Novel fluorescent lapachone-based BODIPY: Synthesis, computational and electrochemical aspects, and subcellular localisation of a potent antitumour hybrid quinone

Article abstract of DOI:10.1039/c6cc07054j

For the first time, a fluorescent lapachone-based BODIPY was synthesised and characterised by NMR and mass spectrometry. Computational and electrochemical aspects, as well as cytotoxic activity and subcellular localisation, were studied. Confocal microscopy experiments indicated that the probe was a specific mitochondria-staining agent. These in-detail analyses were useful in understanding the cytotoxic effects and mechanism of action of this novel hybrid compound. This molecule constitutes a promising prototype owing to its potential biological activities and the new strategies aimed at mechanistic investigations in cells and in vivo, and opens up an interesting avenue of research.

Full text of DOI:10.1039/c6cc07054j

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J. Corrêa, F. D. S. Emery and E. Silva Junior, Chem. Commun., 2016, DOI: 10.1039/C6CC07054J.
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Novel fluorescent lapachone-based BODIPY: Synthesis,
computational and electrochemical aspects, and subcellular
localisation of a potent antitumour hybrid quinone†
Received 00th January 20xx,
Accepted 00th January 20xx
Talita B. Gontijo,a Rossimiriam P. de Freitas,a Guilherme F. de Lima,a Lucas C. D. de Rezende,b
Leandro F. Pedrosa,c Thaissa L. Silva,d Marilia O. F. Goulart,d Bruno C. Cavalcanti,e Claudia
Pessoa,e,f Marina P. Bruno,g José R. Correia,g Flavio S. Emeryb* and Eufrânio N. da Silva Júniora*
DOI: 10.1039/x0xx00000x
www.rsc.org/
For the first time, a fluorescent lapachone-based BODIPY was synthesised and characterised by NMR and mass
spectrometry. Computational and electrochemical aspects, as well as cytotoxic activity and subcellular localisation, were
studied. Confocal microscopy experiments indicated that the probe was a specific mitochondria-staining agent. These in-
detail analyses were useful in understanding the cytotoxic effects and mechanism of action of this novel hybrid compound.
This molecule constitutes a promising prototype owing to the potential biological activities and new strategies aimed at
mechanistic investigations in cells and in vivo, and opens up an interesting avenue of research.
Lapachones have attracted attention within the scientific lower cytotoxicity (i.e., a higher selectivity index).11 The synthesis of
community owing to their biological profiles (Scheme 1A).1 There asymmetric and A-ring-modified quinones are among the great
are examples of lapachone derivatives with trypanocidal,2 challenges associated with quinoidal compounds. Recently, our
leishmanicidal,3 antiviral,4 and antitumour5 activities. β-lapachone group shed light on naphthoquinone chemistry with some examples
(β-lap), one of the most representative compounds of this class, is that overcame these challenges, using Rh-catalysed C-H activation
involved in ongoing clinical trials for several cancers.6
A
reactions and chiral squaramide-catalysed asymmetric synthesis, via
combination of β-lap and docetaxel is currently under evaluation as cascade reactions with Morita-Baylis-Hillman acetates of
a treatment for patients with advanced or metastatic cancer. nitroalkenes (Scheme 1B).12,13 Considering our experience in
Furthermore, phase II clinical trials have been completed for working with lapachone groups, we have identified another
patients with locally advanced, recurrent, or metastatic squamous relevant challenge associated with difficulties in performing
cell cancer of the head and neck.7 The main mechanism of action of bioimaging studies, in both chemical biology or medicinal
β-lap is related to the generation of reactive oxygen species (ROS) chemistry, owing to the lack of fluorescence of fluorophore-labelled
and destruction of cancer cells with elevated endogenous levels of quinones.
