Synthesis and in Vitro antiproliferative activity of new phenylaminoisoquinolinequinones against cancer cell lines

Article abstract of DOI:10.3390/molecules18010721

A variety of phenylaminoisoquinolinequinones were synthesized and tested for their antiproliferative activity against three human-tumor derived cancer cell lines. The new aminoquinones were prepared from 4-methoxycarbonyl-3- methylisoquinoline-5,8- quinone (1) via acid-induced amination and bromination reactions. Remarkable differences in antiproliferative activity were observed depending upon the location and donor capacity of the substituted phenylamino group at the quinone nucleus. The effect of the substituents on the biological activity is discussed in terms of the donor-Acceptor interactions which were evaluated through the redox properties of the aminoquinones.

Full text of DOI:10.3390/molecules18010721

Molecules 2013, 18, 721-734; doi:10.3390/molecules18010721
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and in Vitro Antiproliferative Activity of New
Phenylaminoisoquinolinequinones against Cancer Cell Lines
Virginia Delgado 1, Andrea Ibacache 1, Verónica Arancibia 1, Cristina Theoduloz 2 and
Jaime A. Valderrama 1,3,
*
1
Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago 26094411, 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.: +56-02-2354-4432; Fax: +56-02-2354-4744.
Received: 10 December 2012; in revised form: 27 December 2012 / Accepted: 31 December 2012 /
Published: 8 January 2013
Abstract: A variety of phenylaminoisoquinolinequinones were synthesized and tested for
their antiproliferative activity against three human-tumor derived cancer cell lines. The
new aminoquinones were prepared from 4-methoxycarbonyl-3-methylisoquinoline-5,8-
quinone (1) via acid-induced amination and bromination reactions. Remarkable differences
in antiproliferative activity were observed depending upon the location and donor capacity
of the substituted phenylamino group at the quinone nucleus. The effect of the substituents
on the biological activity is discussed in terms of the donor-acceptor interactions which
were evaluated through the redox properties of the aminoquinones.
Keywords: phenylaminoisoquinoline-5,8-quinones; regioselectivity; half-wave potential;
antiproliferative activity; SAR analysis
1. Introduction
The molecular framework of a variety of naturally occurring antibiotics contains an aminoquinoline-
and aminoisoquinoline-5,8-quinone moiety as the key structural component (i.e., streptonigrin [1,2],
lavendamycin [3,4], cribrostratin 3 [5], caulibugulones A–C [6] and mansouramycins A–C [7]). This
structural array has stimulated the synthesis of novel aminoquinoline- and aminoisoquinoline-5,8-
quinones [8–12]. The main target of these synthetic efforts is to extend the spectrum of antiproliferative

Molecules 2013, 18
722
activity on cancer cells. In the light of these facts we and other authors have reported the preparation
and antiproliferative evaluation of 6,7-substituted aminoquinoline- and aminoisoquinolinequinones.
Evidences arising from these studies demonstrate that insertion and change of location of alkylamino
and halogen substituents in the quinone nucleus of the corresponding N-heterocyclic cores induce
difference in the antiproliferative activity [13–15].
In the search for new aminoquinones endowed with high antiproliferative potency we are interested
in the evaluation of the effect of the insertion and change of location of p-substituted phenylamino and
bromine groups at the 6- and 6,7-positions of an isoquinoline-5,8-quinone chemotype on the
antiproliferative activity. In this paper, we present the synthesis of a series of 6,7-substituted
isoquinolinequinones, which were evaluated in terms of their in vitro antiproliferative properties
against a panel of three cancer cell lines.
2. Results and Discussion
2.1. Chemistry
Isoquinolinequinone 1 was selected as precursor of the designed phenylaminoisoquinolinequinones
taking into account its reactivity with alkylamines, which provides simultaneous access to 6- and
7-aminoisoquinolinequinones [12]. The synthesis of 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 [12] (Scheme 1). The preparation of compounds 2a,b was
initially explored by reaction of quinone 1 with aniline in ethanol at room temperature. The reaction
went to completion in 3.5 h giving a 95:5 mixture of the expected regioisomers 2a,b, albeit in 37%
yield. The ratio between regioisomers 2a,b was evaluated by 1H-NMR using the signals of the protons
at C-1 of each regioisomer, located at 9.24 and 9.29 ppm, respectively. A better result in the
preparation of regioisomers 2a,b was obtained when the amination reaction of 1 was carried out in
ethanol in the presence of 5 mmol % of CeCl3·7H2O. Under these conditions, the reaction was clean
and fast (2 h) to give a 66:34 mixture of isomers 2a,b in 74% yield. Pure samples of compounds 2a,b
were achieved by column chromatography. These results clearly demonstrate that the catalyst
improves the yield of the amination reaction and changes the regioselectivity, favoring the formation of
the minor regioisomer 2b.
Scheme 1. Synthesis of 6- and 7-phenylaminoisoquinolinequinones 2a,b–4a,b.

