New 1,4-anthracenedione derivatives with fused heterocyclic rings: Synthesis and biological evaluation

Article abstract of DOI:10.1039/c4ra11726c

Several terpenylquinones derived from 1,4-anthracenedione (1,4-anthracenequinone, AQ) have been prepared by addition or substitution nucleophilic reactions and further transformed into extended polycyclic systems, which mainly kept the 1,4-quinone moiety fused to different nitrogen-heterocyclic rings (pyrrole, imidazole, pyrazine or quinoxaline) into the structure. The compounds synthesized were evaluated for their antineoplastic, antifungal and antiviral activities. GI₅₀ antineoplastic values remained under μM levels for AQs, while the heterocyclic derivatives showed antifungal MIC values in the low μg mL^-1 range against yeasts and filamentous fungi. Only few compounds displayed a discrete non-selective antiherpetic activity in the μg mL^-1 range. This journal is

Full text of DOI:10.1039/c4ra11726c

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Page 1 of 35
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DOI: 10.1039/C4RA11726C
Table of contents
New 1,4-anthracenediones bearing fused-heterocycle rings were synthesized and
evaluated as cytotoxics, antifungals and antivirals. Some of them showed GI50 at the
µM level.
O
O
O
N
N
X
N
N
O
R
R
R
O
O
R
O
X = C-R, N
O
N
O
O
N
H(Cl)
H(Cl)
N
N
OR
O

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New 1,4-anthracenedione derivatives with fused heterocyclic rings:
View Article Online
DOI: 10.1039/C4RA11726C
synthesis and biological evaluation.
Mª Ángeles Castro#,*, Ana Mª Gamito#, Verónica Tangarife-Castaño¥, Vicky Roa-Linares¥, José Mª
Miguel del Corral#, Ana C. Mesa-Arango¥,*, Liliana Betancur-Galvis¥, Andrés M. Francesch‡,
Arturo San Feliciano#
#
Departamento de Química Farmacéutica, Facultad de Farmacia, CIETUS-IBSAL. Universidad de Salamanca.
Campus Unamuno. E-37007 Salamanca. Spain.
¥
Grupo de Investigación Dermatológica. Facultad de Medicina. Departamento de Medicina Interna. Universidad de
Antioquia, Medellín, Colombia.
‡
PharmaMar S.A. Avda. de los Reyes, P.I. La Mina Norte, E-28770 Colmenar Viejo, Madrid, Spain
*Corresponding authors:
Mª Ángeles Castro
Departamento de Química Farmacéutica. Facultad de Farmacia.
Campus Miguel de Unamuno. Universidad de Salamanca.
37007 Salamanca. SPAIN
Ph. 34 923 294528
Fax 34 923 294515
E-mail: macg@usal.es
Ana Cecilia Mesa Arango
Grupo de Investigación Dermatológica.
Facultad de Medicina. Departamento de Medicina Interna.
Universidad de Antioquia,
Medellín, COLOMBIA
Tel: 574- 2196064
E-mail: amesa@medicina.udea.edu.co

Page 3 of 35
RSC Advances
Abstract.- Several terpenylquinones derived from 1,4-anthracenedione (1,4-anthracenequinone,
View Article Online
DOI: 10.1039/C4RA11726C
AQ) have been prepared by addition or substitution nucleophylic reactions and further transformed
into extended polycyclic systems, which mainly kept the 1,4-quinone moiety fused to different
nitrogen-heterocyclic rings (pyrrole, imidazole, pyrazine or quinoxaline) into the structure. The
compounds synthesized were evaluated for their antineoplastic, antifungal and antiviral activities.
GI50 antineoplastic values attained under µM levels for AQs, while the heterocyclic derivatives
showed antifungal MIC values in the low µg/mL range against yeasts and filamentous fungi. Only
few compounds displayed a discrete non-selective antiherpetic activity in the µg/mL range.
Key words.- 1,4-anthracenequinones; heterocycle-fused quinones; cytotoxicity; antifungal;
antiviral.

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1. Introduction
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The quinone/hydroquinone moiety is a common structural feature in a large numberDoOfI: 1n0a.1t0u39r/aCl4RaAn11d726C
synthetic compounds with a wide range of biological activities such as anticancer,1-4 antiparasitic,5
antimicrobial,6 antifungal,7 antiviral,8 anti-oxidant,1b,9 etc, and it is possible to find agents, derived
from benzoquinone (BQ), naphthoquinone (NQ) or anthracenequinone (AQ), used clinically as
anticancer agents10 such as daunorubicin or mitomycin C (Figure 1A). The mechanisms of the
antineoplastic cytotoxicity of quinone derivatives have been widely studied, and it is known that
they usually participate in important biological redox processes and can also act as intercalating or
alkylating agents.11 The potential of quinones to generate reactive oxygen species (ROS) has been
described in the literature,12 as well as the different targets that might be involved in its molecular
mechanisms of action depending on the tumour cell type.13 For instance, the DNA cross-linking and
monoalkylation by mitomycin C require the reduction of the quinone ring, while
furonaphthoquinones are able to modify the mitochondrial permeability through ROS generation.12c
In this sense, quinones can be considered as multi-target drugs.14 Several of those bioactive
quinones bore a heterocyclic fragment fused to the quinone moiety, including five and six-
membered heterocycles with nitrogen,15 oxygen16 and/or sulphur17 atoms. It has also been reported
that the presence of an N-heterocyclic moiety in a polycyclic planar structure contributes to enhance
their activity,18 being important too the number and position of the nitrogen atoms in the
heterocyclic system.19
Among the bioactive quinones, 1,4-NQs and 9,10-AQs have been extensively studied. In contrast,
AQs with the quinone carbonyls located at other positions are much less frequent and only the
antitumor activity20,21 and the promising anti-trypanosomal profile22 of a limited number of 1,4-AQs
has been described in the last few years. These facts prompted us to design and prepare new 1,4-AQ
derivatives, including a number of heterocycle-fused quinones for evaluating them, not only for
their expected antineoplastic cytotoxicity, but also for their antifungal activity, based on previous
results reported by us for NQs and AQs23 and prospectively, against viruses, based on the
assumption of their wide involvement in bio-redox processes, on their structural novelty and on the
absence of previous evaluations in such field. Complementarily, it can be noted that antifungal and
antiviral activities reported for quinones were often related to their reduction potential,6c,8b,11 and
that recent mechanistic studies revealed that the antifungal activity of several NQs against various
Candida spp strains was related to the generation of ROS,24 in the same way as other antifungals as
amphotericin B, miconazole or histatin 5 also do.25
In our search focused to the induction of antineoplastic and antimicrobial activities on natural
inactive terpenoids, we prepared and evaluated a large number of 1,4-NQs, with the model
structures shown in figure 1B, starting from natural terpenoids such as myrcene and

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myrcecommunic acid.26,27 They were synthetized through Diels-Alder cycloaddition between the
View Article Online
DOI: 10.1039/C4RA11726C
natural terpenoids and p-benzoquinone. The preliminary biological results indicated that
monoterpenylquinones (MTQs) and diterpenylquinones (DTQs) (Figure 1B) were cytotoxic and
good candidates to further structural optimization, becoming our lead compounds in order to
improve their bioactivity profiles. Several structural modifications, dealing with size and oxidation
degree of the quinone ring, were performed on those derivatives, including changes of substituents
in the side chain or in the quinone ring.26,27
From those studies, we deduced that the presence of a terpenyl residue attached to the benzenoid
ring of the NQ moiety improved the cytotoxicity of 1,4-NQ itself, and we attained to obtain some
derivatives with IC50 values in the µM range against several tumour cell lines. Among them, we
also prepared a number of 1,4-AQs by cyclization of the side chain in the MTQs 1 to get MTQs 2
(Fig. 1B), which also showed a very interesting cytotoxicity.21
A)
O
OH
O
OCONH2
OCH3
O
HO
R
H2N
N
NH
CH3O
O
OH
O
O
O
R
OH Doxorubicin
Daunorubicin
Mitomycin C
H
NH2
OH
B)
O
O
X
X
O
O
MTQs 1
MTQs 2
O
O
O
O
H
H
COOCH3
COOCH3
DTQ 1
DTQ 2
Figure 1.- A) Structure of clinically used anticancer
quinones. B) Structure of several monoterpene-
derived (MTQ) and diterpene-derived (DTQ)
antineoplastic quinones.
In a prospective manner, several representative MTQs were evaluated against human pathogenic
yeasts and filamentous fungi, with promising antifungal results found for several chlorinated
derivatives.23
The incidence of opportunistic infections caused by microorganisms including fungi and viruses
have increased significantly in recent decades, mainly in immunocompromised patients as those
under anticancer treatment, who are susceptible to opportunistic infections. Among fungal

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pathogens, the species from Candida, Aspergillus and Cryptococcus genera are the most frequent in
View Article Online
human infections.28 With the increase of these infections, has also increased the occurrence of
DOI: 10.1039/C4RA11726C
secondary resistance by prolonged treatment with antifungals or by using them in prophylactic
treatments.29 Also, infections caused by other emerging fungi as Fusarium spp. and Scedosporium
spp. are increasing within these genera, leading to multiresistant strains or with variable
susceptibility to antifungal drugs.30
Infections by Human Herpes virus type 1 (HHV-1) and Human Herpes virus type 2 (HHV-2) are
also important due to morbidity that they can cause31 in immunocompromised patients. In this
sense, the multitarget approach could be advantageous over drug combination, as it has been
recently discussed,22a,32 and would reinforce the convenience of evaluating the new compounds for
their antifungal and antiviral potential, in addition to the antineoplastic evaluation. Discovering
entities that can be effective against two or more pathologies improves the chance of efficacy, and
could prevent the development of the more and more frequent appearance of multi-drug resistant
strains.33,34
Keeping in mind that quinone systems are involved in a wide range of biological activities and that
the presence of nitrogen atoms can contribute to improve the bioactivity of quinone systems,
together with our previous results with terpenylquinones, we planned to synthesize several new 1,4-
AQs bearing fused nitrogen-heterocyclic rings very common in drug molecules, such as pyrrole,
imidazole, pyrazine or quinoxaline, in order to analyze the influence of the enlargement of the
heteropolycyclic planar system on their bioactivity. The synthesis of such new heterocyle-fused 1,4-
AQ derivatives and the results of their evaluation in the three proposed pathogenic areas are
reported and discussed here.
2. Results and discussion
2.1. Chemistry
The heterocyclic-fused quinones objective of this study included naphthoindole, anthraimidazole,
anthraoxazole, naphthophenazine, naphthoquinoxaline and anthraimidazopyridine derivatives. We
have used as starting compounds the AQs 1, 2 and 3 previously obtained by us through Diels-Alder
cycloaddition between myrcene and p-benzoquinone derivatives as reported21,23 (Figure 2).
O
O
O
Cl
Cl
Cl
O
O
O
1
2
3a
Figure 2.-Anthracene-1,4-quinones used as starting materials

