Synthesis, Cytotoxicity Evaluation in Human Cell Lines and in Vitro DNA Interaction of a Hetero-Arylidene-9(10H)-Anthrone

Article abstract of DOI:10.1002/ejoc.201701500

A new and never before reported hetero-arylidene-9(10H)-anthrone structure (4) was unexpectedly isolated on reaction of 1,2-dimethyl-3-ethylimidazolium iodide (2) and 9-anthracenecarboxaldehyde (3) under basic conditions. Its structure was unequivocally confirmed by X-ray crystallography. No cytotoxicity in human healthy fibroblasts and in two different cancer cell lines was observed, indicating its applicability in biological systems. Compound 4 interacts with CT-DNA by intercalation between the adjacent base pairs of DNA with a high binding affinity [K_b = 2.0 (±0.20) × 10⁵ m^–1], which is 10 × higher than that described for doxorubicin [K_b = 3.2 (±0.23) × 10⁴ m^–1]. Furthermore, compound 4 quenches the fluorescence emission of a GelRed–CT-DNA system with a quenching constant (K_SV) of 3.3 (±0.3) × 10³ m^–1 calculated by the Stern–Volmer equation.

Full text of DOI:10.1002/ejoc.201701500

A Journal of
Accepted Article
Title: Synthesis, cytotoxicity evaluation in human cell lines and in vitro
DNA interaction of a hetero arylidene-9(10H)-anthrone
Authors: Paula S. Branco, Daniela Peixoto, Margarida Figueiredo,
Gabriela Malta, Catarina Roma-Rodrigues, Pedro Viana
Batista, Alexandra R. Fernandes, Sónia Barroso, Ana Luisa
Carvalho, Carlos A. M. Afonso, and Luísa Maria Ferreira
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201701500
Link to VoR: http://dx.doi.org/10.1002/ejoc.201701500
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10.1002/ejoc.201701500
European Journal of Organic Chemistry
COMMUNICATION
Synthesis, cytotoxicity evaluation in human cell lines and in vitro
DNA interaction of a hetero arylidene-9(10H)-anthrone
Daniela Peixoto,† Margarida Figueiredo,† Gabriela Malta,† Catarina Roma-Rodrigues,‡ Pedro V.
Baptista,‡ Alexandra R. Fernandes,‡ Sónia Barroso,‡‡ Ana Luísa Carvalho,‡‡ Carlos A. M. Afonso,§
Luisa M. Ferreira,*,† and Paula S. Branco*,†
Abstract: A new and never yet reported hetero arylidene-9(10H)-
anthrone structure (4) was unexpectedly isolated on reaction of 1,2-
also show good anti-tumor activities with some compounds acting
as inhibitors of tubulin polymerization.[2b, 3] Here, the three rings of
the anthrone system are not co-planar because of steric
interactions with the benzylidene group.[4] The 10-substituted
benzylidene anthrone (1) may be prepared with moderate yields
by reaction of anthrone and substituted benzaldehydes under
dimethyl-3-ethylimidazolium
iodide
(2)
and
9-
anthracenecarboxaldehyde (3) under basic conditions. Its structure
was unequivocally attributed by X-ray crystallography. No cytotoxicity
in human healthy fibroblasts and in two different cancer cell lines was
observed indicating its applicability in biological systems. Compound
4 interacts with CT-DNA by intercalation between the adjacent base
pairs of DNA with a high binding affinity (Kb = 2.0(± 0.20) x 105 M-1)
which is 10x higher than that described for doxorubicin (Kb = 3.2
acidic (gaseous HCl) or basic (pyridine/piperidine) conditions
2b]
(Scheme 1).[2a,
Notwithstanding the extensive work on
imidazolium-based ionic liquids (IL), only but a few references
exist concerning the reactivity of these azolium salts when
functionalized at the C2-position.[5] In the presence of a base the
N-heterocyclic olefin (NHO)[6] those formed act as a nucleophile
reacting with aldehydes,[7] with CO2,[8] as catalyst in the trans-
(±0.23)
× 4 quenches the
104 M-1). Furthermore, compound
fluorescence emission of GelRed-CT-DNA system with a quenching
constant (KSV) of 3.3(±0.3) x 103 M-1 calculated by the Stern-Volmer
equation.
