 179.30, 180.65; HRMS (M+): m/z calcd for
C12H10N2O4: 246.06406; found: 246.06207.
7-Amino-6-bromo-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (13). Prepared from 12 and
NBS (20 min, 86%): mp 146–148 °C; IR max 3423 and 3326 (N-H), 1726 (C=O ester), 1683

Molecules 2012, 17
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(C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.67 (s, 3H, Me), 4.06 (s, 3H, CO2Me), 5.45 (br s, 1H,
13
NH), 6.26 (br s, 1H, NH), 9.22 (s, 1H, 1-H); C-NMR (100 MHz): δ 23.18, 53.48, 105.07, 121.08,
126.91, 134.81, 147.20, 148.50, 163.58, 168.26, 174.53, 177.24; HRMS (M+): m/z calcd for
C12H9N2O4Br: 323.97455; found: 323.97419.
3.4. Anticancer Assay
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
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%, as
assessed by trypan blue exclusion test. Cells were sub-cultured once a week and the medium was
changed every two days. Cells were grown in the following media: MRC-5, SKMES-1, and J82 in
Eagle's minimal essential medium (EMEM) and AGS cells in Ham F-12. The EMEM medium
contained 2 mM L-glutamine, 1 mM sodium pyruvate and 1.5 g/L sodium hydrogen carbonate. Ham F-12
was supplemented with 2 mM L-glutamine and 1.5 g/L sodium hydrogen carbonate. All media were
supplemented with 10% heat-inactivated FBS, 100 IU/mL penicillin and 100 g/mL streptomycin in a
humidified incubator 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 at concentrations ranging from 0 up to 100 M during 3 days and finally the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (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 experiment was carried out in sextuplicate.
4. Conclusions
We have carried out the synthesis of 4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (1)
from 2,5-dihydroxybenzaldehyde, methyl aminocrotonate and silver (I) oxide by employing a highly
efficient one-pot procedure. A variety of new isoquinolinequinones substituted with amino, alkylamino
and halogen groups were prepared from quinone 1. The members of this series expressed moderate
to high in vitro cytotoxic activity against MRC-5 (healthy lung fibroblasts) and four human cancer
cell lines: AGS (gastric), SK-MES-1 (lung), J82 (bladder), and HL-60 (leukemia) cell lines. From
the current investigation, structure activity relationships of the aminoisoquinolinequinone series
demonstrate that nitrogen and halogen substituents at the quinone nucleus of pharmacophore show
increased antitumor activity compared with precursor 1. The effect of such substitutions is more
significant in enhancing the antitumor activity for those members containing the cyclohexylamino
group at C-6 (compound 4b), the amino group at C-7 (compound 12) and the amino and bromine
substituents at the 6- and 7-positions (compound 13). Due to the high incidence of undesirable side-
effects induced by the majority of current anticancer drugs and by considering the selectivity index values
of aminoquinones 4b (SI: 0.7–3.7), 12 and 13 (Figure 3), they appear as promising and interesting leads

Molecules 2012, 17
7054
endowed with potential anticancer activity. These results prompt us to design and synthesize
more new members of the aminoisoquinoline-5,8-quinones series in order to discover more active
and selective anticancer agents. Studies aimed to understand the mechanisms of bioactivity of
aminoisoquinolinequinones at cellular level are currently in progress.
Figure 3. Structures of the isoquinolinequinones selected as the most significant antitumor agents.
O
O
O
H2N
Br
H2N
N
N
N
N
Me
CO2Me
Me
CO2Me
Me
CO2Me
H
O
O
O
12
4b
13
Acknowledgments
This research were supported by FONDECYT (Grants N°: 1060591 and 1100376) and the
Vicerrectoria de Investigación (VRI) of the Pontificia Universidad Católica de Chile (V.D.;
POSTDOC/N°1/2010).
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Sample Availability: Samples of all the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).