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The actual precursors of the heterocyclic systems were obtained through nucleophilic processes,35
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DOI: 10.1039/C4RA11726C
either addition or substitution reactions, using N-nucleophiles as it is represented in Schemes 1-9.
As the starting quinones are non-symmetrically substituted, the corresponding reaction products
consisted of approximately 1:1 mixtures of the two expected regioisomers. However, in several
cases they were separated by column chromatography in quantities enough to facilitate their
independent spectroscopic characterization, and to perform further chemical transformations and
1
their biological evaluation. NMR spectra of representative compounds and complete H and 13C
NMR data assignments are included in the supplementary material section.
2.1.1. Pyrrole fused systems.
Michael addition of aliphatic or aromatic amines to AQ 1 gave the monosubstituted AQs 4-8 as 1:1
mixtures of regioisomers, which were separated by CC to allow independent characterization and
biological evaluation23 (Scheme 1). The regioisomers 4a and 4b, which were unequivocally
identified in our previous work,23 were used to obtain the naphtho[2,3-f]indole-4,11-diones 9 and 10
by condensation with β-dicarbonyl compounds via a cerium(IV) mediated oxidative free radical
reaction.36 The condensation reaction was carried out with each regioisomer independently. Thus,
4a was treated with 2,4-pentanedione or ethyl acetoacetate and CAN in methanol at room
temperature to yield 9a and 10a respectively. When AQ 4b was used as the starting material,
derivatives 9b and 10b were obtained in moderate yields. The formation of the pyrrole ring fused to
1
the quinone system was deduced from the analysis of H NMR spectra, in which the signal at 5.6
ppm corresponding to the hydrogen H-3 in compounds 4, disappeared from the spectrum and new
singlets at 2.4 and 2.7 (and 3.9) ppm were observed respectively for compounds 9 (and 10) (see
supp. material). The correlations observed in two-dimensional NMR spectra obtained for 9a and 9b
confirmed the structures assigned to both naphthoindolediones.
O
O
O
O
O
10
9
6
R
N1
R1
MeOH, CAN, rt
O
4a CH3-CH2
5a 4-CH3O-Ph
6a 4-HO-Ph
7a 4-AcO-Ph
8a 3,4,5-(CH3O)3-Ph
H
N
1
2
8
5
R
4
2
R1
R1
CH3
OCH3
O
O
9a
10a
O
O
8
5
9
9a
4a
R-NH2
EtOH
1
4
+
O
O
O
R1
6
9
10
4
R
O
R1
MeOH, CAN, rt
2
4b CH3-CH2
5b 4-CH3O-Ph
6b 4-HO-Ph
7b 4-AcO-Ph
8b 3,4,5-(CH3O)3-Ph
1
5
8
N
1
11
R
1
2 N
H
O
R1
CH3
OCH3
O
9b
10b
Scheme 1. Preparation of aminoanthracenediones 4-8 and naphtho[2,3-f]indole-4,11-diones 9 and 10

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2.1.2. Imidazole-fused systems
View Article Online
DOI: 10.1039/C4RA11726C
The imidazole ring can be formed through the condensation of an acylamino and an alkylamino
groups attached to the quinone fragment. Such groups would be previously introduced by
substitution of the two chlorine atoms on the dichloro-AQ 2 as stated in the retrosynthetic Scheme
2.
R1
O
O
O
O
R1
NH
N
N
O
R
N
H
R
O
O
Cl
Cl
AQ 2
Scheme 2. Retrosynthetic scheme for imidazole-fused AQs.
Dichloro-AQ 2 was obtained from 1,23 and both halogen atoms were subsequently substituted using
N-nucleophiles. It is known that aliphatic amines and anilines smoothly substitute one chlorine
atom, but such substitution induces electronic enrichment of the quinone system and makes difficult
the replacement of the second chlorine atom.35,37 The second reaction step could be favoured if a
substituent with electron-withdrawing effect is attached to the quinone ring. Due to that, we chose
to acylate the monoaminoquinone, previously to the introduction of the alkylamino substituent. For
such proposes, two different acyl groups, acetyl and benzoyl, were chosen.
Thus, treatment of 2 with sodium azide in acetic acid, followed by reduction with NaBH4 yielded
the mixture of regioisomers 11 in low yield (data not shown). However, when ammonia was used as
nucleophile, AQs 11 were obtained in 95% yield (Scheme 3). Reaction of 11 with acetic anhydride,
in the presence of sulphuric acid at 0 ºC, afforded the monoacylated AQs 12 in moderate yield. In
order to improve the result, the acylation reaction was performed at room temperature and the imide
derivatives 13 were obtained in good yield. When the monoacylated AQs 12 were treated in the
same conditions, the same imide analogues 13 were obtained together with the oxazoles 14. The
formation of the later can be easily explained through the intramolecular displacement of the
halogen by the oxygen atom of the tautomerized amide function.
When chloroquinones 11 were treated with benzoyl chloride in basic media, only the dibenzoylated
AQs 15 were detected (Scheme 3). The next step was the substitution of the second chlorine atom
using alkylamines and anilines. The corresponding monoacylated derivatives 16-18 were obtained
when AQs 13 and 15 were submitted to the substitution reaction, in which one acyl group was
transferred to the amine, used in excess in the reaction, as can be deduced from the acetyl-p-
anisidine isolated during the chromatographic purification of the reaction products. In the case of
15, the imides 19 were also formed and the reaction of 12 with p-anisidine also gave the

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monoacylated analogues 17. Aiming to obtain the diacylated analogues from 16 and 17, both were
View Article Online
DOI: 10.1039/C4RA11726C
treated with acetic anhydride in the presence of sulphuric acid but instead the expected products, the
oxazoles 14 were formed again as the main products (up to 60 % yield).
O
Cl
O
N(Ac)2
O
O
O
O
1
Cl
O
O
Cl
Cl
Cl
8
5
NH4OH 30 %
EtOH
13a/13b
O
Ac2O/H2SO4
rt
2
Ac2O/H2SO4
0 ºC
+
NH2
NH
1
11
O
O
9
2
12a/12b
11a/11b
6
N
3
4
O
p-anisidine
EtOH
14a/14b
A or B
A: Ac2O/H2SO4, rt
Ac2O/H2SO4
rt
!
B: NaH / THF, Ph-COCl
R1
Ph-OCH3
NH
O
O
O
R1-NH2
EtOH
!
Cl
N
NH
8
5
O
8
5
O
O
2
2
+
4
4
R
N
H
N
O
R
Ph
Ph
O
O
O
R
R
R1
O
R
CH3
Ph
CH3 CH3-CH2
16a/16b
17a/17b
18a/18b
19a/19b
13a/13b
15a/15b
4-CH3O-Ph
4-CHO3-Ph
CH3
Ph
Scheme 3. Preparation of intermediates and heterocycle-fused 1,4-anthraquinones
The reactions performed until this point gave mixtures of the two regioisomers in a 1:1 ratio. In
some cases they were separated by CC, but the exact position of the substituents in the AQ ring was
undetermined, so that the same reactions were applied to the major regioisomer 3a, obtained
previously at our laboratory.21b When 3a was treated with ammonia followed by acetic anhydride,
1
the imide 13a was obtained in quantitative yield. The H NMR spectrum of 13a showed only two
singlet signals over 6 ppm (δ 7.83 and 8.04 ppm) indicating that an addition reaction took place at
the position C-3, instead of the substitution reaction of the chlorine atom. Further substitution with
primary amines afforded 16a and 17a, which allowed us to identify regioisomer “a” as the one with
the acyl group on the same side as the geminal methyl groups (Scheme 4).
O
O
Cl
Cl
NH4OH 30 %
EtOH
1
8
5
2
NH2
O
O
3a
11a
Ac2O/H2SO4
rt
R1
O
O
O
O
NH
Cl
N
R1-NH2
O
O
EtOH
!
N
H
R1
CH3-CH2
4-CH3O-Ph
13a
O
16a
17a
Scheme 4. Synthesis of AQ regioisomers 16a and 17a

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Once in our hands the disubstituted AQs 16-18, the regioisomers 16a, 16b, 17a, 17b separately and
View Article Online
DOI: 10.1039/C4RA11726C
the mixture 18a/18b were treated with NaOH to give the imidazole derivatives 20-22 in good yields
(Scheme 5).
R1
R1
O
O
NH
NH
O
O
N
H
N
H
R
R
O
O
R1
R1
R
R
16b
17b
18b
CH3-CH2
4-CH3O-Ph
4-CH3O-Ph
16a
17a
18a
CH3
CH3
Ph
CH3-CH2
4-CH3O-Ph
4-CH3O-Ph
CH3
CH3
Ph
NaOH 4%
EtOH
!
NaOH 4%
EtOH
!
O
11
O
11
R1
R1
1
N
1
N
9
6
9
6
R
R
N
N
4
4
3
3
O
O
R1
CH3-CH2
4-CH3O-Ph
4-CH3O-Ph
R1
CH3-CH2
4-CH3O-Ph
4-CH3O-Ph
R
R
20b
21b
22b
20a
21a
22a
CH3
CH3
Ph
CH3
CH3
Ph
Scheme 5. Preparation of anthra[2,3-d]imidazole-
4,11-diones.
2.1.3. Other heterocycle-fused systems
In a previous work21b we found that 1,2-naphthoquinones (1,2-NQs) fused to a furan ring were more
cytotoxic than the corresponding 1,4-NQs. Similar results were obtained with 1,2-
naphthohydroquinone diacetates38 and after that, we explored the formation of 1,2-quinone
analogues fused to nitrogenated rings. To get such type of compounds, we chose 2-aminopyridine
as nucleophile to react with the dichloro-AQ 2. It was expected that the aromatic nitrogen promoted
first the nucleophilic substitution of one chlorine atom, and then the reaction could evolve in two
different ways, depending on if the amino group would produce the substitution of the second
chlorine atom or the condensation with the close quinone carbonyl. In the later case, the remaining
chlorine atom would be replaced by a hydroxyl group, which should favour the formation of the o-
quinone.39 Both possible products are represented in Scheme 6.