Molecules 2013, 18
723
Based on the above Lewis acid-induced procedure, phenylaminoisoquinolinequinones 3a,b and
4a,b were prepared by amination reaction of quinone 1 with p-anisidine and p-aminophenol,
respectively. In these experiments, pure samples of the corresponding regioisomers 3a,b and 4a,b were
isolated by column chromatography.
The structures of the new compounds were 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. The
location of the nitrogen group at C-7 in compounds 2a, 3a and 4a was deduced by the 3JC,H couplings
between the carbon at C-8 with the protons at C-1, at C-6 and that of the NH group. In the case of
3
aminoquinones 2b, 3b and 4b the location of the nitrogen substituent at C-6 was established by JC,H
coupling between the carbon at C-5 with the proton at C-7 and the proton of the NH group [12,16].
The insertion of a bromine atom in phenylaminoisoquinolinequinones 2–4 was attempted by
reaction of aminoquinones 2–4 with N-bromosuccinimide (NBS). The substitution reaction of
aminoquinones 2a,b and 3a,b proceeded under mild conditions to give the expected bromoquinones
5a,b and 6a,b (Table 1). Regarding the bromination attempts of arylaminoquinone 4a, by TLC was
observed the disappearance of 4a with concomitant appearance of a yellow spot. However, after the
aqueous work-up of the mixture reaction, aminoquinone 4a was recovered and no reaction products
1
were detected (TLC, H-NMR). A plausible explanation for the lack of bromination of compound 4a
could be attributed to the oxidation of the p-hydroxyphenylamino group of 4a with NBS [13–15] to
give the corresponding electron-attracting iminoquinoyl group, which probably inhibits the
bromination reaction at the quinone double bond and, under aqueous media, undergoes a reversible
reduction to the hydroxyphenylamino group. In order to avoid the interference of the phenylamino
substituent on the bromination reaction at the 6-position of 4a, we attempted to prepare the target
brominated regioisomer 9 through a three step sequence, which involved the protection of the hydroxyl
group in 4a, as the acetate, followed by bromination of 7a with NBS and further base-induced
deprotection of acetyl group in 8, of the hydroxyl group (Scheme 2). This strategy was successful and
the expected bromine derivative 9 was isolated in 33% total yield (Table 1).
Scheme 2. Synthesis of the bromine derivative 9 from phenylaminoquinone 4a.

Molecules 2013, 18
Table 1. Preparation of phenylaminobromine derivatives 5a,b, 6a,b, 8 and 9.
724
Bromine derivative
N° (Yield %) a
Bromine derivative
N° (Yield) a
Substrate
Substrate
2a
2b
5a (87)
5b (74)
6a (80)
3b
7a
8
6b (78)
8 (56)
9 (64)
3a
a Isolated by column chromatography.
2.2. Electrochemical Results
The redox potentials of the new compounds were measured by cyclic voltammetry in acetonitrile as
solvent, at room temperature, using a platinum electrode and 0.1 M tetraethylammonium tetrafluoroborate
as the supporting electrolyte [16]. Well-defined quasi-reversible waves were observed for the
compounds, the cathodic peak related to the reduction of quinone, and the anodic one due to its
reoxidation. The voltammograms were run in the potential range 0.0–2.0 V versus non-aqueous
Ag/Ag+. The first half-wave potential values, EI1/2, evaluated from the voltammograms obtained at a
sweep rate of 100 mVs−1, are summarized in Table 2.
Table 2. Half wave potentials and quinone-proton chemical shifts of 1 and the new compounds.
Compound N° −EI1/2 (mV) −EII1/2 (mV)
logP a 6 or 7-H b
1
352
563
494
551
482
555
525
373
518
426
373
521
544
386
431
1140
874
1035
1083
1040
869
881
932
906
795
953
1058
711
944
777
0.10
0.74
0.74
0.61
0.61
0.35
0.35
1.05
1.05
0.92
0.92
0.32
0.32
0.63
0.66
7.04
6.39
6.38
6.21
6.20
6.20
6.18
-
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8
-
-
-
6.34
6.34
-
9
-
a Determined by the ChemBioDraw Ultra 11.0 software; b Recorded in CDCl3.