[9]
esterification reaction,[6] is susceptible to alkylation or, can be
involved in coupling reactions.[10]
Anthraquinones, whose principal structural features is a tricyclic
planar ring system are a group of functionally diverse aromatic
compounds displaying a wide range of important pharmaceutical
properties.[1] Although historically employed as a natural dye the
discovery of various anthraquinone derivatives with medicinal
value such as anticancer, antibacterial, anti-inflammatory,
antioxidant, antidiabetic, antiviral among others make this scaffold
a promising candidate to be studied.[1b, 1c] Doxorubicin,[1b] dauno-
rubicin and carminomycin[1c] are successful examples of
anticancer agents resulting from their good DNA-intercalating
action in result of the flat aromatic anthraquinone core. The
anthrone derivatives, 10-substituted benzylidene anthrones,[2]
Scheme 1. Synthesis of 10-substituted benzylidene anthrones (1).
Recently,[11] we have disclosed an unusual reactivity of 1,2-
dimethyl-3-ethylimidazolium salt (2). We have observed that on
reaction with aldehydes, an unpredicted result with the oxidation
to the corresponding carboxylic acid took place. Here we present
our unexpected and singular result, obtained when on the above
[†]
Dr. D. Peixoto, G.Malta, M. Figueiredo, Prof. L. M. Ferreira, Prof. P.
S. Branco
LAQV-REQUIMTE, DQ, 2829-516, Caparica, Portugal
Faculdade de Ciꢀncias e Tecnologia, Universidade NOVA de Lisboa
2829-516, Caparica, Portugal
E-mail: lpf@fact.unl.pt
(https://www.dq.fct.unl.pt/pessoas/docentes/luisa-maria-da-silva-
pinto-ferreira)
paula.branco@fct.unl.pt
(https://www.dq.fct.unl.pt/pessoas/docentes/paula-cristina-de-serio-
branco)
Dr. C. Roma-Rodrigues, Prof. A. R. Fernandes, Prof. P. V. Batista
UCIBIO-REQUIMTE, DCV
Faculdade de Ciꢀncias e Tecnologia, Universidade NOVA de Lisboa
2829-516, Caparica, Portugal
Dr. S. Barroso, Dr. A. L. Carvalho
UCIBIO-REQUIMTE, DQ
Faculdade de Ciꢀncias e Tecnologia, Universidade NOVA de Lisboa
2829-516, Caparica, Portugal
Prof. C. A. M. Afonso
iMed.ULisboa
Faculdade de Farmácia, Universidade de Lisboa
Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
described
reaction
the
substrate
is
the
9-
anthracenecarboxaldehyde.
Applying the reaction conditions described previously,[11] 2,
cesium carbonate and 9-anthracenecarboxaldehyde (3a) in dry
THF, the hetero arylidene-9(10H)-anthrone (4) was obtained after
72h of reaction, in 70% yield (Table 1, entry 2). The structural
assignment of 4 (see SI) was unequivocally confirmed by single-
crystal X-ray crystallography (Figure 1). Under the reactions
conditions an isomeric structure 4a was isolated, which is
interconverted to 4 in DMSO solution, presents the chemical shifts
for the imidazolium part slightly shielding in result of the
asymmetric boat conformation of the anthrone system. The
central ring of the anthrone unit is not co-planar (observed in other
substituted anthrones.[4]) with the two outer benzene rings due to
steric interactions between the anthrone moiety and the
imidazolium group. The outer benzene rings form a dihedral angle
of 20.7(3)˚ and atoms C2, C7 C9 and C14 in the central ring are
[‡]
‡‡
[
]
[§]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201701500
European Journal of Organic Chemistry
COMMUNICATION
co-planar (Rms = 0.0138 Å), with atoms C1 and C8 deviating from
the plane by 0.139(10) and 0.297(10) Å, respectively (Figure 1).
To elucidate the mechanism for the formation of 4, other 10-
substituted 9-anthracenecarboxaldehyde derivatives (3b-3d)
were subject to the reaction conditions (Table 1) and, surprisingly,
compound 4 was also isolated upon reaction with 10-chloro-9-
a)
b)
anthracenecarboxaldehyde
(3b)
and
9,10-
anthracenedicarboxaldehyde (3c) in 90% and 37% yields
respectively (entries 10 and 16) but absent upon reaction with 10-
methyl-9-anthracenecarboxaldehyde (3d) (entry 20). To elucidate
the mechanism of the reaction it was crucial to identify minor
compounds isolated in the ethereal fraction from the washing of
the crude reaction mixture. Common to all reaction is the
formation of anthraquinone (5), although the amount is dependent
on the reaction time and substrate. We postulate its formation
through fragmentations of the endoperoxides (6a-c) (Scheme 2)
Figure 1. a) MERCURY diagram of compound 4, using 30% probability level
ellipsoids; b) the asymmetric boat conformation of the central ring in the
anthrone unit. Hydrogen atoms and the outer aromatic rings are omitted for
clarity
Table 1. Reaction conditions of 2 and 3.