NAD(P)H:quinone oxidoreductase 1 (NQO1).8 Recently, Ohayon and
coworkers discussed the hypothesis of β-lap being able to act as an coumarin-naphthoquinones (Scheme 1C).14 The authors observed
irreversible inhibitor of deubiquitinases.9
and discussed, based on experimental and computational evidence,
Vargas and co-workers attempted to obtain fluorescent hybrid
Recently, our group has reported important advances related to that the strong π-electron acceptor character of the
the synthesis and discovery of novel antitumour lapachones.10 We naphthoquinone moiety was able to quench the fluorescence of the
have conducted strategies such as redox centre modulation and hybrid compounds. Recently, we described the synthesis of
A/C-ring modifications for chemical transformations of quinones, lapachone-based 2,1,3-benzothiadiazole-triazole (Scheme 1C), for
aimed at the preparation of compounds with higher activity and the preparation of fluorescent lapachones,15 owing to the
luminescent properties of the 2,1,3-benzothiadiazole (BTD) nucleus,
to be used as bioimaging probes.16
A similar fluorescence
a.Institute of Exact Sciences, Department of Chemistry, Federal University of Minas
Gerais, Belo Horizonte, 31270-901, MG, Brazil. E-mail: eufranio@ufmg.br; Tel: +55
31 34095720
suppression was observed for hybrid quinone-based BTD. We
therefore studied the association of lapachone with fluorescent
boron-dipyrromethene (BODIPY). These boron complexes are
among the most representative and widely used fluorophores in
studies involving imaging of biomolecules in living cells.17 To our
surprise, we obtained the first fluorescent lapachone-based BODIPY
with the photophysical features required for subcellular studies. In
addition, we evaluated the cytotoxic activity of this compound
against 17 cancer cell lines and normal cell lines (PBMC), besides
their electrochemical and computational aspects.
b.Faculty of Pharmaceutical Sciences at Ribeirao Preto, University of São Paulo, CEP
14040-903, Ribeirão Preto, SP, Brazil
c. Institute of Exact Sciences, Department of Chemistry, Fluminense Federal
University, CEP 27213-145, Volta Redonda, RJ, Brazil
d.Institute of Chemistry and Biotechnology, Federal University of Alagoas, CEP
57072-970, Maceió, AL, Brazil
e. Department of Physiology and Pharmacology, Federal University of Ceará, CEP
60180-900, Fortaleza, CE, Brazil
f. Fiocruz-Ceará, CEP 60180-900, Fortaleza, CE, Brazil
g.Institute of Chemistry, University of Brasília, CEP 70904970, Brasília, DF, Brazil
†Electronic supplementary informaꢀon (ESI) available with full descriptions of all
experimental procedures, the synthesis and characterisation of lapachone-based
BODIPY 7 and all intermediates, NMR and HRMS spectra, photophysical
parameters, cellular studies, computational and electrochemical details.
This journal is © The Royal Society of Chemistry 2016
Chem. Commun., 2016, 00, 1-3 | 1
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Scheme 2. Synthesis of lapachone-based BODIPY 5.
Photophysical properties of dye 5 were evaluated by means of
absorption and emission spectra (see Supplementary Information).
The obtained data is summarised in Table S1. Compound 5
presented good stability and fluorescence suitable for further
imaging studies. Our strategy was efficient since the quinone
moiety was not capable of quenching the intrinsic fluorescence of
the BODIPY portion.
To evaluate the stabilising effect of the molecular architecture on
the stability of lapachone-based BODIPY (5), computational studies
were performed to determine the contour plots of the frontier
orbitals (HOMOs and LUMOs), which are important features related
to the fluorescence presented by 5. The structure of 5 was fully
optimised at the B3LYP21,22/TZVP level of theory using the RI
approach and the dispersion correction proposed by Grimme et al.
(D3).23 The convergence criteria was set to 10–8 Hartree in the SCF
calculation and 10–6 Hartree in the geometry optimisation.
Vibrational analyses were performed to confirm that the optimised
structure was a minimum in the potential energy surface. All
calculations were carried out in Orca 3.0.2.24 The frontier orbitals
were plotted using an isovalue of 0.008. Our results indicated that
the HOMO was concentrated on the BODIPY moiety of the probe,
whereas the LUMO was centred on the quinoidal moiety. HOMO-1
and HOMO-2 were located over both moieties (Figure S31). This
was an important characteristic of this new lapachone-based
BODIPY hybrid, and could explain the lack of quenching, which was
different to that observed in other quinones coupled with
fluorescent compounds.14,15
Scheme 1. Overview.
The synthesis of the naphthoquinoidal probe was accomplished
by a convergent synthetic route, using a classical click chemistry
reaction18 to join the quinone and BODIPY moieties. Initially, we
prepared 3-azido-nor-β-lapachone (4) from lapachol in four steps
with good to excellent yields, by a well-established methodology
developed by our group (Scheme S1, SI). This method allowed us to
access a potent antitumour clickable azido-lapachone prototype.