Molecules 2013, 18
725
The EI1/2 values for the first electron, which is related to the formation of the semiquinone radical
anion, are in the potential range −563 to −352 mV. The data of Table 2 indicate that the presence of
phenylamino substituents at the quinone ring in precursor 1 induces the displacement of the half wave
potential of 1 from −352 mV to an upper potential zone located in the range: −563 to −373 mV. The
position of the potential values within this range evidently depends on the nature and location of the
nitrogen substituents.
Comparison of the first half wave potentials between the pair of regioisomers 2, 3, 4 and 7, reveals
that the reduction for the 7-substituted isomers, as 2a, 3a and 4a, occurs at more negative EI1/2
potentials than that of the corresponding 6-substituted isomers, except for the pair 7. According to
reported precedents on the electronic effect of substituents in 1,4-naphthoquinones on EI1/2 potentials [17],
it can be deduced that for each pair of regioisomers, that containing the nitrogen substituent at
7-position has a greater electron-donor capacity. It is interesting to note that comparison of the
chemical shifts of the quinone-proton of compounds 2a,b, 3a,b, and 4a,4b (Table 2) indicate that a
nitrogen substituent at the 7-position induces a large shielding effect (lower chemical shift values) on
the proton at C-6 compared to the effect of the nitrogen substituent at the 6-position on the proton at
C-7. This effect is consistent with the differences in half-wave potentials among the analog pairs.
2.3. In Vitro Antiproliferative Activity of Phenylaminoisoquinolinequinones again Cancer Cell Lines
Isoquinolinequinones 1, 2a,b, 3a,b, 4a,b, 5a,b, 6a,b, 7a,b (prepared by acetylation of 4b), 8 and 9
were evaluated for their in vitro antiproliferative activity on a panel of three human cancer cell lines:
AGS (gastric), SK-MES-1 (lung), and J82 (bladder) using the conventional microculture tetrazolium
reduction assay [18–20]. The broad variety of the synthesized compounds was designed in order to gain
insight into the influence of nitrogen and halogen groups at the quinone nucleus of the isoquinolinequinone
chromophore on the biological activity. Table 3 summarizes the data from these evaluations.
According to the IC50 values collected in Table 3, it is evident that the insertion of nitrogen
substituents at the quinone double bond of compound 1, as in 2a,b, 3a,b, 4a,b, and 7a,7b induces a
remarkable increase of the antiproliferative activity in all the evaluated cell lines, compared to those of
precursor 1, reaching, in some cases, submicromolar IC50 values on the tested cancer cell lines. Among
the members of this series, compounds 3b, 4b, 7b and 5a, exhibit higher antiproliferative activities
than etoposide, used as the reference drug in the screening.
The initial structure-activity relationship (SAR) of the series was focused on the nature and location
of the phenylamino group at the quinone nucleus of the isoquinolinequinone pharmacophore. The data
of Table 3 for compounds 2a,b, 3a,b, 4a,b, and 7a,7b reveal that the nature of the substituent at the
para-position in the phenylamino group (H, OMe, OH, OAc) has a significant influence on the
antiproliferative activity on the tested tumor cell lines. Concerning the effect of the location of the
phenylamino groups on the biological activity, it is noteworthy that in the 6-regioisomers 2b, 3b, 4b,
and 7b, the nitrogen substituent exerts a greater effect on the antiproliferative activity of the
pharmaco-phore than the corresponding 7-regioisomers 2a, 3a, 4a, and 7a on the gastric and lung
cancer cell lines. This biological effect is opposite on bladder tumor cell line for the pair 2a,b and 7a,b
where it was observed that the 7-regioisomers were more potent than the respective 6-regioisomers.

Molecules 2013, 18
726
Table 3. Antiproliferative activity and half-wave potentials of 1 and their 6,7-substituted derivatives.
IC50 ± SEM a (μM)
N°
Structure
AGS b
SK-MES1 c
J82 d
−EI1/2 (eV)
1
17.34 ± 1.64 25.90 ± 1.60 14.81 ± 0.74
352
2a
2b
3a
3b
1.10 ± 0.03
0.57 ± 0.20
1.10 ± 0.10
0.32 ± 0.04
4.40 ± 0.09
2.60 ± 0.30
3.40 ± 0.60
1.10 ± 0.09
1.50 ± 0.70
1.60 ± 0.70
3.80 ± 0.70
1.90 ± 0.2 0
563
494
551
482
4a
4b
7a
7b
5a
5b
6a
0.72 ± 0.19
0.24 ± 0.01
1.01 ± 0.04
0.29 ± 0.01
0.70 ± 0.04
1.84 ± 0.14
1.11 ± 0.06
2.40 ± 0.12
0.89 ± 0.05
0.98 ± 0.05
0.53 ± 0.04
0.31 ± 0.01
4.72 ± 3.23
1.07 ± 0.04
2.06 ± 0.10
0.76 ± 0.04
0.72 ± 0.19
1.34 ± 0.05
2.30 ± 0.10
4.49 ± 0.31
9.72 ± 0.58
555
525
521
544
373
518
426
6b
1.80 ± 0.09
1.79 ± 0.11
1.98 ± 0.13
373

Molecules 2013, 18
727
Table 3. Cont.
IC50 ± SEM a (μM)
N°
8
Structure
AGS b
SK-MES1 c
J82 d
−EI1/2 (eV)
1.73 ± 0.12
3.91 ± 0.27
3.34 ± 0.23
386
9
1.10 ± 0.06
0.58 ± 0.02
2.87 ± 0.14
1.83 ± 0.09
2.35 ± 0.19
3.49 ± 0.16
431
-
-
etoposide
a
b
Data represent mean average values for six independent determinations; Human gastric adenocarcinoma
cell line; c Human lung cancer cell line; d Human bladder carcinoma cell line.
Next, the SAR analysis was focused on the effects of insertion of a bromine atom at the quinone
ring in the aminoquinones 2a,b, 3a,b, 4a and 7a. Comparison of the IC50 values of bromine derivatives
5a,b, 6a,b, 8 and 9 with those of their corresponding precursors indicates that the insertion decreases
the biological activity with respect to their precursors, except in the case of compound 5a, which
showed a weak increase respect to 2a in the activity on gastric and lung cancer cell lines.
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 (C=O
1
ester), 1668 and 1575 (C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.65 (s, 3H, Me), 4.03 (s, 3H,
CO2Me), 7.04 (s, 2H, 6- and 7-H), 9.22 (s, 1H, 1-H); 13C-NMR (100 MHz): δ 22.7, 53.1, 122.2, 125.2,
133.4, 138.4, 138.6, 148.6, 161.8, 167.7, 183.38, 183.6; HRMS (M+): m/z calcd for C12H9NO4:
231.05315; found: 231.05229.