9-anthracene
aldehyde (3)
(1 equiv)
Time
(h)
2
Recovered
Entry
Base
4 (%) 4a (%) 5 (%) 6a (%) 8 (%)
(equiv)
3 (%)
1a
1b
2
3a)
4 b)
5
6
7
8
9
Cs2CO3 (1.2 equiv)
NEt3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
NaH (1.2 equiv)
NaOH (1.2 equiv)
NEt3(1.2 equiv)
Piperidine (1.2 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
NaH (1.2 equiv)
0
0
1
1
1
1
1
1
1
0
1
1
1
1
1
0
1
2
1
0
1
48
48
72
72
72
20
100
-
-
70
20
5.2
vestg.
-
-
-
-
-
-
-
3.2
-
13
45
5
46
27
-
5
15
5
12
10
-
3.5
5
19.6
-
6
3.2
vestg.
-
-
6
10
-
-
-
-
-
-
-
-
-
-
-
-
-
30
88
-
3a
72
-
72
-
-
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
120
120
72
48
72
-
90
79
40
5
23
-
99
89
75
10
vestg
35
35
25
-
10
11
90
8.8
82
-
-
-
37
-
9
-
72
3b
12
13
14
15
16
17
18b)
19
20
NaOH (1.2 equiv)
NEt3 (1.2 equiv)
120
120
72
48
72
168
72
48
168
Piperidine (1.2 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (2.4 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
Cs2CO3 (1.2 equiv)
40
60
14.6
22
45
3c
-
-
-
-
-
-
32.7
20
3d
-
a) THF saturated with O2; b) THF degassed
under basic conditions formed due to the presence of residual
oxygen on the solvent. Bauch et al. have recently[12] reported
that the decomposition of anthracene endoperoxides proceeds
via various intermediates, as for example the 9,10-
dihydroxyanthracene (7). We also proposed the formation of
intermediate 7, which is subsequently oxidized to 5 (Scheme
2). We isolate compound 6a and its structure was attributed by
1H NMR. As expected, the mass spectra only shows the [M-
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10.1002/ejoc.201701500
European Journal of Organic Chemistry
COMMUNICATION
32]+ ion. The formation of the endoperoxides intermediates
justifies that upon reaction with 3d the anthraquinone 5 and the
10-hydroxy-10-methylanthracen-9(10H)-one (8) (Scheme 2)
were the major products isolated (entries 19 and 20). Since in
the reaction with 3d, the dialdehyde 3c was identified in
residual amount by GC-MS, we postulate the formation of the
hydroperoxide 9 and endoperoxide 6d as intermediates to 5
and 8 respectively. To clarify the formation of 4 and 5 additional
experiments were conducted, namely in the absence or
presence of O2 and with other bases besides Cs2CO3 such as,
NEt3, NaOH or NaH. Only nucleophilic bases, such as NaOH
(entries 6 and 12) or Cs2CO3 (entries 2, 10 and 16) allowed the
formation of 4, and small amounts were observed with NaH
(entry 5 and 11). With NEt3 as base, the starting materials 3a
was recovered and 6a observed in 6% yield (entries 7 and 13),
which reinforces the need of a stronger base to remove the
proton from 6a (Scheme 2).No reaction was observed between
5 and 2 in the presence of Cs2CO3 in THF, which led to exclude
5 as the intermediate for the formation of 4. We have also
observed the release of CO in the reaction medium and a
decrease of the yield, from 70% to 5.2% when the reaction was
performed in absence of O2 (entries 2 vs 4) which prompted us
to present the mechanism in Scheme 3. Since only nucleophilic
bases allowed the formation of 4, we proposed a 1,6
conjugated addition of the base with formation of 10-
hydroxyanthracene-9-carbaldehyde (10). This intermediate
upon addition of the NHO (11) and after water elimination
followed by reaction with O2 gave the endoperoxide 12 through
the anthracene derivative intermediate 13.The CO release
(observed qualitatively in higher amount after 72h by GC-TCD),
led to the formation of 4. A higher amount of CO was detected
when the reaction was performed with 3c. The reduced specie
(14) previously observed by us [11], was here identified which
supports the proposed mechanism involving the oxidation to
13. Although the reaction was performed with dry THF, no
particular caution was taken to exclude the contact with air,
with the concomitant presence of oxygen with time. This, and
the presence of moisture, can explain the formation of 4 in
small amounts when NaH was used as base (entry 11). We
would expected, taking in consideration the above proposed
mechanism that the yield for the formation of 4 would increase
in the presence of an O2 atmosphere and decrease in the
exclusion of O2. Indeed, vestigial amounts were observed in
the absence of O2 but with an O2 atmosphere, the parallel
reaction of the 3 with O2 (scheme 3) lead to the increase
amount of 5 (entries 2 vs 3).