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O
O
O
Cl
Cl
Cl
Cl
N
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DOI: 10.1039/C4RA11726C
N
NH2
O
H2N
2
O
O
O
N
N
N
or
O
N
Scheme 6. Possible reaction products of
anthracenequinone 2 with 2-aminopyridine
Thus, when AQ 2 was treated with 2-aminopyridine in methanol or ethanol, in the presence of
K2CO3, complex reaction products were obtained from which the major products were the
dimethoxy and diethoxy derivatives 23 and 24 respectively, resulting from the substitution of both
chlorine atoms by the alcohol used as solvent, along with a small amount of the corresponding o-
quinones 25 (Scheme 7). The formation of the alkoxy derivatives 23 and 24 should be due to the
presence of base, so that the reaction was repeated without base to obtain in moderate yield only the
o-quinone derivatives 25. Both regioisomers were separated by CC and their spectroscopic
characterization was done on the basis of two-dimensional COSY and HMBC NMR experiments
(see supplementary material). Representative correlations observed between aromatic hydrogens
and some carbonyl and quaternary carbons are shown in Fig. 3. These correlations served to
unequivocally assign the structures of 25a and 25b. The presence of the 1,2-quinone system was
also demonstrated through its reaction with 4,5-dimethyl-o-phenylenediamine to give compounds
26a and 26b. In order to achieve the formation of 1,2-quinones with six-membered fused-
heterocyclic rings, we used aliphatic and aromatic 1,2-diamines in the addition and substitution
reactions.
O
O
O
O
O
O
9
7
6
R
R
O
O
N
Cl
Cl
N
NH2
+
+
12
5
N
K2CO3
3
N
N
MeOH( EtOH)
1
R
OCH3
OCH2CH3
2
25b
25a
23
24
NH2
NH2
EtOH, AcOH
!
2
1
N
N
16
13
5
N
N
N
6
N
7
N
N
11
26a
26b

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Scheme 7. Preparation of 1,2-AQ derivatives 25a/b and extended condensation products
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DOI: 10.1039/C4RA11726C
7.80 ppm
7.84 ppm
1
O
6
H
3
H
N
9
12
N
O
7
5
6
12
9
7
5
O
N
3
H
N
H
O
1
8.10 ppm
8.10 ppm
25a
25b
Figure 3. Selected long-range H/C connectivities
for the heterofused ortho-quinones 25a and 25b.
When ethylenediamine was added to the AQ 1 in short times (10 min) only the addition products 27
were detected (Scheme 8); however with longer reaction times (30 min) or under microwave
irradiation, mixtures of the addition products 27 and the further condensation/aromatization
products 28 were obtained, giving rise to a polycyclic naphtho[2,3-a]quinoxaline system in low
yield. The same quinoxaline arrangement was formed when dichloroquinone 2 was treated with
ethylenediamines. In these cases, the compounds first formed were the substitution products of one
chlorine atom, 30 or 31, followed by the condensation of the second amine group with one of the
carbonyl groups of the quinone system, leading to compounds 32 and 33. The presence of the
phenolic hydroxyl group was confirmed by acetylation to obtain the acetates 34 and 35. A similar
result was attained when 4,5-dimethyl-o-phenylenediamine was used as the nucleophile, and only
the naphthophenazines 36 were obtained after acetylation of the reaction product.
1
N
O
O
H
N
4
8
5
NH2
N
8
5
9
1
9a
4a
H2N
11
8
NH2
1
4
+
5
4
3
EtOH, rt
or
K-10, MW
10
OR
O
O
1
R
H
Ac
27a/27b
28a/28b
29a/29b
Ac2O/Py
R
O
NH2
O
O
H
N
N
H2N
Cl
Cl
N
Cl
NH2
R
R
+
Cl
EtOH, !
O
OR1
2
R
H
CH3
R
H
CH3
H
R1
H
H
Ac
NH2
30a/30b
31a/31b
1)
32a/32b
33a/33b
34a/34b
35a/35b
NH2
Ac2O/Py
CH3 Ac
2) Ac2O/Py
3
1
N
5
N
12
9
6
Cl
36a/36b
OAc
Scheme 8. Preparation of angular naphthoquinoxaline and naphthophenazine derivatives

Page 13 of 35
RSC Advances
Finally, linear naphthophenazines retaining the p-quinone system were prepared in two steps by
View Article Online
DOI: 10.1039/C4RA11726C
reaction of the dichloroquinone 2 with arylamines to obtain the mononitrogenated quinones 37-39,
in very good yields,21 from which further reaction with sodium azide afforded the expected
naphthophenazinediones 40-43 in moderate to low yields (Scheme 9).
O
O
O
O
4
1
Cl
Cl
NH
Cl
R1
R2
N5
N
2
8
6
11
8
NaN3
Ar-NH2
2
+
11
DMF
!
N
R2
N
EtOH
!
13
1
14
5
O
O
O
R1
O
R1
H
R2
CH3O
R1
H
R2
CH3O
2
37a/37b
38a/38b
39a/39b
40a/40b
41a/41b
42a/42b
43
CH3O CH3O
CH3 CH3
CH3O CH3O
CH3 CH3
Scheme 9. Preparation of linear naphthophenazinequinones
2.2. Antineoplastic cytotoxicity
Most of the compounds prepared were evaluated in vitro to establish their cytotoxicity against the
following tumour cell lines: A-549 lung carcinoma, HT-29 colon adenocarcinoma and SK-BR3
breast carcinoma using a colorimetric cell growth inhibition assay.40 The results are shown in Table
1, expressed as GI50 values (drug concentration in µM causing 50% growth inhibition). The
common anticancer quinone drug doxorubicin was included in the assays as reference. Most of the
compounds tested were cytotoxic and from the results obtained, some general observations can be
made for each class of heterocycles in comparison with the parent compounds 1 and 2.
Table 1. Cytotoxicity data (GI50 µM)a for AQ and
AQ-heterofused derivatives.
Compound
1
A-549
>12.5
-
HT-29
9.16
9.64
>10.9
1.64
2.07
0.15
0.14
0.19
1.09
0.18
3.92
5.42
7.22
9.39
28.2
29.1
11.5
>17.1
3.71
3.54
19.7
21.6
SK-BR3
9.95
6.27
>10.9
9.88
>10.6
5.70
>8.30
>8.64
0.38
>7.12
2.47
4.22
3.74
3.81
>29.4
13.2
2
3a
4a
4b
5a
5b
6a/6b
7a/7b
8a/8b
12a
12b
13a
15a/15b
16a
16b
>10.9
6.28
6.81
0.17
8.31
4.52
>7.7
5.72
2.98
4.82
4.55
6.09
>29.4
>29.4
15.3
>17.1
2.79
4.04
10.1
12.7
17a
10.5
19a/19b
30a/30b
37a/37b
38a/38b
39a/39b
>17.1
>30.1
3.03
9.39
11.4

RSC Advances
Page 14 of 35
9a
9b
10a
10b
14a
14b
20a
20b
21a
14.0
>27.2
>26.4
>26.4
1.79
1.93
8.68
12.1
9.24
23.0
5.10
>27
>27
1.06
8.17
>23.2
>23.2
13.1
11.8
12.1
11.6
>26.4
5.76
6.09
11.5
13.0
10.2
>25
15.7
17.1
>26.4
17.1
1.93
1.93
52.7
14.9
9.74
12.2
4.83
>27
15.7
19.4
>30.3
>23.2
>23.2
13.1
13.5
>27.2
>23.1
0.09
View Article Online
DOI: 10.1039/C4RA11726C
21b
40a/40b
42a/42b
43
25a
25b
4.83
>27
>27
12.4
>30.3
16.3
>23.2
13.1
9.02
>27.2
>23.1
0.10
26a
26b
32a/32b
34a/34b
35a/35b
36a/36b
Doxorubicin
11.3
>27.2
>23.1
0.07
a
GI50 values are expressed as the mean from triplicate
determinations on A-549 lung carcinoma, HT-29 colon
adenocarcinoma and SK-BR3 breast carcinoma. Values
under 1 µM are bold-faced to facilitate comparisons.
If we considered the 1,4-AQ derivatives without any fused-heterocycle at the quinone ring (Table
1), the presence of an alkyl/aryl/acylamino groups at position C-2 with or without a chlorine atom at
C-3, in general improved the cytotoxicity in comparison with the unsubstituted AQ 1 as we have
already described for similar 1,4-NQs.27b
The better cytotoxicity results, on one or more tumour cell lines tested, were found for those
derivatives bearing an arylamino group at C-2, AQs 5, 6, 7 and 8, which showed GI50 values under
de µM level (bold-faced in Table 1), very close to those of doxorubicin. When the quinone ring was
disubstituted at C-2 and C-3 with an alkyl/arylamino group and a chlorine atom, the cytotoxicity
slightly decreased with respect to those monosubstituted (37 vs 5), although they were more
cytotoxic than the unsubstituted 1, keeping the GI50 values in the µM range. Additionally, the
simultaneous presence of an alkyl/arylamino and an acylamino groups at neighbouring positions led
to a decrease in cytotoxicity, as in AQs 16, 17 and 19.
Some of these derivatives were several times more potent on the HT-29 cell line than on the other
cell lines tested, thus indicating a certain degree of selectivity, which is always an objective in the
design of better drugs. When possible, the regioisomers were tested separately, but no significant
differences were observed between them, except on the A-549 cell line for AQ 5, whose 5a isomer
was nearly fifty times more potent than the 5b.

Page 15 of 35
RSC Advances
Those analogues with a heterocycle fused to the quinone ring at positions C-2/C-3 (linearly fused-
View Article Online
DOI: 10.1039/C4RA11726C
heterocycle, Table 1) showed from moderate to poor cytotoxicity values, with a certain dependence
on the ring size and on the number and type of heteroatoms present. Those with pyrrole- or
imidazole-fused rings were cytotoxic in the same level than the parent compound 1, giving slightly
better results with the imidazole-fused than with the pyrrole-fused rings. However, the presence of
two different heteroatoms, as in compound 14, which bear an oxazole ring, led to a potency increase
in comparison with the parent AQ 1.
Considering the six-membered fused rings, the nature of the substituent present in the additional
aromatic rings seemed important for the bioactivity, and the presence of a methoxy group was much
better than the presence of methyl groups (42, 43 vs 40).
Heterocycles fused at C-1/C-2 positions of the quinone ring (angularly fused-heterocycle, Table 1)
did not improve at all the cytotoxicity, and only compound 25a, which contains an o-quinone
system, showed better cytotoxicity, though only against the A-549 cell line, as compared with the
imidazole linearly-fused 1,4-quinones 20 and 21, being worth to notice the difference in potency
observed for both regioisomers 25a and 25b. The other analogues in this group did not contain the
1,2-quinone system and this could explain their poor cytotoxicity. Again, the presence of methyl
groups on the added heterocyclic system led to lesser potent compounds.
2.3. Antifungal activity
Recently we have described the antifungal activity of several 1,4-NQs and 1,4-AQs related to those
reported here.23 Continuing this line of research, and in order to analyse the influence of the fused-
heterocycles on the antifungal activity of 1,4-AQs, several representative compounds obtained in
this work were also evaluated against Candida spp., Aspergillus spp., dermatophytes, and Fusarium
oxysporum. The minimum inhibitory concentration 90 (MIC90), was determined following the
standard microdilutions method AFST-EUCAST and CLSI M38-A,41,42 with some modifications,
for yeast and filamentous fungi, respectively. Minimum inhibitory concentrations 90 were
expressed as the Geometric mean (GM-MIC90). Only those derivatives that showed MIC values
lower than 32.0 µg/mL (~100 µM) against one or more strains were considered active. The selected
results are shown in Table 2. Three standard drugs, itraconazole (ITZ), amphotericin B (AMB) and
terbinafine (TRB), were used as reference drugs to cover the fungal spectrum. Complete evaluation
data for all the compounds tested are given as supplementary material. Also cytotoxicity was
evaluated in Vero cells (African green monkey kidney-Cercopithecus aethiops ATCC: CCL 81
line) and the cytotoxic concentrations 50% (IC50) were calculated and included in Tables 2 and 3.
Seven of seventeen evaluated compounds were actives against Trichophyton spp while five
compounds showed activity against Candida spp and three against A. fumigatus. The other