Molecules 2013, 18
728
3.3. General Procedure for the Synthesis of 6 and 7-Aminoisoquinolinequinone Derivatives
A suspension of isoquinolinequinone 1 (1 mmol), the required amine (2 mmol), CeCl3.7H2O
(0.05 mmol) and ethanol (25 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 (85:15 CH2Cl2/AcOEt) to yield the corresponding the mixture of
1
regioisomers. These were analysed by H-NMR to evaluate the proportion between the 6- and
7-aminoisoquinolinequinone derivatives. Column chromatography of the mixture, over silica gel
(95:5 CH2Cl2/ AcOEt), provided pure samples of the regioisomers.
6- and 7-Phenylamino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (2a,b). The mixture of
regioisomers was prepared from 1 and aniline (2 h, 74%); red solid; isomer proportion 2a:2b = 66:34.
Compound 2a (less polar, 47%): red solid, mp 197.4–199.3 °C; IR max 3449 (N-H), 1718 (C=O ester),
1680 and 1633 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.65 (s, 3H, Me), 4.03 (s, 3H, CO2Me),
6.39 (s, 1H, 6-H), 7.25 (m, 3H, arom), 7.43 (m, 2H, arom), 7.70 (br s, 1H, NH), 9.24 (s, 1H, 1-H);
13C-NMR (100 MHz): δ 23.1, 53.0, 103.7, 121.9, 123.1 (2C), 126.2, 126.6, 130.0(2C), 135.8, 136.8,
144.9, 148.4, 163.3, 168.7, 180.9, 181.5; HRMS (M+): m/z calcd for C18H14N2O4: 322.09534; found:
322.09529.
Compound 2b (23%): red solid, mp 171.0–172.5 °C; IR max 3442 (N-H), 1729 (C=O ester), 1692 and
1
1626 (C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.67 (s, 3H, Me), 4.09 (s, 3H, CO2Me), 6.38
(s, 1H, 7-H), 7.25 (m, 3H, arom), 7.42 (m, 2H, arom), 7.45 (br s, 1H, NH), 9.29 (s, 1H, 1-H);
13C-NMR (100 MHz): δ 22.8, 53.4, 103.7, 122.8, 123.2 (2C), 125.2, 126.6, 130.1 (2C), 132.4, 137.0,
144.9, 148.8, 160.6, 168.4, 181.7, 182.6; HRMS (M+): m/z calcd for C18H14N2O4: 322.09534;
found: 322.09485.
6- and 7-(4-Methoxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (3a,b).
The mixture of regioisomers was prepared from 1 and p-anisidine (1.3 h, 70%); purple solid; isomer
proportion 3a:3b = 60:40.
Compound 3a (less polar, 36%): purple solid, mp 164.0–165.5 °C; IR max 3349 (N-H), 1738 (C=O
ester), 1631(C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.66 (s, 3H, Me), 3.83 (s, 3H, OMe), 4.02
(s, 3H, CO2Me), 6.21 (s, 1H, 6-H), 6.94 (d, 2H, J = 8.9 Hz, 3'- and 5'-H), 7.18 (d, 2H, J = 8.9 Hz, 2'-
13
and 6'-H), 7.58 (br s, 1H, NH), 9.23 (s, 1H, 1-H); C-NMR (100 MHz): δ 21.1, 53.6, 55.6, 102.8,
115.1 (2C), 122.0, 125.1, 127.0, 129.3, 136.0, 145.7, 148.2, 158.2, 163.0, 168.7, 171.2, 180.9, 181.1;
HRMS (M+): m/z calcd for C19H16N2O5: 352.10593; found: 352.10505.
Compound 3b (22%): purple solid, mp 162.5–163.4 °C; IR max 3440 (N-H), 1731 (C=O ester), 1613
1
(C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.67 (s, 3H, Me), 3.83 (s, 3H, OMe), 4.07 (s, 3H,
CO2Me), 6.20 (s, 1H, 7-H), 6.94 (d, 2H, J = 8.8 Hz, 3'- and 5'-H), 7.18 (d, 2H, J =8.8 Hz, 2'- and 6'-H),
13
7.32 (br s, 1H, NH), 9.28 (s, 1H, 1-H); C-NMR (100 MHz): δ 22.7, 53.7, 55.7, 102.8, 115.2 (2C),
122.9, 125.0, 125.2, 129.3, 129.5, 132.4, 145.7, 148.7, 158.2, 160.3, 171.3, 181.7, 182.3; HRMS (M+):
m/z calcd for C19H16N2O5: 352.10593; found: 352.10449.