Compound 4 cytotoxicity was assessed by the MTS
assay on representative human cancer cell lines: ovarian
carcinoma (A2780) and colorectal carcinoma (HCT116), and
compared to normal human primary fibroblasts (see SI Figure
S1). No impairment of cell viability is observed after the
incubation of the compound in all cell lines for the low
micromolar range. Compound 4 shows no toxicity to human
fibroblasts contrary to doxorubicin.[13] This demonstrates that
compound 4 may be used in biological applications due to the
lack of cytotoxicity in those human cell lines. Also, exposure of
A2780 cells for 6 h to a high concentration of compound 4
(1000 μM) or to 250 μM (IC50) for 48 h does not induce reactive
oxygen species (ROS) (SI Figure S2).
The UV-visible absorption spectra of compound 4 after a
24 h incubation period in Tris-HCl 0.5 mM pH 7.0 with 50 mM
NaCl may be observed in SI.
Scheme 2. Proposed mechanism for the formation of 5 and 8.
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10.1002/ejoc.201701500
European Journal of Organic Chemistry
COMMUNICATION
Scheme 3. Proposed mechanism for the formation of 4.
1.6
Since DNA is one of the cellular targets of therapeutic
anthraquinones (e.g. doxorubicin), whose flat aromatic
y = 3343.9x + 1.0132
1.5
R² = 0.9964
1.4
1.3
1.2
1.1
1.0
anthraquinone core allows intercalation between the DNA
1c]
bases,[1b,
we evaluated the interaction in vitro between
compound 4 and CT-DNA. Following incubation of CT-DNA
(15 µM) with increasing concentrations of compound 4, UV-
spectra were acquired (Figure 2). The absorption intensity of
CT-DNA decreases (hypochromism) with increasing
concentration of compound, a characteristic of intercalation
usually attributed to the interaction between the electronic
states of anthraquinones and those of DNA nucleobases.
Corroborating this hypothesis, the hypochromism of CT-DNA
in the presence of compound 4 is still observed under higher
ionic strength conditions (SI Figure S3).
[compound 4] (M)
compound 4:CT-DNA:GelRed
120
100
80
0:10:1
0.5:10:1
1:10:1
2:10:1
3:10:1
4:10:1
5:10:1
6:10:1
60
40
20
0
500
550
600
650
700
750
-20
7:10:1
0.82
0.8
Wavelength (nm)
0.78
0.9
0.8
0.7
0.76
0.74
Figure 3. Absorption spectra of the competitive reaction between different
ratios of compound 4 and GelRed bonded to CT-DNA (different lines - ratios
compound 4: CT-DNA: GelRed), GelRed = 20 µM and CT-DNA = 200 µM
Tris-HCl 0.5 mM pH 7.0 with NaCl 50 mM. The black arrow indicates the
quenching effect observed with increasing concentrations of compound 4.
Inset: plot of (I0 /I) versus [Compound 4] to calculate the quenching constant.
0.72
0.7
250 255 260 265 270
0.6
Wavelength (nm)
0.5
0.4
0.3
0.2
0.1
0
0 mM
10 mM
20 mM
30 mM
This is consistent with the combination of compound 4 π
electrons and π electrons of DNA nucleobases with the
consequent decrease of the energy level of the π-π* electron
transition.[14] The calculated intrinsic binding constant Kb is
2.0(± 0.20) x 105 M-1. The Kb values described in the literature
for classical intercalators (e.g. ethidium bromide–DNA, 7 × 107
M−1),[15] are at least 100× higher than that of compound 4.
235
255
275
295
315
335
Wavelength (nm)
Figure 2. Absorption spectra of CT-DNA (15 µM) in the presence of
increasing concentrations of compound 4 (0-30 µM). Inset: maximum
absorbance peak hypochromism due to the increase of compound 4
concentrations – indicated by the black arrow.