RSC Advances
Page 16 of 35
Aspergillus spp and F. oxysporum were less susceptible. Only compound 2 resulted active against
View Article Online
DOI: 10.1039/C4RA11726C
A. flavus and 3a and 12a against F. oxysporum. These two later compounds showed fairly wide
antifungal spectra. The most potent compounds against Candida spp were 1, 2, 3a, 12a and 14. The
AQ 12a was the best against both tested strains of C. albicans. This yeast is the most important in
infections caused by fungi. AQ 12a was also active against other pathogenic yeasts like C.
parapsilosis, C. krusei, C. lusitaniae and C. tropicalis with GM-MIC90 values from 2.2 to 8.0
µg/mL.
Compound 2 showed important activity against C. parapsilosis, C. krusei and C. lusitaniae. These
results are important considering the intrinsic resistance to fluconazole and echinocandin of C.
krusei and C. parapsilosis, respectively and also because C. lusitaniae has the ability to rapidly
develop resistance to AMB.34 This antifungal is the most important available agent for treating
candidiasis and actually the only antifungal that has a broad spectrum of activity.
AQs 2, 3a and 12a and oxazole 14, showed moderate activity against Aspergillus fumigatus. The
compound 2 was the most active against this filamentous fungus and also was active against A.
flavus. None of the tested compounds were active against A. terreus and A. niger. A. terreus is
intrinsically resistant to AMB and less susceptible to other antifungals43 for this reason it would be
important to find new active molecules against this fungus.
The AQ 3a, in addition to be fairly active against Candida spp and A. fumigatus, showed moderate
activity against F. oxysporum. This result is relevant because, compared with other filamentous
fungi, Fusarium species are intrinsically resistant to ITZ and echinocandins and the response to
AMB and voriconazole is variable depending on the particular species.44
Table 2. In vitro susceptibilities of Candida spp and filamentous fungi (GM-MIC90, µg/mL) and Vero cell
cytotoxicity (IC50, µg/mL) for selected AQs and heterofused derivatives
HSI
HSI
HSI
Comp Cp Ck
Cl Ct1 Ct2 Ca1 Ca2
Afu Afl At An Fo
Tm Tr
Vero
(C. spp.)
(Asp. spp.)
(Tric. spp.)
1
2
4.8 4.0
2.0 1.0
5.0 12.7 5.0 18.0 22.6 2.2 (Ck) >32 >32 >32 >32 >32
na
1.3 4.0 6.6 (Tm) 8.6
2.0 >32 5.0 >32 8.0 42.2 (Ck) 8.0 4.8 >32 >32 >32 8.8 (Afl) 6.1 8.0 6.9 (Tm) 42.2
5.0 12.7 8.0 20.2 16.0 54.5 (Ck) 16 >32 >32 >32 16 6.8 (Afu) 3.2 5.0 34.1 (Tm) 109
3a
4b
4.0 2.0
>32 >32 >32 >32 >32 >32 >32
8.0 2.8 2.2 6.4 4.0 4.0 8.0 10.1 (Cl) 22.6 >32 >32 >32 32 0.9 (Afu) 3.2 5.0 7.0 (Tm) 22.3
6.7 19.0 20.2 >32 32 >32 >32 0.7 (Cp) 16 >32 >32 >32 >32 0.3 (Afu) 32 8.0 0.6 (Tr)
na
>32 >32 >32 >32 >32
na
5.0 3.2 11.1 (Tr) 35.6
12a
14
4.6
50.5
>50
28.6
38
>32 >32 >32 >32 >32 >32 >32
na
>32 >32 >32 >32 >32
na
25.4 25.4
2.0
ITZ 0.25 0.5
AMB
>100 (Ck)
2.0 4.0
7.1 (Afl)
TRB
0.03 0.08 386 (Tr) 30.9
Minimun Inhibitory Concentrations 90 (MICs90) values are expressed as the geometric mean (GM). Cytotoxic
Concentrations 50% (IC50. Vero cells) found in triplicate experiments for each compound. Cp: Candida parapsilosis;
Ck: C. krusei; Cl: C. lusitaniae; Ct1 and Ct2: C. tropicalis; Ca1 and Ca2: C. albicans; Afu: Aspergillus fumigatus; Afl:

Page 17 of 35
RSC Advances
A. flavus; At: A. terreus; An: A. niger; Fo: Fusarium oxysporum; Tm: Trycophyton mentagrophytes; Tr: T. rubrum. Vero
View Article Online
cells (African green monkey kidney-Cercopithecus aethiops). na: not applicable. ITZ: itraconzole; AMB: amphotericin
DOI: 10.1039/C4RA11726C
B; TRB: terbinafine. HSI: Highest Selectivity Index (IC50 Vero / lowest MIC90 found for each compound) rounded
values. MIC values under 10 µg/mL and HSI over 8 are bold-faced to facilitate comparisons. Complete table with MIC
values ±SD are given in supplementary material and for fungal strain characterizations, see experimental part.
All compounds listed in Table 2 were active against both dermatophytes strains. AQs 3a, 4 and 12a,
showed similar good activity against both Trichophyton species. AQ 1 was the most active against
T. rubrum and T. mentagrophytes. This compound may be of interest as a lead compound for
further studies in order to develop new molecules with activity against this group of fungi.
According to this data and the previous results with similar NQs,23 we can conclude that, in general,
the precursor 1,4-AQs showed better antifungal activity than their heterofused derivatives. Only the
oxazole-fused compound 14 was active against several strains, although its selectivity was poor.
The MIC value for compound 14 against C. parapsilosis (MIC90 = 6.7 µg/mL) was approximately
three times lower than for the other Candida species. These data are important because C.
parapsilosis exhibits a consistent trend of decreasing fluconazole susceptibility over the treatment
time.45 AQ 14 also showed activity against A. fumigatus, but not against other Aspergillus spp or F.
oxysporum. However, it was also active against both Trycophyton species.
The viability of the Vero cell line in the presence of the seven 1,4-AQ derivatives selected was
evaluated and the corresponding cytotoxicity calculated (Tables 2 and 3). All the tested compounds
produced a dose-dependent inhibition on the Vero cell line, with linear regression coefficients
higher than 0.7 (Supplementary data). According to the results obtained, the compounds reported
here should be considered as low cytotoxic for normal monkey cells and the highest selectivity
index (HSI = IC50 for Vero cells/lowest GM-MIC90 value of a compound against any pathogenic
fungus) was calculated in order to quantify their respective selectivity and to facilitate comparisons.
According to literature, SI values higher than 8 are considered indicative of a potential therapeutic
agent that merit further attention.46 To this respect, AQs 2, 3a and 12a were the most selective
against Candida spp. (HSI = 10.1–54.5), AQ 2 was also selective against A. flavus and compounds
3a and 4b were selective against dermatophytes. At this point, it is important to note that HSI
values should be higher than those calculated and included in Table 2 if we have in mind that MIC
values refer to nearly total (90 - 100%) fungal growth inhibition and IC50 values refer to the
inhibition of only 50 % of Vero cells.
2.4. Antiviral activity
It has been reported that the most effective drugs against herpetic infections, acyclovir and its
related prodrugs and analogues, have developed a number of drug-resistant strains after prolonged

RSC Advances
Page 18 of 35
or inadequate use.33,47 Consequently, the search for new therapeutic agents, with non-related
View Article Online
DOI: 10.1039/C4RA11726C
structures and different mechanisms of action, seems necessary to maintain efficacious treatments,
and can justify the antiviral evaluation of some representative AQs synthesised in this work.
The antiviral activity was evaluated against the 50% Tissue-Culture Infective Dose, 1TCID50 and
10TCID50, of Human Herpes virus 1 (HHV-1 CDC Atlanta acyclovir-sensitive strain) and Human
Herpes virus 2 (HHV-2 VR-734-G acyclovir-sensitive strain) respectively, by determining the
reduction factor (Rf), which corresponds to the value obtained by dividing the viral titer in the
absence of compound over the viral titer obtained in the presence of compound.48 Two positive
controls, dextran sulfate and acyclovir, were included in this assay. Dextran sulfate was included
because this sulfated polysaccharide can inhibit viral entry by a competitive effect with heparan
sulfate receptors present on the cell surface. In addition, acyclovir was included because is effective
in the inhibition of viral DNA polymerase. Compounds that showed reduction of viral titer respect
to control (Rf) at concentrations lower or equal than 50 µg/mL, were considered active and are
collected in Table 3. Complete evaluation data for all the compounds tested are given as
supplementary material.
In this study, compounds that showed mild activity (Rf ≤1x101) against HHV-1 at a concentration
of 50 µg/mL were 2, 37, 38 and 39, while AQ 1 attained the same Rf at a concentration of 6.25
µg/mL. Notably, AQ 3a showed better anti HHV-1 activity (Rf =1x102) at a concentration of 6.25
µg/mL. The AQ 10a and the naphthophenazine 43 showed mild activity against HHV-2 at a
concentration of 50 µg/mL. Moreover, compounds 12a and 14 were active at a concentration of
12.5 and 25 µg/mL, respectively. Only AQ 2 showed moderate anti HHV-2 activity (Rf =1x102) at a
concentration of 25 µg/mL.
Table 3. Antiviral activity of selected AQ derivatives.
Vero ATCC CCL-81a
Vero
cells
b
1 TCID50 HHV-1
Antiviral
10 TCID50c HHV-2
Antiviral
Compound
IC50
(µg/mL)
(Rf)d activity e
(µg/mL)
SIf
(Rf)d
activity e
(µg/mL)
>50
SIf
1
2
3a
10a
12a
10 0.5
10 0.5
10 2
nd
6.25
50
1.37
0.85
17.5
nd
nd
nd
1.42
1.01
≤0.5
nd
nd
10 2
nd
nd
1.7
nd
8.6
42.2
109.3
69.5
22.3
4.6
71.0
50.5
≤25
25
>50
6.25
>50
>50
>50
50
50
50
10 1
10 1
10 1
nd
50
12.5
25
1,39
1.78
0,18
nd
nd
14
nd
37a/b
38a/b
39a/b
43
10 1
10 1
10 1
nd
>50
nd
>50
nd
nd
>50
nd
>50
10 1
50
0.33
16.4
Dextran
sulphate
Acyclovir
10 2
10 4
0.5
1.5
nd
nd
10 2
10 4
0.5
1.5
nd
nd