Molecules 2013, 18
729
6- and 7-(4-Hydroxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (4a,b). The
mixture of regioisomers was prepared from 1 and 4-aminophenol (1.3 h, 75%); purple solid; isomers
proportion 4a:4b = 65:35.
Compound 4a (less polar, 42%): purple solid, mp 195.6–197.4 °C; IR max 3331 (N-H), 1677 (C=O
1
ester), 1629 (C=O quinone); H-NMR (400 MHz, CDCl3+DMSO): δ 2.77 (s, 3H, Me), 3.93 (s, 3H,
CO2Me), 6.07 (s, 1H, 6-H), 6.81 (d, 2H, J = 8.8 Hz, 2'- and 6'-H), 7.02 (d, 2H, J = 8.8 Hz, 3'- and 5'-H),
13
8.18 (br s, 1H, NH), 9.12 (s, 1H, OH), 9.14 (s, 1H, 1-H); C-NMR (100 MHz): δ 22.6, 52.8, 102.1,
116.27 (2C), 121.9, 125.2 (2C), 125.7, 127.8, 135.9, 146.1, 147.7, 156.0, 162.3, 168.5, 180.5, 180.7;
HRMS (M+): m/z calcd for C18H14N2O5: 338.09027; found: 338.08967.
Compound 4b (19%): purple solid, mp 198.2–199.8 °C; IR max 3334 (N-H), 1670 (C=O ester), 1625
1
(C=O quinone); H-NMR (400 MHz, CDCl3+DMSO): δ 2.59 (s, 3H, Me), 4.05 (s, 3H, CO2Me), 6.10
(s, 1H, 7-H), 6.82 (d, 2H, J = 8.8 Hz, 2'- and 6'-H), 7.01 (d, 2H, J = 8.8 Hz, 3'- and 5'-H); 7.91 (br s,
1H, NH), 9.02 (s, 1H, OH), 9.18 (s, 1H, 1-H); 13C-NMR (100 MHz): δ 22.9, 53.0, 102.4, 116.5, 122.0,
125.3 (2C), 125.9, 128.0 (2C), 136.0, 146.1, 148.4, 156.2, 162.7, 168.7, 180.8, 180.9; HRMS (M+):
m/z calcd for C18H14N2O5: 338.09027; found: 338.08972.
7-(4-Acetyloxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (7a). A solution of
4a (1 mmol), acetic anhydride (1.5 mmol) and pyridine (5 mL) was stirred at rt for 2.3 h. The mixture
was diluted with H2O and extracted with EtOAc. The organic phase was washed with saturated
aqueous solution of KHSO4, dried (MgSO4) and the solvent was removed under reduced pressure. The
residue was column cromatographed over silica gel (90:10 CH2Cl2/AcOEt) to yield 7a (92%) as a red
1
solid, mp 186.2–187.5 °C; IR max 3222 (N-H), 1746 (C=O ester), 1683 and 1625 (C=O); H-NMR
(400 MHz, CDCl3): δ 2.32 (s, 3H, OCOMe), 2.66 (s, 3H, Me), 4.02 (s, 3H, CO2Me), 6.34 (s, 1H, 6-H),
7.16 (d, 2H, J = 8.8 Hz, 2'- and 6'-H), 7.28 (d, 2H, J = 8.8 Hz, 3'- and 5'-H), 7.70 (br s, 1H, NH), 9.23
13
(s, 1H, 1-H); C-NMR (100 MHz): δ 21.2, 22.7, 53.4, 103.6, 122.6, 123.2 (2C), 124.2 (2C), 125.1,
132.2, 134.4, 144.8, 148.6, 148.7, 160.6, 168.3, 169.4, 181.5, 182.4; HRMS (M+): m/z calcd for
C20H16N2O6: 380.10084; found: 380.10043.
6-(4-Acetyloxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (7b). According to
the procedure for the preparation of compound 7a, quinone 7b was synthesized in 89% yield from 4b.
Compound 7b was isolated as a pure red solid, mp 144.1–145.8 °C; IR max 3309 (N-H), 1732 (C=O
ester), 1681 and 1631 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.32 (s, 3H, OCOMe), 2.67 (s,
3H, Me), 4.07 (s, 3H, CO2Me), 6.34 (s, 1H, 7-H), 7.17 (d, 2H, J = 8.8 Hz, 2'- and 6'-H), 7.27 (d, 2H,
13
J = 8.8 Hz, 3'- and 5'-H), 7.41 (br s, 1H, NH), 9.29 (s, 1H, 1-H); C-NMR (100 MHz): δ 21.1, 23.0,
53.2, 103.6, 121.8, 123.1 (2C), 124.2 (2C), 126.0, 134.2, 135.6, 144.9, 148.3, 148.6, 163.2, 168.6,
169.4, 180.7, 181.3; HRMS (M+): m/z calcd for C20H16N2O6: 380.10084; found: 380.10015.
3.4. General Procedure for the Synthesis of 6- and 7-Bromo Derivatives
A solution of 6- or 7-phenylaminoisoquinolinequinone (1 mmol), the corresponding N-bromosuccinimide
(NBS) (1 mmol) and methanol (15 mL) was left with stirring at rt after completion of the reaction as