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10.1002/ejoc.201701500
European Journal of Organic Chemistry
COMMUNICATION
However, the intrinsic binding constant is 10x higher than that
described for doxorubicin (Kb = 3.2 (±0.23) × 104 M-1).[16]
The binding of compound 4 to CT-DNA as a competitive
intercalative binding probe with GelRed[17] was further
analyzed. In the competitive binding experiments, GelRed was
first incubated with CT-DNA for 30 min to ensure sufficient
binding sites between GelRed and DNA (concentration ratio
was set at [GelRed]/[CT-DNA] = 1:10). The emission spectra
of GelRed–CT-DNA system in the absence and presence of
increasing concentrations of compound 4 are shown in Figure
3. As expected, when excited at 350 nm, the GelRed–CT-DNA
system presented a characteristic fluorescence emission at
around 590 nm, indicating intercalation of GelRed within the
Keywords: imidazolium salt • anthracenecarboxaldehyde •
arylidene anthrone • decarbonylation • DNA intercalation
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adjacent nucleobases. The presence of compound
4
considerably quenched the GelRed’s fluorescence emission
(Figure 3 – indicated by the black arrow) with no observed
saturation state until a [compound 4]/[GelRed] ratio of 7. The
quenching constant (KSV) for compound 4 bound to GelRed-
DNA system was determined 3.3(±0.3) x 103 M-1 by Stern-
Volmer equation.
Circular dichroism (CD) was also used to clarify the
nature of the interactions between compound 4 and CT-DNA,
i.e. electrostatic interaction or minor groove binding or
intercalation.[18] Results showed a modification on both positive
and negative bands of the B-form of CT-DNA when compound
4 is added (SI Figure S4), which corroborates that compound
4 intercalates within adjacent nucleobases.
Here we report for the first time an unusual hetero
arylidene-9(10H)-anthrone salt (4). Its structure was
unequivocally attributed by X-ray crystallography. A detailed
mechanistic study was carried out to provide insights to the
unexpected reactivity of the imidazolium salt and its oxidative
role. Alongside decarbonylation and the involvement of
endoperoxide species are proposed in the pathway for the
formation of the anthrone derivative 4. Compound 4 showed
no cytotoxicity in human normal healthy fibroblasts or in two
different cancer cell models, indicating it might be suitable for
several applications in biological systems. Compound 4
strongly interacts with CT-DNA by intercalation (Kb = 2.0(±
0.20) x 105 M-1), and is able to displace GelRed from the DNA
strands, thus quenching fluorescence of GelRed-CT-DNA
system (KSV=3.3 ±0.3 x 103 M-1).
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Acknowledgements
This work was supported by the LAQV, which is financed by
national funds from FCT/MEC(UID/QUI/50006/2013) and co-
financed by the ERDF under the PT2020 Partnership
Agreement (POCI-01-0145- 710-FEDER-007265), and by
UCIBIO
by
national
funds
from
FCT/MEC
(UID/Multi/04378/2013) and co-financed by the ERDF under
the PT2020 Partnership Agreement (POCI-01-0145-FEDER-
007728). The National NMR Facility and the SCXRD facility are
supported by Fundação para
a Ciência e Tecnologia
(RECI/BBB-BQB/0230/2012 and RECI/BBB-BEP/0124/2012,
respectively). We thank Dr. R. Pandian for the crystallographic
data collection. We acknowledge the Laboratório de Análises
REQUIMTE for the technical support for the mass
spectrometry analyses.
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European Journal of Organic Chemistry
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Entry for the Table of Contents (Please
choose one layout)
Layout 1:
COMMUNICATION
Synthesis, cytotoxicity evaluation in
human cells lines and in vitro DNA
interaction of a hetero arylidene-
9(10H)-anthrone
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
Daniela Peixoto,† Margarida
Cytotoxicity
evaluation
Mechanism
elucidation
Figueiredo,† Gabriela Malta,† Catarina
Roma-Rodrigues,‡ Pedro V. Baptista,‡
Alexandra R. Fernandes,‡ Sónia
Barroso,‡ Carlos A. M. Afonso,§ Luisa
M. Ferreira,*,† and Paula S. Branco*,†
In vitro DNA
interaction
X-ray
Crystallography
compound 4:CT-DNA:GelRed
120
0:10:1
0.5:10:1
1:10:1
2:10:1
3:10:1
4:10:1
5:10:1
6:10:1
7:10:1
100
80
60
40
20
0
500
550
600
650
700
750
-20
Wavelength (nm)
Binding affinity to CT-DNA is 10x
higher than that described for
doxorubicin
A hetero arylidene-9(10H)-anthrone structure
(4) was unexpectedly isolated on reaction of
imidazolium salt (2) and 9-
anthracenecarboxaldehyde (3) which revealed
no cytotoxicity and was able to interact with
CT-DNA by intercalation. Its structure was
unequivocally attributed by X-ray
crystallography. Decarbonylation and the
involvement of endoperoxide species are
proposed on mechanistic studies.
Key Topic: N-Heterocyclic olefin reactivity
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