Page 19 of 35
RSC Advances
a
Vero Cercopithecus aethiops African green monkey kidney cell line ATCC CCL
b
c
View Article Online
DOI: 10.1039/C4RA11726C
81; 1TCID50:1 Cell Culture Infectious Dose Fifty percent. 10TCID50:10 Cell
Culture Infectious Dose Fifty percent; dRf: Value obtained by dividing the viral titer
in the absence of the compound on the titer obtained in presence of the compound;
eMaximum non-toxic concentration of compound which had the highest factor of
f
reduction of viral titer; SI: Selectivity Index (IC50 Vero/TCID50); nd: Not
determined.
In our study, the parameter established to define antiviral selectivity was the selectivity index (SI)
value. SI is defined as the relative effectiveness of a product to inhibiting viral replication
compared to the capacity for inducing cell death. It is desirable to have a high therapeutic index
(SI ≥ 10) giving maximum antiviral activity with minimal cell toxicity.48 The antiviral SI for each
active compound was calculated and listed in Table 3. Our results showed that only compound 3a
showed a high antiviral SI (17.5) against HHV-1 strain. This value could be considered indicative
of a potential lead compound for further antiviral studies.49
3. Conclusions
As a global consideration, the best biological results were principally found for 1,4-AQs and their
substituted derivatives rather than for their heterocycle-fused analogues. Among the 1,4-AQs, those
bearing an arylamino group at C-2 were the most cytotoxic against the tumour cell lines tested,
while the presence of a chlorine atom at the quinone moiety seemed to improve the antifungal and
antiviral activities. Though the heterofused AQs were generally less potent than their corresponding
AQ precursors and intermediates, the oxazole-fused compound 14 showed positive results on the
three biological tests.
The relevance of the antifungal activity varies with the pathogenicity of the fungus. In general,
filamentous fungi are more resistant than yeasts to antifungal agents. Then, the discovery of AQ 2,
active against A. flavus, and A. fumigatus is considered enough relevant. Furthermore, the finding of
several molecules (AQs 1, 2, 3a, and 12a and the oxazole-fused AQ 14), active against yeasts as C.
parapsilosis and C. krusei, with lower susceptibility to one or several standard antifungal drugs,
could support further studies to establish their mechanism(s) of action.
Finally, among the compounds tested as antiviral, the chlorinated 1,4-AQ 3a, as an active
compound against HHV-1 with the highest selectivity index, could be considered as a lead
compound for further research.
At the current level of our research, we cannot formulate consistent proposals for possible
mechanisms or targets to the new type of compounds, with novel structural arrangements directly
attached to the quinone ring. Further experimental research will be planned with such objectives.
4. Experimental section

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Page 20 of 35
4.1. Chemistry
1
Vie1w3Article Online
DOI: 10.1039/C4RA11726C
NMR spectra were recorded on a Bruker AC 200 at 200 MHz for H and 50.3 MHz for C in
deuterochloroform with TMS as internal standard. Chemical shift (δ) values are expressed in ppm
followed by multiplicity and coupling constants (J) in Hz. IR spectra were obtained on a Nicolet
Impact 410 spectrophotometer in NaCl film, and wavenumbers are given in cm-1. UV spectra were
determined on a Hitachi 100-60 spectrophotometer in CHCl3 solution, and λmax are given in nm.
HRMS were run in a VG-TS-250 spectrometer working at 70 eV. Column chromatography (CC)
was performed on silica gel (Merck No 9385) and TLC was carried out on silica gel 60 F245 (Merck,
0.25 mm thick). Solvents and reagents were purified by standard procedures as necessary.
1
1H NMR data are given for the new compounds described here and complete H and 13C NMR
assignments are included in the supplementary material.
Anthracene-1,4-diones 1-8, 12, 24, and 37-39 were obtained by means of previously described
procedures.21, 23 Other compounds were prepared as follows.
4.1.1. 3-Acetyl-1-ethyl-2,6,6-trimethyl-1H-6,7,8,9-tetrahydronaphtho[2,3-f]indole-4,11-dione
9a. To a solution of 4a (22 mg, 0.08 mmol) and 2,4-pentanedione (32 µL, 0.29 mmol) in MeOH (1
mL) and dichloromethane (0.2 mL) stirred at rt was added CAN (154 mg, 0.28 mmol) in four
portions at 10 min intervals. The reaction mixture was stirred for another 30 min and then diluted
with EtOAc, washed with sat aq sodium bicarbonate and brine, dried over Na2SO4 and and vacuum-
evaporated to yield a product, which was purified by column chromatography (eluent: Hex/EtOAc
8:2) affording 9a (12 mg, 43%): mp 128-132 ºC (from CH2Cl2); UV λmax(log ε): 270 (4.6), 297
(4.2), 388 (3.9); IR νmax (film): 1657, 1596, 1495, 1463, 1265, 1026 cm-1; 1H NMR (CDCl3) δ: 1.34
(6H, s), 1.39 (3H, t, J = 7.1), 1.70-1.82 (4H, m), 2.42 (3H, s), 2.72 (3H, s), 2.88 (2H, t, J = 6.3),
4.52 (2H, m), 7.78 (1H, s), 8.09 (1H, s); HRMS (ES, M+Na) calcd: 386.1726, found: 386.1736.
4.1.2. 3-Acetyl-1-ethyl-2,9,9-trimethyl-1H-6,7,8,9-tetrahydronaphtho[2,3-f]indole-4,11-dione
9b. Following the above procedure, treatment of 4b and 2,4-pentanedione with CAN, gave 9b
(47%): mp 137-140 ºC (from CH2Cl2); IR νmax (film): 1660, 1596, 1495, 1465, 1265, 1017 cm-1; 1H
NMR (CDCl3) δ: 1.35 (6H, s), 1.41 (3H, t, J = 7.1), 1.71-1.83 (4H, m), 2.43 (3H, s), 2.70 (3H, s),
2.88 (2H, t, J = 6.3), 4.52 (2H, m), 7.79 (1H, s), 8.09 (1H, s); HRMS (ES, M+Na) calcd: 386.1726,
found: 386.1734.
4.1.3. Methyl 1-ethyl-2,6,6-trimethyl-4,11-dioxo-1H-6,7,8,9-tetrahydronaphtho[2,3-f]indole-3-
carboxylate 10a. Following the above procedure, treatment of 4a and methyl acetylacetate with
CAN, gave 10a (40%): mp 160-163 ºC (from CH2Cl2); IR νmax (film): 1711, 1660, 1597, 1464,

Page 21 of 35
RSC Advances
1
1263, 1018 cm-1; H NMR (CDCl3) δ: 1.32 (6H, s), 1.40 (3H, t, J=7.1), 1.69-1.81 (4H, m), 2.48
View Article Online
(3H, s), 2.86 (2H, t, J = 6.2), 3.95 (3H, s), 4.52 (2H, m), 7.78 (1H, s), 8.07 (1H, sD)O; IH: 10R.10M39S/C4(REAS11,726C
M+Na) calcd: 402.1675, found: 402.1691.
4.1.4. Methyl 1-ethyl-2,9,9-trimethyl-4,11-dioxo-1H-6,7,8,9-tetrahydronaphtho[2,3-f]indole-3-
carboxylate 10b. Following the above procedure, treatment of 4b and methyl acetylacetate with
CAN, gave 10b (22%): mp 150-154 ºC (from CH2Cl2); IR νmax (film): 1710, 1668, 1597, 1465,
1
1270, 1108 cm-1; H NMR (CDCl3) δ: 1.33 (6H, s), 1.39 (3H, t, J = 7.1), 1.72-1.82 (4H, m), 2.48
(3H, s), 2.87 (2H, t, J = 6.0), 3.97 (3H, s), 4.52 (2H, m), 7.77 (1H, s), 8.11 (1H, s).
4.1.5. 3-Amino-2-chloro-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones 11a(11b).
To a solution of AQ 2 (1.82 mmol) in EtOH (30 mL), 30 % NH4OH (17 mL) was added dropwise
and kept stirring at room temperature for 1 h. Then, the solvent was vacuum-evaporated and the
residue was redissolved in ethyl acetate. The organic layer was washed with 2 N HCl and brine,
dried over Na2SO4, filtered and evaporated till dryness, affording a 1:1 mixture of 11a/11b, that was
used in the next steps without purification.
Treatment of 3a (0.71 mmol) with 30 % NH4OH (7 mL), following the above procedure, afforded
11a.
4.1.6.
3-Acetamido-2-chloro-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones
12a(12b). AQ 11a/11b (0.36 mmol) were acetylated at 0 ºC with Ac2O-H2SO4 (6 mL taken from a
previously solution prepared with 50 mL of Ac2O and 0.15 mL of H2SO4) for 30 min. The reaction
was quenched with ice and extracted with EtOAc. The organic layer was washed with satdaq
NaHCO3, and brine, dried over anhydrous Na2SO4 and vacuum-evaporated yielding a crude
product, which was purified by column chromatography (eluent: Hex/EtOAc 8:2) to afford
12a/12b.23
4.1.7.
2-Chloro-3-diacetylamino-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones
13a(13b). AQ 11a/11b (1.73 mmol) were acetylated at rt with Ac2O-H2SO4 (31 mL taken from a
solution previously prepared with 50 mL of Ac2O and 0.15 mL of H2SO4) for 1h. The reaction was
worked up as described above to afford 13a/13b (80%): IR νmax (film): 1670, 1599, 1493, 1288,
1253, 1048 cm-1; 1H NMR (CDCl3) δ: 1.34 (6H, s), 1.71-1.86 (4H, m), 2.28/2.36 (6H, s), 2.90/2.91
(2H, t, J = 6.2), 7.83/8.17 (1H, s), 8.04/7.82 (1H, s); HRMS (ES, M+Na) calcd: 396.0973, found:
396.0986.
Treatment of 11a with Ac2O-H2SO4, in the same way described above yielded 13a (96%).