Molecules 2013, 18
730
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 haloisoquinolinequinone
derivative.
6-Bromo-7-phenylamino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (5a). Prepared from 2a
and NBS (15 min, 87%): dark red solid, mp 188.1–190.5 °C; IR max 3311 (N-H), 2955 and 2926 (C-H),
1
1732 (C=O ester), 1683 and 1641 (C=O quinone); H-NMR (400 MHz, CDCl3): δ 2.68 (s, 3H, Me),
4.06 (s, 3H, CO2Me), 7.05 (m, 2H, arom), 7.33 (m, 3H, arom), 7.88 (s, 1H, NH), 9.25 (s, 1H, 1-H);
13C-NMR (100 MHz): δ 23.2, 53.6, 106.6, 121.2, 125.4 (2C), 126.8, 128.8 (2C), 131.9, 134.6, 136.8,
144.5, 148.8, 163.5, 168.2, 175.6, 178.8; HRMS (M+): m/z calcd for C18H13N2O4Br: 400.00586;
found: 400.00553.
7-Bromo-6-phenylamino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (5b). Prepared from
2b and NBS (15 min, 74%): purple solid, mp 175.4–176.6 °C; IR max 3263 (N-H), 2953 and 2922
(C-H), 1734 (C=O ester), 1686 and 1633 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.67 (s, 3H,
Me), 4.01 (s, 3H, CO2Me), 7.01 (m, 2H, arom), 7.33 (m, 3H, arom), 7.62 (s, 1H, NH), 9.33 (s, 1H,
13
1-H); C-NMR (100 MHz): δ 22.8, 53.4, 108.3, 122.2, 124.8 (2C), 126.3, 128.8 (2C), 131.8, 132.4,
137.1, 144.4, 149.3, 161.2, 167.8, 176.0, 179.4; HRMS (M+): m/z calcd for C18H13N2O4Br: 400.00586;
found: 400.00585.
6-Bromo-7-(4-methoxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (6a). Prepared
from 3a and NBS (30 min, 80%): purple solid, mp 160.2–162.5 °C; IR max 3263 (N-H), 2956 and
2836 (C-H), 1724 (C=O ester), 1684 and 1637 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.67 (s,
3H, Me), 3.83 (s, 3H, OMe), 4.06 (s, 3H, CO2Me), 6.88 (d, 2H, J = 8.8 Hz, 3'- and 5'-H), 7.06 (d, 2H,
13
J = 8.8 Hz, 2'- and 6'-H), 7.88 (br s, 1H, NH), 9.23 (s, 1H, 1-H); C-NMR (100 MHz): δ 23.2, 53.4,
55.6, 105.1, 113.9 (2C), 121.2, 126.8, 127.2 (2C), 129.5, 134.8, 144.3, 148.8, 158.6, 163.5, 168.3,
175.4, 178.8; HRMS (M+): m/z calcd for C19H15N2O5Br: 430.01642; found: 440.01596.
7-Bromo-6-(4-methoxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (6b). Prepared
from 3b and NBS (30 min, 78%): purple solid, mp 163.3–164.5 °C; IR max 3238 (N-H); 2923 and
2854 (C-H), 1729 (C=O ester); 1685 and 1641 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.70 (s,
3H, Me), 3.86 (s, 3H, OMe), 4.05 (s, 3H, CO2Me), 6.87 (d, 2H, J = 8.8 Hz, 3'- and 5'-H), 7.03 (d, 2H,
13
J = 8.8 Hz, 2'- and 6'-H), 7.58 (br s, 1H, NH), 9.35 (s, 1H, 1-H); C-NMR (100 MHz): δ 22.8, 53.5,
55.6, 106.5, 114.0 (2C), 122.3, 125.4, 126.9 (2C), 129.8, 132.2, 144.4, 149.4, 158.4, 161.1, 168.0,
176.0, 179.6; HRMS (M+): m/z calcd for C19H15N2O5Br: 430.01642; found: 430.01517.
6-Bromo-7-(4-acetyloxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (8). Prepared
from 7a and NBS (20 min, 56%): red solid, mp 179.5–180.8 °C; IR max 3282 (N-H), 1753 (C=O
ester), 1731 (C=O acetyl), 1679 and 1629 (C=O quinone); 1H-NMR (400 MHz, CDCl3): δ 2.32 (s, 3H,
OCOMe), 2.68 (s, 3H, Me), 4.06 (s, 3H, CO2Me), 7.12 (s, 4H, arom), 7.84 (br s, 1H, NH), 9.25 (s, 1H,
13
1-H); C-NMR (100 MHz): δ 21.3, 23.2, 53.5, 106.6, 121.2, 121.9 (2C), 126.4 (2C), 126.8, 134.2,
134.6, 144.1, 148.9, 149.1, 163.6, 168.2, 169.3, 175.6, 178.7; HRMS (M+): m/z calcd for C20H15N2O6Br:
458.01135; found: 458.01006.