Meso-(2,6-dichlorophenyl)-3-chloro BODIPY (2) was synthesised
from 1, according to a previously published method.19 Compound 2
was selected owing to the sterically hindered rotation of the meso-
(2,6-dichlorophenyl) group, which is associated with high
fluorescence quantum yields. The 3-halogenated position in
compound 2 is known to be very reactive towards nucleophiles20
and, after a nucleophilic substitution reaction with 2.5 equivalents
of propargylamine in acetonitrile at room temperature, desired
BODIPY 3 was isolated in 65% yield (Scheme 2A). The wavelength of
the absorption peak of BODIPY 3 in THF appeared at 498 nm, while
the fluorescence emission peak was seen at 528 nm. As expected,
the fluorescence quantum yield of 3 was high, at around 0.71,
which enabled its application as a fluorescent probe. It is worth
noting that the fluorescence quantum yields of meso-phenyl-3-
amino BODIPY published in the literature are considerably lower,
usually under 0.13. This observation reiterates the benefits of
working with 2,6-disubstituted meso-BODIPY 3, for the synthesis of
fluorescent probes.
The electrochemical profile of 5 was also investigated by cyclic
voltammetry, as presented in Figure 1. Compound 5 was composed
of two non-conjugated main reducible systems, ortho-dihydrofuran-
naphthoquinone and boron-dipyrromethene. Reduction was
represented by two main redox systems (Ic and IIIc) and some
shoulders (a broad IIc). The oxidation counterpart had two main
waves (Ia and IIIa), with an ill-defined IIa. The first system (Ic/Ia) was
well-defined, diffusional, and had
a quasi-reversible electron
transfer nature, at potentials of -0.535 V and -0.407 V, while the
second was ill-defined and better visualised at higher scan rates (1
V s–1) (IIc/IIa). This was representative of reduction of the quinone
system, where disproportionation occurs, at slow scan rates,
leading to a hydroquinone dianion (red). The other reduction wave
(IIIc), at -1.901 V (blue) was related to reduction of the heterocyclic
BODIPY system and led to an aromatic ring and a very stabilised
Following our strategy, and with azido-quinone 4 and alkyne
BODIPY 3 in hand, we easily prepared, for the first time, fluorescent
lapachone-based BODIPY (5) in a good 79% yield (Scheme 2B).
2
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anion, which was further oxidised at potential IIIa (–0.009 V), as Therefore, 5 was less cytotoxic against normal cells than against the
cancer cell lines evaluated (Table S2). The selectivity indexes
observed for 5 were close to those found for doxorubicin, the
positive control, against several lineages. Doxorubicin is used
clinically and is considered one of the most potent antitumour
quinones.
The known ability of the quinoidal redox centre of lapachones
to generate reactive oxygen species (ROS) is intrinsically associated
with cell death. Therefore, as expected, the current preliminary
studies of cytotoxicity mechanisms of action for 5 showed the
generation of ROS as an important characteristic of cell toxicity.27 In
order to clarify the ROS contributions to cellular toxicity, we
evaluated the content of endogenous non-enzymatic antioxidant
reduced glutathione (GSH) and the levels of lipid peroxidation
(TBARS assay). Our data, as seen in Table S3, regarding intracellular
GSH content and lipid peroxidation (MDA production), using MX-1
shown by the inversion of potentials (Eλ ~2.0 V). Investigation of
BODIPY system 3, helped to clarify the mechanism (SI, Figure S30).
Compound 3 was also investigated through cyclic voltammetry. Its
cyclic voltammograms displayed two cathodic and one anodic wave.
The first wave (Ic/Ia) was quasi-reversible, whereas the second was
irreversible, as seen for other derivatives.25 This was an interesting
and complex redox system, as suggested by Scheme 3, and
corroborates the computational studies, with systems Ic/Ia and
IIc/IIa related to quinone reduction (LUMO) and IIIc related to
LUMO +1. The oxidation profile was more complex and also
corroborated the computational studies, with the benzylic double
bond, the oxidisable region, generating a benzylic cation.
10
10
IIIa
IIIa
Ia
Ia
A
B
5
5
IIa
IIa
0
0
breast cancer cells as
a cellular model, showed a positive
-5
-5
IIc
IIc
Ic
correlation between cytotoxicity and ROS formation after 24 h
exposure at ~7 and 15 µM, in a concentration-dependent manner (p
<0.05 compared with vehicle control).
Ic
-10
-15
-20
-10
-15
-20
IIIc
IIIc
Finally, we studied the potential of the fluorescent compound 5
as a probe in subcellular localisation studies. Compound 5 produced
an intense dual fluorescent signal with green and red fluorescence
emissions; the best emission results were obtained at wavelengths
of 520–560 nm (green). No photobleaching was detected under
standard operational conditions during the image acquisition
process. The fluorescence emission was also stable during 12 h of
photobleaching analyses (Figure S22). Compound 5 showed a
distinctive pattern of fluorescence distribution in cell cytoplasm.