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DOI: 10.1039/C4RA11726C
4.1.8. 2-Chloro-3-dibenzoylamino-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones
15a(15b). AQ 11a/11b (0.83 mmol) and NaH (1.8 mmol) were stirred at room temperature until
compound 12 disappeared by TLC control. Then, benzoyl chloride (1.23 mmol) was added and
stirred for another 5 min. The reaction mixture was quenched with water and extracted with EtOAc.
The organic layer was washed with satd aq NaHCO3, and brine, dried over anhydrous Na2SO4 and
vacuum-evaporated yielding a crude product, which was purified by column chromatography
1
(eluent: Hex/EtOAc 8:2) to afford AQs 15a/15b (30%): H NMR (CDCl3) δ: 1.30/1.33 (6H, s),
1.71-1.81 (4H, m), 2.83-2.93 (2H, m), 7.27-7.47 (6H, m), 7.73-7.77 (4H, m), 7.84/8.12 (1H, s),
8.07/7.78 (1H, s); HRMS (ES, M+Na) calcd: 520.1286, found: 520.1277.
4.1.9. General procedure for the nucleophilic substitution to obtain compounds 16-19. AQ 12,
13 or 15 and the corresponding amine in EtOH were stirred at reflux for a specify time. Then, the
solvent was vacuum-evaporated and the residue was redissolved in ethyl acetate. The organic layer
was washed with 2N HCl and brine, dried over Na2SO4, filtered and evaporated till dryness,
affording a crude product, which was purified through column chromatography over silica gel.
3-Acetamido-2-ethylamino-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones
16a(16b). Following the above procedure, treatment of 13a/13b (0.33 mmol) with ethylamine (0.8
mmol) for 45 min, gave out, after preparative TLC (eluent EtOAc): (a) 15 mg (13%) of 16a: mp
160-165 ºC (from MeOH); IR νmax (film): 3300, 1674, 1594, 1558, 1505, 1341, 1257, 1013, 809
cm-1; 1H NMR (CDCl3) δ: 1.24 (2H, t, J = 7.2), 1.31 (6H, s), 1.69-1.80 (4H, m), 2.23 (3H, s), 2.83
(2H, t, J = 6.1), 3.45 (2H, m), 7.68 (1H, s), 7.99 (1H, s); HRMS (ES, M+Na) calcd: 363.1679,
found: 363.1689. (b) 9 mg (8%) of 16a/16b. (c) 16 mg (14%) of 16b: mp 154-159 ºC (from
1
MeOH); IR νmax (film): 3283, 1673, 1600, 1595, 1515, 1341, 1300, 1005, 919 cm-1; H NMR
(CDCl3) δ: 1.24 (2H, t, J = 7.0), 1.31 (6H, s), 1.66-1.80 (4H, m), 2.22 (3H, s), 2.85 (2H, t, J = 6.1),
3.45 (2H, m), 7.69 (1H, s), 7.97 (1H, s); HRMS (ES, M+Na) calcd: 363.1679, found: 363.1666.
Following the same procedure, treatment of 13a (0.7 mmol) with ethylamine (0.7 mmol) afforded
16a (48%) after CC.
3-Acetamido-2-(p-methoxyanilino)-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones
17a(17b). Following the above procedure, treatment of 13a/13b (0.2 mmol) with p-anisidine (0.5
mmol) for 20 h at rt, gave out, after two consecutive preparative TLC (eluents Hex/EtOAc 3:7 and
benzene/methanol 9:1): (a) 3 mg (4%) of 17a: 1H NMR (CDCl3) δ: 1.33 (6H, s), 1.62 (3H, s), 1.67-
1.85 (4H, m), 2.87 (2H, t, J = 6.4), 3.80 (3H, s), 6.81 (2H, d, J = 9.1), 6.89 (2H, d, J = 9.1), 7.75
(1H, s), 8.02 (1H, s). (b) 30 mg of 17a/17b impurified with N-acetylanisidine. (c) 7 mg (9%) of

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1
17b: H NMR (CDCl3) δ: 1.34 (6H, s), 1.61 (3H, s), 1.67-1.85 (4H, m), 2.88 (2H, t, J = 6.4), 3.80
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DOI: 10.1039/C4RA11726C
(3H, s), 6.80 (2H, d, J = 9.0), 6.90 (2H, d, J = 9.0), 7.76 (1H, s), 8.04 (1H, s).
Treatment of 12a/12b (0.08 mmol) with p-anisidine (0.11 mmol) following the general procedure
for 1.5 h, afforded the mixture 17a/17b in quantitative yield.
Treatment of 13a (0.3 mmol) with p-anisidine (0.7 mmol) following the same procedure, afforded
17a impurified with N-acetylanisidine.
3-Benzamido-2-(p-methoxyanilino)-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-diones
18a(18b) and 3-dibenzoylamino-2-(p-methoxyanilino)-5,5(8,8)-dimethyl-5,6,7,8-tetrahydro
anthracene-1,4-diones 19a(19b). Following the above procedure, treatment of 15a/15b (0.26
mmol) with p-anisidine (0.56 mmol) for 1 h, gave out, after column chromatography (eluent:
toluene/EtOAc 8:2): (a) 29 mg of 19a/19b with N-acetylanisidine, which were submitted to PTLC
(eluent: benzene/EtOAc 8:2) to yield 14 mg (11%) of 19a/19b: IR νmax (film): 3346, 1664, 1599,
1508, 1474, 1334, 1249, 1029 cm-1; 1H NMR (CDCl3) δ: 1.30/1.33 (6H, s), 1.67-1.82 (4H, m), 2.86
(2H, m), 3.66 (3H, s), 6.56 (2H, d, J = 8.8), 6.89 (2H, d, J = 8.8), 7.21-7.76 (10H, m), 7.76 (1H, s),
8.03/8.04 (1H, s); HRMS (ES, M+Na) calcd: 607.2203, found: 607.2200. (b) 19 mg of 18a/18b
1
(19%): IR νmax (film): 3300, 1667, 1596, 1515, 1472, 1343, 1284, 1031 cm-1; H NMR (CDCl3) δ:
1.30/1.33 (6H, s), 1.68-1.83 (4H, m), 2.88 (2H, t, J = 6.2), 3.59 (3H, s), 6.64 (2H, d, J = 8.7), 6.91
(2H, d, J = 8.7), 7.30-7.80 (5H, m), 7.75/7.77 (1H, s), 8.05/8.07 (1H, s); HRMS (ES, M+Na) calcd:
503.1941, found: 503.1924.
4.1.10. 2,6,6(2,9,9)-Trimethyl-6,7,8,9-tetrahydroanthra[2,3-d]oxazole-4,11-dione 14a(14b).
Treatment of 16a/16b (0.06 mmol) with Ac2O-H2SO4 as described above for 12a/12b, yielded a
reaction product that was chromatographed on PTLC (eluent Hex/EtOAc 1:1) to give: (a) 4 mg
(24%) of 14a: mp 154-156 ºC (from CH2Cl2); IR νmax (film): 1674, 1588, 1453, 1381, 1240, 1089,
1031, 803 cm-1; 1H NMR (CDCl3) δ: 1.35 (6H, s), 1.71-1.83 (4H, m), 2.63 (3H, s), 2.82 (2H, d, J =
6.2), 7.57 (1H, s), 7.81 (1H, s); HRMS (ES, M+Na) calcd: 318.1100, found: 318.1041. (b) 6 mg
(36%) of 14b: mp 135-138 ºC (from CH2Cl2); IR νmax (film): 1681, 1592, 1462, 1420, 1264, 1091,
1
1032, 736 cm-1; H NMR (CDCl3) δ: 1.31 (6H, s), 1.67-1.86 (4H, m), 2.61 (3H, s), 2.82 (2H, m),
7.32 (1H, s), 8.10 (1H, s); HRMS (ES, M+Na) calcd: 318.1100, found: 318.1096.
Treatment of 17a/17b (0.09 mmol) with Ac2O-H2SO4 as described above, yielded 14a/14b (40%).
Treatment of 12a/12b (0.09 mmol) with Ac2O-H2SO4 as described above, yielded a complex
reaction product containing 13a/13b and 14a/14b.
4.1.11. General procedure for the preparation of compounds 20-22. The corresponding AQ was
dissolved in EtOH (1-3 mL) and 4 % NaOH (3-6 mL) was added dropwise and kept under reflux

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for 30 min. Then, the organic solvent was evaporated off, the residue was redissolved in EtOAc,
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DOI: 10.1039/C4RA11726C
washed with 2N HCl and brine, and dried over Na2SO4 to give the corresponding imidazole.
1-Ethyl-2,6,6-trimehyl-6,7,8,9-tetrahydro-1H-anthra[2,3-d]imidazole-4,11-dione
20a.
Following the above general procedure, treatment of 16a (0.71 mmol) with 4 % NaOH (3 mL)
yielded 20a (77%): mp 104-108 ºC (from CH2Cl2); UV λmax(log ε): 259 (4.5), 267 (4.6), 299 (4.1),
347 (3.4); IR νmax (film): 1665, 1594, 1517, 1473, 1436, 1291, 1243, 1172, 1091, 1012, 922 cm-1;
1H NMR (CDCl3) δ: 1.34 (6H, s), 1.43 (3H, t, J = 7.2), 1.70-1.82 (4H, m), 2.57 (3H, s), 2.88 (2H, t,
J = 6.3), 4.43 (2H, m), 7.77 (1H, s), 8.17 (1H, s); HRMS (ES, M+Na) calcd: 345.1578, found:
345.1593.
1-Ethyl-2,9,9-trimehyl-6,7,8,9-tetrahydro-1H-anthra[2,3-d]imidazole-4,11-dione
20b.
Following the above general procedure, treatment of 16b (0.04 mmol) with 4 % NaOH (3 mL)
yielded 20b (63%): mp 158-161 ºC (CH2Cl2); IR νmax (film): 1662, 1594, 1517, 1473, 1436, 1289,
1244, 1172, 1088, 921 cm-1; 1H NMR (CDCl3) δ: 1.34 (6H, s), 1.45 (3H, t, J = 7.1), 1.70-1.82 (4H,
m), 2.56 (3H, s), 2.87 (2H, t, J = 6.3), 4.43 (2H, m), 7.89 (1H, s), 8.08 (1H, s); HRMS (ES, M+Na)
calcd: 345.1578, found: 345.1562.
1-(p-Methoxyphenyl)-2,6,6-trimehyl-6,7,8,9-tetrahydro-1H-anthra[2,3-d]imidazole-4,11-dione
21a. Following the above general procedure, treatment of 17a (0.03 mmol) with 4 % NaOH (3 mL)
yielded 21a (86%): mp 98-102 ºC (from CH2Cl2); IR νmax (film): 1670, 1597, 1515, 1454, 1251,
1171, 1027, 802 cm-1; 1H NMR (CDCl3) δ: 1.34 (6H, s), 1.65-1.79 (4H, m), 2.38 (3H, s), 2.82 (2H,
t, J = 6.2), 3.89 (3H, s), 7.06 (2H, d, J = 8.8), 7.24 (2H, d, J = 8.8), 7.65 (1H, s), 8.18 (1H, s);
HRMS (ES, M+1) calcd: 401.1859, found: 401.1879.
1-(p-Methoxyphenyl)-2,9,9-trimethyl-6,7,8,9-tetrahydro-1H-anthra[2,3-d]imidazole-4,11-dione
21b. Following the above general procedure, treatment of 17b (0.03 mmol) with 4 % NaOH (3 mL)
yielded 21b (97%): mp 130-133 ºC (from CH2Cl2); IR νmax (film): 1671, 1596, 1515, 1456, 1253,
1171, 1025, 800 cm-1; 1H NMR (CDCl3) δ: 1.27 (6H, s), 1.61-1.84 (4H, m), 2.39 (3H, s), 2.87 (2H,
t, J = 6.3), 3.90 (3H, s), 7.07 (2H, d, J = 6.8), 7.25 (2H, d, J = 6.8), 7.91 (1H, s), 7.99 (1H, s);
HRMS (ES, M+1) calcd: 401.1859, found: 401.1869.
1-(p-Methoxyphenyl)-2-phenyl-6,6(9,9)-dimethyl-6,7,8,9-tetrahydro-1H-anthra[2,3-
d]imidazole-4,11-dione 22a(22b). Following the above general procedure, treatment of 18a/18b
(0.04 mmol) with 4 % NaOH (3 mL) yielded 22a/22b (93%): IR νmax (film): 1670, 1596, 1514,
1
1429, 1246, 1174, 1030, 840 cm-1; H NMR (CDCl3) δ: 1.28/1.36 (6H, s), 1.67-1.84 (4H, m), 2.86
(2H, m), 3.88 (3H, s), 6.96-7.59 (9H, m), 7.69/8.03 (1H, s), 8.23/7.95 (1H, s); HRMS (ES, M+Na)
calcd: 485.1835, found: 485.1832.