Molecules 2013, 18
731
6-Bromo-7-(4-hydroxyphenyl)amino-4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (9).
A
suspension of 8, K2CO3 (15 mg) in MeOH (5 mL) was stirred at rt for 1 h, the reaction mixture was
partitioned between EtOAc/water and the organic layer was washed with water (3 × 15 mL). The dried
extract was evaporated under reduced pressure and the residue was column cromatographed over silica
gel (90:10 CH2Cl2/AcOEt) to give compound 9 (64%) as purple solid, mp 190.5–192.1 °C; IR max
1
3328 (N-H), 1753 (C=O ester), 1618 (C=O quinone); H-NMR (400 MHz, (CD3)2CO): δ 2.58 (s, 3H,
Me), 3.96 (s, 3H, CO2Me), 6.83 (d, 2H, J = 8.7 Hz, 3'- and 5'-H), 7.11 (d, 2H, J = 8.7 Hz, 2'- and 6'-H),
13
8.52 (s, 1H, OH), 8.79 (br s, 1H, NH), 9.13 (s, 1H, 1-H); C-NMR (100 MHz): δ 22.9, 53.2, 105.1,
115.8 (2C), 123.0, 127.1, 128.2 (2C), 130.8, 135.6, 146.6, 149.0, 156.9, 162.9, 168.8, 175.7, 179.8;
HRMS (M+): m/z calcd for C18H13N2O5Br: 416.00078; found: 415.99527.
3.5. Antiproliferative 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 μg/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 μM during 3 days. The
concentrations used to calculate the IC50 values were: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39,
0.195 and 0.00 µM. The compounds were dissolved in DMSO (1% final concentration) and complete
medium. Untreated cells (medium containing 1% DMSO) were used as controls. At the end of the
incubation, the MTT reduction (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
was carried out to determine cell viability. The final concentration of MTT was 1 mg/mL. MTT
metabolite was dissolved adding 100 µL of ethanol (acidified with HCl). The plates were shaken for
10 min and the absorbance was measured at 550 nm using a Universal Microplate Reader (ELX 800,
Bio-Tek Instruments Inc., Winnoski, VT, USA). Six replicates for each concentration were used and
the values were averaged. The results were transformed to percentage of controls and the IC50 values
were graphically obtained from the dose-response curves. The IC50 value was obtained adjusting the
dose-response curve to a sigmoidal model (a + (b – a)/1 + 10(x−c)), where c = log IC50.

Molecules 2013, 18
732
4. Conclusions
We have reported the synthesis and biological evaluation as antiproliferative agents of a broad
variety of isoquinoline-5,8-quinones substituted at the quinone nucleus with phenylamino and bromine
groups. The new substituted isoquinolinequinones were prepared through efficient amination and
bromination substitution reactions from 4-methoxycarbonyl-3-methylisoquinoline-5,8-quinone (1).
The members of this series expressed moderate to high in vitro antiproliferative activity against three
human cancer cell lines: AGS (gastric), SK-MES-1 (lung) and J82 (bladder) cell lines. From the
current investigation, structure-activity relationships of the phenylaminoisoquinolinequinone series
demonstrate that a phenylamino group substituted at the quinone nucleus of the isoquinolinequinone
pharmacophore promotes a high increase of the antiproliferative activity on gastric, lung and bladder
cancer cell lines. The substitution effects are more significant in enhancing the antiproliferative
activity for those members containing the phenylamino group at C-6. The insertion of a bromine atom
at the quinone nucleus of the phenylaminoquinones decreases the antiproliferative activity compared to
that of their precursors, except for aminoquinone 2a.
The large antiproliferative potencies of the 6-substituted regioisomers 2b, 3b, 4b and 7b, with
higher half-wave potentials (less negative EI1/2), compared to the 7-substituted derivatives 2a, 3a, 4a
and 7a on gastric and lung cancer cell lines suggest that their biological activity involves a redox
cycling process. Compounds 3b, 4b, 7b and 5a are the most significant antiproliferative active
members of the series. Among these substances, the member 4b is the most potent congener by its
submicromolar IC50 values against the three tested cell lines.
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).
References
1. Rao, K.V.; Biemann, K.; Woodward, R.B. The structure of streptonigrin. J. Am. Chem. Soc. 1963,
85, 2532–2533.
2. Gould, S.J.; Weimeb, S.M. Streptonigrin. Fortschr. Chem. Org. Naturst. 1982, 41, 77–111.
3. Balitz, D.M.; Bush, J.A.; Bradner, W.T.; Doyle, T.W.; O’Herron, F.A.; Nettleton, D.E. Isolation
of Lavendamycin, a New Antibiotic from Streptomyces Lavendulae. J. Antibiot. (Tokyo) 1982, 35,
259–265.
4. Doyle, T.W.; Balitz, D.M.; Grulich, R.E.; Nettleton, D.E.; Gould, S.J.; Tann, C.H.; Mews, A.E.
Structure Determination of Lavendamycin—A New Antitumor Antibiotic from Streptomyces
Lavendulae. Tetrahedron Lett. 1981, 22, 4595–4598.
5. Pettit, G.R.; Knight, J.C.; Collins, J.C.; Herald, D.L.; Pettit, R.K.; Boyd, M.R.; Young, V.G.
Antineoplastic Agents 430. Isolation and Structure of Cribrostatins 3, 4, and 5 from the Republic
of Maldives Cribrochalina Species. J. Nat. Prod. 2000, 63, 793–798.