This molecule produced an intense fluorescent signal, accumulated
near to the cell nuclei, in a sharply defined region (Figures 3A and
3B, and S23–S25). This fluorescent pattern was consistent in all
assays and might demonstrate mitochondria-specific staining. No
fluorescent staining was detected inside the cell nuclei, which were
represented as black voids inside the cells. Although this compound
had dimensions that allowed passive traffic through the nuclear
pores, no nuclei staining was detected. This result indicated no
affinity between compound 5 and nuclear chemical constituents.
Notably, the fluorescence emissions obtained in living cells for all
samples were much more intense than those obtained in fixed cell
samples (Figure 3A). This indicates an association between the
-2.5 -2.0 -1.5 -1.0 -0.5
0.0
0.5
-2.5 -2.0 -1.5 -1.0 -0.5
0.0
0.5
E / V vs. Ag|AgCl|Cl- (sat.)
E / V vs. Ag|AgCl|Cl- (sat.)
Figure 1. Cyclic voltammetry (CV) of 5 (1 mM) in DMF + TBAPF6 (0.1
M), glassy carbon electrode, cathodic direction, potential range: 0.5
V up to –2.5 V, ν = 0.1 V s–1. A) Successive CVs (red arrows:
reduction of the quinone function; blue arrows: reduction of the
BODIPY system and oxidation of the electroreduced product). B)
Several inversion potentials in the CV of 5 (Eλ).
Scheme 3. Proposal for the reduction of 5. Quinone reduction
related to waves Ic and IIc is shown in red, and reduction of the
BODIPY system, occurring in IIIc, leading to the oxidation wave IIIa,
is shown in blue.
photon emission of
mitochondria.
5 and the activity or product in the
The fluorescent signal pattern observed in mammal cells for β-
lap derivative 5 was identical to the fluorescent pattern obtained
from the Mitotracker staining assay. Both markers were
accumulated near the cell nuclei and slightly distributed in the cell
cytoplasm, representing the typical mitochondria distribution in this
line cell model. The results obtained by Mitotracker cell staining
Lapachone-based BODIPY (5) was evaluated in vitro by MTT
assay (IC50 µM, CI 95%) for 17 cancer cell lines. We separated cancer
cell lineages into seven types of cancer: prostate, colon, leukaemia,
glioblastome, breast, melanoma, and lung. As previously
described,26 compound 5 was classified, according to its potency
against cancer cells, as highly active (IC50 < 2 µM), moderately active
(2 µM < IC50 < 10 µM), or inactive (IC50 > 10 µM). Lapachone 5 was
highly active against prostate, colon, and leukaemia cancer cell
lines. Leukaemia cells are more sensitive to 5 than prostate and
colon lineages, with IC50 values in the range 0.34-0.37 µM. In
comparison with previously described lapachones,10 compound 5
presented similar activity to compounds without the BODIPY moiety
and luminescence. The results of cytotoxicity against breast,
glioblastome, melanoma, and lung cancer cell lines showed that 5
was moderately active against these lineages, with IC50 values in the
range 1.71–2.17 µM. We also evaluated 5 against normal peripheral
blood mononuclear cells (PBMC), which gave an IC50 of 3.26 µM.
confirmed our previous findings, that compound
mitochondria. Figures 3B (Images A and C) show the cell cytoplasm
fluorescence distribution when stained with (Green) and
5 stains
5
Mitotracker (red), respectively. It was not possible to perform
fluorescence channel overlay analyses because of the dual
wavelength emission of compound 5. The arrows indicate the
lapachone region of accumulation in the cell cytoplasm, which was
precisely the same as observed for Mitotracker staining. These
results demonstrated that compound
5 appeared as a new
mitochondria marker permeable to the cell membrane. Phase
contrast images for all samples were also generated in order to
verify the cells morphological aspects, and no morphological
alteration was observed in all cell models used in this work.
This journal is © The Royal Society of Chemistry 2016
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B and D show the normal
In order to compare lapachone-based BODIPY (5) and non-
fluorescent lapachones, we studied the computational and
electrochemical characteristics, besides biological activities and
subcellular localization (see SI for complete data) of β-lapachone,
nor-β-lapachone and 3-azido-nor-β-lapachone (4). As expected
compound 5 presented similar behaviour as observed for the
precursor lapachones plus the fluorescence feature arising from
BODIPY moiety.
To the best of our knowledge, the present study reports the
first synthesis of a fluorescent lapachone-based BODIPY, which
presents potent antitumour activity. Aspects related to their
mechanism of action and subcellular localisation were also studied
in detail. In broader terms, the strategy adopted here opens a new
avenue for biological studies involving lapachones. With the present
investigation, we could overcome the drawback related to
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