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4.1.12. 12,12(9,9)-Dimethyl-9,10,11,12-tetrahydroanthra[1',2':4,5]imidazo[1,2-a]pyridine-6,7-
View Article Online
DOI: 10.1039/C4RA11726C
dione 25a(25b). A mixture of AQ 2 (0.65 mmol) and 2-aminopyridine (1.3 mmol) in EtOH (8 mL)
was stirred under reflux for 72 h. The solvent was vacuum evaporated and the residue was
redissolved in EtOAc, washed with 2N HCl and brine, dried over Na2SO4, filtered and evaporated
till dryness yielding a reaction product that was purified by column chromatography (eluent
Hex/EtOAc 1:1) to afford: (a) 22 mg (10%) of 25a: mp 270-275 ºC (from CH2Cl2): UV λmax(log ε):
1
283 (4.4), 351 (3.7), 402 (3.7): IR νmax (film): 1644, 1455, 1428, 1391, 1249, 1039, 913 cm-1: H
NMR (CDCl3) δ: 1.39 (6H, s), 1.70-1.85 (4H, m), 2.82 (2H, t, J = 6.4), 7.16 (1H, dd, J = 6.2 and
7.6), 7.60 (1H, dd, J = 7.6 and 8.8), 7.79 (1H, s), 7.80 (1H, d, J = 8.9), 8.10 (1H, s), 9.29 (1H, d, J =
6.2): HRMS (ES, M+Na) calcd: 353.1260, found: 353.1260. (b) 26 mg (12%) of 25a/25b. (c) 7 mg
(3%) of 25b: mp 279-282 ºC (from CH2Cl2): IR νmax (film): 1641, 1457, 1427, 1388, 1250, 1111,
1
1041, 769 cm-1: H NMR (CDCl3) δ: 1.34 (6H, s), 1.70-1.85 (4H, m), 2.89 (2H, t, J = 6.4), 7.17
(1H, dd, J = 6.3 and 7.4), 7.61 (1H, dd, J = 7.4 and 8.7), 7.78 (1H,d, J = 8.7), 7.84 (1H, s), 8.10
(1H, s), 9.29 (1H, d, J = 6.3); HRMS (ES, M+Na) calcd: 353.1260, found: 353.1275.
A mixture of AQ 2 (0.43mmol), 2-aminopyridine (0.95mmol) and K2CO3 (64 mg) in MeOH (2 mL)
was stirred under reflux for 19 h. Following the procedure described above and after column
chromatography (eluent Hex/EtOAc 8:2 and 1:1) of the complex reaction product, 18 mg (14%) of
1
23 were obtained: IR νmax (film): 1665, 1602, 1455, 1338, 1296, 1041, 917, 734 cm-1; H NMR
(CDCl3) δ: 1.32 (6H, s), 1.71-1.80 (4H, m), 2.86 (2H, t, J = 6.2), 4.08 (6H, s), 7.71 (1H, s), 8.00
(1H, s).
A mixture of AQ 2 (0.65 mmol), 2-aminopyridine (1.3 mmol) and K2CO3 (200 mg) in EtOH (5 mL)
was stirred under reflux for 14 h. Following the procedure described above and after column
chromatography (eluent Hex/EtOAc 8:2 and 1:1) of the reaction product, the following compounds
were obtained: 44 mg (20%) of 2423 and 22 mg (10%) of 25a/25b.
4.1.13. 2,3,13,13-Tetramethyl-13,14,15,16-tetrahydronaphtho[2,3-a]pyrido[1',2':1,2]imidazo
[4,5-c]phenazine 26a. A mixture of 25a (0.07 mmol) and glacial acetic acid (400 µL) in EtOH (1.5
mL) was stirred at rt for 30 min. Then, 4,5-dimethyl-1,2-phenylenediamine (0.07 mmol) was added
and heated under reflux for 6 h. The reaction mixture was diluted with water and extracted with
EtOAc. The organic phase was washed with brine, dried over Na2SO4, filtered and evaporated off to
yield a reaction product, which was purified by column chromatography (eluent Hex/EtOAc 1:1)
affording 26a (50%): mp 240-245 ºC (from CH2Cl2); IR νmax (film): 1450, 1334, 1264, 1114, 1028,
735 cm-1; 1H NMR (CDCl3) δ: 1.48 (6H, s), 1.84-1.97 (4H, m), 2.46 (3H, s), 2.49 (3H, s), 3.14 (2H,
t, J = 6.2), 7.10 (1H, dd, J = 6.7 and 7.0), 7.51 (1H, dd, J = 7.0 and 8.2), 7.80 (1H, s), 7.90 (1H, s),

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7.94 (1H, d, J = 8.2), 8.70 (1H, s), 8.94 (1H, s), 10.12 (1H, d, J = 6.7); HRMS (ES, M+H) calcd:
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DOI: 10.1039/C4RA11726C
431.2230, found: 431.2260.
4.1.14. 2,3,16,16-Tetramethyl-13,14,15,16-tetrahydronaphtho[2,3-a]pyrido[1',2':1,2]imidazo
[4,5-c]phenazine 26b. A mixture of 25b (0.05 mmol) and glacial acetic acid (300 µL) in EtOH (1.5
mL) was stirred at rt for 30 min. Then, 4,5-dimethyl-1,2-phenylenediamine (0.06 mmol) was added
and heated under reflux for 2 h. The reaction mixture was diluted with water and extracted with
EtOAc. The organic phase was washed with brine, dried over Na2SO4, filtered and evaporated off to
yield a reaction product, which was purified by column chromatography (eluent Hex/EtOAc 1:1)
affording 26b (32%): mp 205-210 ºC (from CH2Cl2); IR νmax (film): 1454, 1336, 1261, 1116, 1043,
735 cm-1; 1H NMR (CDCl3) δ: 1.57 (6H, s), 1.75-1.95 (4H, m), 2.49 (3H, s), 2.52 (3H, s), 3.11 (2H,
t, J = 6.2), 7.08 (1H, dd, J = 6.7 and 7.0), 7.48 (1H, m), 7.85 (1H, d, J = 8.4), 7.85 (1H, s), 7.98 (1H,
s), 8.39 (1H, s), 9.31 (1H, s), 10.13 (1H, d, J = 6.7); HRMS (ES, M+H) calcd: 431.2230, found:
431.2225.
4.1.15. General procedure for the preparation of compounds 27-36.
Method A: The AQs 1 or 2 and the corresponding 1,2-diamine were dissolved in EtOH and kept
stirring for the specified time. Then, the organic solvent was evaporated off and the residue was
chromatographed over silica-gel to give the corresponding derivative.
Method B: The AQs 1 or 2 and the corresponding 1,2-diamine were dissolved in dichloromethane
and mortmorillonite K10, as solid support (3 times the reactants weight), was added and well
mixed. Then, the organic solvent was evaporated under vacuum and the dried material was
subjected to microwave irradiation in a domestic microwave oven for a specified time. The solid
mixture was extracted with EtOAc and MeOH and after evaporation of the organic solvent, the
reaction product was obtained and purified by column chromatography.
2-(2-Aminoethylamino)-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-dione
27a(27b)
and 8,8(11,11)-dimethyl-8,9,10,11-tetrahydronaphtho[2,3-f]quinoxalin-6-yl acetate 29a(29b).
Following the above method A, a mixture of 1 (1.19 mmol) and 1,2-ethylenediamine (1.20 mmol)
dissolved in EtOH, was stirred at rt for 10 min. Column chromatography of the reaction product
(eluent Hex/EtOAc 1:1) afforded a 1:1 mixture of 27a/27b (30%): IR νmax (film): 3296, 3246, 1609,
1
1572, 1543, 1458, 1335, 1297, 1104, 732 cm-1; H NMR (CDCl3) δ: 1.30 (6H, s), 1.72-1.80 (4H,
m), 2.81 (2H, t, J = 6.1), 3.28 (2H, t, J = 6.2), 4.02 (2H, t, J = 6.2), 5.63 (1H, s), 7.73 (1H, s), 7.99
(1H, s); HRMS (ES, M+Na-H2O) calcd: 303.1473, found: 303.1483.
Following the method A, a mixture of 1 (1.21 mmol) and 1,2-ethylenediamine (1.73 mmol)
dissolved in EtOH, was stirred at rt for 30 min. Column chromatography of the reaction product

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(eluent Hex/EtOAc 7:3) afforded: (a) a 1:1 mixture of 28a/28b (8%). (b) a 1:1 mixture of 27a/27b
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DOI: 10.1039/C4RA11726C
(6%). Acetylation of 28a/28b with acetic anhydride and pyridine yielded the acetates 29a/29b
1
(61%): IR νmax (film): 1766, 1605, 1441, 1371, 1299, 1194, 1093, 733 cm-1; H NMR (CDCl3) δ:
1.42/1.44 (6H, s), 1.78-1.95 (4H, m), 2.54/2.53 (3H, s), 3.05/3.12 (2H, t, J = 6.3), 7.64/7.63 (1H, s),
7.90/7.62 (1H, s), 8.84 (1H, s), 8.86 (1H, s), 8.89/9.22 (1H, s); HRMS (ES, M+H) calcd: 321.1603,
found: 321.1616.
Following the method B, a mixture of 1 (0.96 mmol) and 1,2-ethylenediamine (1.76 mmol)
adsorbed on mortmorillonite K10 was irradiated at 700 W for 3 min at 1 min intervals. Column
chromatography of the reaction product (eluent Hex/EtOAc 8:2 and 7:3) afforded: (a) a 1:1 mixture
of 28a/28b (13%). (b) a 1:1 mixture of 27a/27b (4%).
2-(2-Aminoethylamino)-3-chloro-5,5(8,8)-dimethyl-5,6,7,8-tetrahydroanthracene-1,4-dione
30a(30b),
5-chloro-8,8(11,11)-dimethyl-8,9,10,11-tetrahydronaphtho[2,3-f]quinoxalin-6-ol
32a(32b) and 5-chloro-8,8(11,11)-dimethyl-8,9,10,11-tetrahydro-naphtho[2,3-f]quinoxalin-6-yl
acetate 34a(34b). Following the above method A, a mixture of 2 (0.45 mmol) and 1,2-
ethylenediamine (0.48 mmol) dissolved in EtOH, was stirred at reflux for 16 h. Column
chromatography of the reaction product (eluent Hex/EtOAc 7:3) afforded a 1:1 mixture of 30a/30b
1
(30%): IR νmax (film): 3290, 1605, 1567, 1554, 1433, 1337, 1287, 1110, 734 cm-1; H NMR
(CDCl3) δ: 1.33/1.34 (6H, s), 1.60-1.82 (4H, m), 2.85 (2H, m), 3.45 (2H, m), 4.14 (2H, m),
7.80/8.10 (1H, s), 8.12/7.81 (1H, s); HRMS (ES, M-H2O+H) calcd: 315.1258, found: 315.1272.
Following the method A, a mixture of 2 (0.65 mmol) and 1,2-ethylenediamine (0.68 mmol)
dissolved in EtOH, was stirred at reflux for 96 h. Column chromatography of the reaction product
(eluent Hex/EtOAc 8:2) afforded a 1:1 mixture of 32a/32b (10%): IR νmax (film): 3306, 1582, 1434,
1
1375, 1331, 1151, 1118, 1082, 844, 735 cm-1; H NMR (CDCl3) δ: 1.44/1.46 (6H, s), 1.75-1.93
(4H, m), 3.06 (2H, m), 8.28/7.99 (1H, s), 8.77/9.11 (1H, s), 8.84 (1H, d, J = 2.2), 8.74 (1H, d, J =
2.2); HRMS (ES, M+H) calcd: 313.1108, found: 313.1148. Acetylation of 32a/32b with acetic
anhydride and pyridine yielded the acetates 34a/34b: IR νmax (film): 1779, 1594, 1447, 1369, 1174,
1
1098, 1010, 871, 736 cm-1; H NMR (CDCl3) δ: 1.46/1.40 (6H, s), 1.76-1.92 (4H, m), 2.59/2.57
(3H, s), 3.03/3.08 (2H, t, J = 6.3), 7.83/7.55 (1H, s), 8.83/9.17 (1H, s), 8.91/8.88 (1H, d, J = 2.1),
8.95 (1H, d, J = 2.1); HRMS (ES, M+H) calcd: 355.1207, found: 355.1216.
Following the method B, a mixture of 2 (0.3 mmol) and 1,2-ethylenediamine (0.47 mmol) adsorbed
on mortmorillonite K10 was irradiated at 500 W for 1 min and at 750 W for 2 min (1 min intervals).
Column chromatography of the reaction product (eluent Hex/EtOAc 8:2) afforded: (a) 2 (11%). (b)
a 1:1 mixture of 32a/32b (24%). (c) a 1:1 mixture of 30a/30b (20%).
2-((2-Amino-1-methyl)ethylamino)-3-chloro-5,5(8,8)-dimethyl-5,6,7,8-tetrahydro-anthracene-
1,4-dione
31a(31b),
5-chloro-3,8,8(3,11,11)-trimethyl-8,9,10,11-tetrahydro-naphtho[2,3-