Molecules 2013, 18
733
6. Milanowski, D.J.; Gustafson, K.R.; Kelley, J.A.; McMahon, J.B. Caulibugulones, A–F. Novel
antiproliferative isoquinoline quinones and iminoquinones from the marine bryozoan caulibugula
intermis. J. Nat. Prod. 2004, 67, 70–73.
7. Hawas, U.W.; Shaaban, M.; Shaaban, K.A.; Speitling, M.; Maier, A.; Kelter, G.; Fiebig, H.H.;
Meiners, M.; Helmke, E.; Laatsch, H. Mansouramycins A–D, cytotoxic isoquinolinequinones
from a marine streptomycete. J. Nat. Prod. 2009, 72, 2120–2124.
8. Boger, D.L.; Yasuda, M.; Mitscher, L.A.; Drake, S.D.; Kitos, P.A.; Thompson, S.C. Streptonigrin
and lavendamycin partial structures. Probes for the minimum, potent pharmacophore of streptonigrin,
lavendamycin, and synthetic quinoline-5,8-diones. J. Med. Chem. 1987, 30, 1918–1928.
9. Fang, Y.; Linardic, C.M.; Richardson, D.A.; Cai, W.; Behforouz, M.; Abraham, R.T.
Characterization of the cytotoxic activities of novel analogues of the antitumor agent,
lavendamycin. Mol. Cancer Ther. 2003, 2, 517–526.
10. Hassani, M.; Cai, W.; Holley, D.C.; Lineswala, J.P.; Maharjan, B.R.; Ebrahimian, G.R.; Seradj, H.;
Stocksdale, M.G.; Mohammadi, F.; Marvin, C.C.; et al. Novel Lavendamycin Analogues as
Antitumor Agents: Synthesis, in Vitro Cytotoxicity, Structure-Metabolism, and Computational
Molecular Modeling Studies with NAD(P)H: Quinone Oxidoreductase. J. Med. Chem. 2005, 48,
7733–7749.
11. Verniest, G.; Wang, X.; de Kimpe, N.; Padwa, A. Heteroaryl Cross-Coupling as an Entry toward
the Synthesis of Lavendamycin Analogues: A Model Study. J. Org. Chem. 2010, 75, 424–433.
12. Delgado, V.; Ibacache, A.; Theoduloz, C.; Valderrama, J.A. Synthesis and in vitro Cytotoxic
Evaluation of Aminoquinones Structurally Related to Marine Isoquinolinequinones. Molecules
2012, 17, 7042–7056.
13. Boyd, V.A.; Sulikowski, G.A. Total Synthesis of the Angucycline Antibiotics Urdamycinone B
and 104-2 via a Common Synthetic Intermediate. J. Am. Chem. Soc. 1995, 117, 8472–8473.
14. Kim, D.W.; Choi, H.Y.; Lee, K.-J.; Chi, D.Y. Facile Oxidation of fused 1,4-dimethoxybenzenes
to 1,4-quinones using NBS: Fine-tuned Control over Bromination and Oxidation Reactions. Org. Lett.
2001, 3, 445–447.
15. Bukhtoyarova, D.; Ektova, L.V.; Alekseev, S.N.; Beregovaya, I.V. Synthesis and Spectral
Properties of N-Aryl-5-hydroxy-1,4-naphthoquinone 4-Imines. Russ. J. Org. Chem. 2003, 39,
1309–1315.
16. Valderrama, J.A.; Ibacache, J.A.; Arancibia, V.; Rodriguez, J.; Theoduloz, C. Studies on
quinones. Part 45: Novel 7-aminoisoquinoline-5,8-quinone derivatives with antitumor properties
on cancer cell lines. Bioorg. Med. Chem. 2009, 17, 2894–2901.
17. Aguilar-Martinez, M.; Cuevas, G.; Jimenez-Estrada, M.; González, I.; Lotina-Hennsen, B.;
Macias-Ruvalcaba, N. An Experimental and Theoretical Study of the Substituent Effects on the
Redox Properties of 2-[(R-phenyl)amine]-1,4-naphthalenediones in Acetonitrile. J. Org. Chem.
1999, 64, 3684–3694.
18. Alley, M.C.; Scudiero, D.A.; Monks, A.; Hursey, M.L.; Czerwinski, M.J.; Fine, D.L.; Abbott, B.J.;
Mayo, J.G.; Shoemaker, R.H.; Boyd, M.R. Feasibility of drug screening with panels of human
tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988, 48, 589–601.

Molecules 2013, 18
734
19. Van de Loosdrecht, A.A.; Beelen, R.H.; Ossenkoppele, G.J.; Broekhoven, M.G.; Langenhuijsen,
M.M. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated
cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia.
J. Immunol. Methods 1994, 174, 311–320.
20. Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.;
Seniff, D.; Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and
drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 1988, 48, 4827–4833.
Sample Availability: Samples of the compounds are available from the authors.
© 2013 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/).