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Page 28 of 35
f]quinoxalin-6-ol
33a(33b)
and
5-chloro-3,8,8(3,11,11)-trimethyl-8,9,10,11-
View Article Online
DOI: 10.1039/C4RA11726C
tetrahydronaphtho[2,3-f]quinoxalin-6-yl acetate 35a(35b). Following the method A, a mixture
of 2 (0.45 mmol) and 1,2-propanediamine (0.48 mmol) dissolved in EtOH, was stirred at reflux for
7 days. Column chromatography of the reaction product (eluent Hex/EtOAc 8:2) afforded: (a) a 1:1
1
mixture of 33a/33b (11%): H NMR (CDCl3) δ: 1.45/1.49 (6H, s), 1.76-1.94 (4H, m), 2.80/2.82
(3H, s), 3.08 (2H, m), 8.28/7.99 (1H, s), 8.82/9.14 (1H, s), 8.73 (1H, s). (b) a 1:1 mixture of
1
31a/31b (20%): IR νmax (film): 3311, 1641, 1605, 1570, 1436, 1272, 1175, 1093, 734 cm-1; H
NMR (CDCl3) δ: 1.32/1.34 (6H, s), 1.65-1.93 (4H, m), 1.66/1.69 (3H, d, J = 3.3) 2.85 (2H, t, J =
6.0), 3.13 (2H, m), 3.51 (1H, m) 7.80/8.10 (1H, s), 8.14/7.83 (1H, s); HRMS (ES, M-H2O+H)
calcd: 329.1421, found: 329.1427. Acetylation of 33a/33b with acetic anhydride and pyridine
yielded the acetates 35a/35b: IR νmax (film): 1779, 1595, 1457, 1362, 1190, 1096, 966, 869, 735
1
cm-1; H NMR (CDCl3) δ: 1.41/1.48 (6H, s), 1.77-1.94 (4H, m), 2.59/2.58 (3H, s), 2.85/2.87 (3H,
s), 3.04/3.10 (2H, t, J = 6.3), 7.81/7.54 (1H, s), 8.88/9.20 (1H, s), 8.83/8.82 (1H, s); HRMS (ES,
M+H) calcd: 369.1364, found: 369.1373.
6-Chloro-2,3,9,9(2,3,12,12)-tetramethyl-9,10,11,12-tetrahydronaphtho[2,3-a]phenazin-7-yl
acetate 36a(36b). Following the method B, a mixture of 2 (0.3 mmol) and 4,5-dimethyl-1,2-
phenylenediamine (0.47 mmol) adsorbed on mortmorillonite K10 was irradiated at 650 W for 0.5
min and an additional 1 min. The reaction product was acetylated and column chromatography of
the acetylation product (eluent Hex/EtOAc 8:2) afforded a 1:1 mixture of 36a/36b (22%): IR νmax
(film): 1778, 1492, 1357, 1179, 1097, 1015, 921, 869 cm-1; 1H NMR (CDCl3) δ: 1.42/1.52 (6H, s),
1.78-1.94 (4H, m), 2.56/2.58 (3H, s), 2.59 (6H, s), 3.04/3.13 (2H, t, J = 6.2), 7.52/7.81 (1H, s), 8.07
(1H, s), 8.13 (1H, s), 9.34/9.01 (1H, s); HRMS (ES, M+H) calcd: 433.1683, found: 433.1687.
4.1.16. General procedure for the preparation of compounds 40-43. To a solution of the
corresponding arylamino-AQ 37-3923 in DMF, sodium azide, suspended in a little amount of water,
was added and heated under reflux overnight. The reaction mixture was diluted with EtOAc and the
organic phase was washed with brine, dried over Na2SO4 and the solvent evaporated off, affording a
crude product that was purified by column chromatography to yield the corresponding phenazines.
2-Methoxy-11,11(8,8)-dimethyl-8,9,10,11-tetrahydronaphtho[2,3-b]phenazine-6,13-dione
40a(40b). From 37a/37b (0.53 mmol) in DMF (5 mL) and NaN3 (1.07 mmol) and following the
general procedure, a 1:1 mixture of 40a/40b (21%) was obtained: IR νmax (film): 1685, 1616, 1487,
1
1417, 1334, 1248, 1127, 1097, 1020, 841, 731 cm-1; H NMR (CDCl3) δ: 1.41 (6H, s), 1.76-1.87
(4H, m), 2.97 (2H, t, J = 6.5), 4.03 (3H, s), 7.62/7.60 (1H, d, J = 9.4/9.1), 7.69/7.71 (1H, s),

Page 29 of 35
RSC Advances
8.33/8.31 (1H, d, J = 9.1/9.4), 8.13/8.41 (1H, s) 8.41/8.13 (1H, s); HRMS (ES, M+Na) calcd:
View Article Online
DOI: 10.1039/C4RA11726C
395.1366, found: 395.1363.
1,2-Dimethoxy-11,11(8,8)-dimethyl-8,9,10,11-tetrahydronaphtho[2,3-b]phenazine-6,13-dione
41a(41b). From 38a/38b (0.35 mmol) in DMF (4 mL) and NaN3 (0.77 mmol) and following the
general procedure, a 1:1 mixture of 41a/41b (13%) was obtained: IR νmax (film): 1686, 1599, 1494,
1465, 1384, 1261, 1071, 1018, 732 cm-1; 1H NMR (CDCl3) δ: 1.41/1.42 (6H, s), 1.41-1.80 (4H, m),
2.99 (2H, t, J = 6.2), 4.14 (3H, s), 4.32/4.33 (3H, s), 7.83 (1H, d, J = 9.3), 8.13/8.42 (1H, s),
8.25/8.26 (1H, d, J = 9.3), 8.42/8.15 (1H, s); HRMS (ES, M+H) calcd: 403.1652, found: 403.1670.
1,2,11,11(1,2,8,8)-Tetramethyl-8,9,10,11-tetrahydronaphtho[2,3-b]phenazine-6,13-dione
42a(42b) and 2,3,8,8-tetramethyl-8,9,10,11-tetrahydro-naphtho[2,3-b]phenazine-6,13-dione 43.
From 39a/39b (0.60 mmol) in DMF (6 mL) and NaN3 (1.29 mmol), following the general
procedure, and after column chromatography, the following compounds were separated: (a) a 1:1
mixture of 42a/42b (13%): IR νmax (film): 1686, 1597, 1458, 1334, 1252, 1018, 753 cm-1; 1H NMR
(CDCl3) δ: 1.42 (6H, s), 1.64-1.77 (4H, m), 2.60 (3H, s), 2.91 (3H, s), 2.99 (2H, m), 7.79 (1H, d, J
= 8.6), 8.20/8.21 (1H, d, J = 8.6), 8.12/8.41 (1H, s), 8.42/8.14 (1H, s); HRMS (ES, M+H) calcd:
371.1754, found: 371.1759. (b) compound 43 (8%): mp 104-107 ºC (from CH2Cl2); UV λmax(log ε):
312 (4.6), 351 (4.0); IR νmax (film): 1688, 1596, 1481, 1363, 1334, 1311, 1248, 1022, 873, 734 cm-
1; 1H NMR (CDCl3) δ: 1.41 (6H, s), 1.73-1.90 (4H, m), 2.56 (6H, s), 2.98 (2H, t, J = 6.3), 8.13 (1H,
s), 8.18 (1H, s), 8.20 (1H, s), 8.41 (1H, s); HRMS (ES, M+H) calcd: 371.1754, found: 371.1766.
4.2. Cytotoxicity studies on tumour cell lines.
A colourimetric assay using sulforhodamine B (SRB) was adapted for a quantitative measurement
of cell growth and viability, following a previously described method.40 Cells were seeded in 96-
well microtitre plates, at 5 x 103 cells per well in aliquots of 195 µL of RPMI medium, and they
were allowed to attach to the plate surface by growing in a drug–free medium for 18 h. Afterwards,
samples were added in aliquots of 5 µL (dissolved in DMSO/H2O, 3:7). After 72 h exposure, the
antitumor effect was measured by the SRB methodology: cells were fixed by adding 50 µL of cold
50% (wt/vol) trichloroacetic acid (TCA) and incubating for 60 min at 4 ºC. Plates were washed
with deionised water and dried; 100 µL of SBR solution (0.4% wt/vol in 1% acetic acid) was added
to each microtitre well and incubated for 10 min at room temperature. Unbound SRB was removed
by washing with 1% acetic acid. Plates were air-dried and bound stain was solubilized with Tris
buffer. Optical densities (OD) were read on an automated spectrophotometer plate reader at single
wavelength of 490 nm. Data analyses were generated automatically by the LIMS implementation.
Using control OD values (C), test OD values (T) and time zero OD values (To), the drug