On the synthesis of quinone-based BODIPY hybrids: New insights on antitumor activity and mechanism of action in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2017.08.007

Fluorescent quinone-based BODIPY hybrids were synthesised and characterised by NMR analysis and mass spectrometry. We measured their cytotoxic activity against cancer and normal cell lines, performed mechanistic studies by lipid peroxidation and determina

Full text of DOI:10.1016/j.bmcl.2017.08.007

Accepted Manuscript
On the Synthesis of Quinone-based BODIPY Hybrids: New Insights on Anti-
tumor Activity and Mechanism of Action in Cancer Cells
Talita B. Gontijo, Rossimiriam P. de Freitas, Flavio S. Emery, Leandro F.
Pedrosa, José B. Vieira Neto, Bruno C. Cavalcanti, Claudia Pessoa, Aaron King,
Fabio de Moliner, Marc Vendrell, Eufrânio N. da Silva Júnior
PII:
S0960-894X(17)30794-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.007
BMCL 25201
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
2 August 2017
3 August 2017
Please cite this article as: Gontijo, T.B., de Freitas, R.P., Emery, F.S., Pedrosa, L.F., Vieira Neto, J.B., Cavalcanti,
B.C., Pessoa, C., King, A., de Moliner, F., Vendrell, M., da Silva Júnior, E.N., On the Synthesis of Quinone-based
BODIPY Hybrids: New Insights on Antitumor Activity and Mechanism of Action in Cancer Cells, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.007
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On the Synthesis of Quinone-based BODIPY Hybrids: New Insights on
Antitumor Activity and Mechanism of Action in Cancer Cells
Talita B. Gontijo,a Rossimiriam P. de Freitas,a Flavio S. Emery,b Leandro F. Pedrosa,c
José B. Vieira Neto,d Bruno C. Cavalcanti,d Claudia Pessoa,d,e Aaron King,f Fabio de
Moliner,f Marc Vendrellf* and Eufrânio N. da Silva Júniora*
aInstitute 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; bFaculty of Pharmaceutical Sciences at Ribeirao Preto, University of São
Paulo, CEP 14040-903, Ribeirão Preto, SP, Brazil; cInstitute of Exact Sciences,
Department of Chemistry, Fluminense Federal University, CEP 27213-145, Volta
Redonda, RJ, Brazil; dDepartment of Physiology and Pharmacology, Federal
University of Ceará, CEP 60180-900, Fortaleza, CE, Brazil; eFiocruz-Ceará, CEP
60180-900, Fortaleza, CE, Brazil; fMRC/UoE Centre for Inflammation Research, The
University of Edinburgh, EH16 4TJ Edinburgh, United Kingdom. E-mail:
mvendrel@staffmail.ed.ac.uk; Tel: +44 (0)131 242 6685. Webpage:
www.dynafluors.co.uk
Abstract: Fluorescent quinone-based BODIPY hybrids were synthesised and
characterised by NMR analysis and mass spectrometry. We measured their cytotoxic
activity against cancer and normal cell lines, performed mechanistic studies by lipid
peroxidation and determination of reduced (GSH) and oxidized (GSSG) glutathione,
and imaged their subcellular localisation by confocal microscopy. Cell imaging
experiments indicated that nor-β-lapachone-based BODIPY derivatives might
preferentially localise in the lysosomes of cancer cells. These results assert the potential
of hybrid quinone-BODIPY derivatives as promising prototypes in the search of new
potent lapachone antitumor drugs.
Keywords: Quinone; BODIPY; Lapachone; Cancer; Subcellular localization.
3

1. Introduction
Over the years, medicinal chemists have been improving several chemical
strategies for the discovery of new drugs.1 For example, enzymatic targets have been
used in molecular docking studies for computational drug design to predict the chemical
structure of potential inhibitors.2 Looking for new directions, late stage
functionalization of drug-like molecules arises as a modern and elegant strategy in drug
discovery programs, as recently reviewed by Cernak and coworkers.3 Another strategy
stems from the inspiration of natural products. Compounds isolated from diverse natural
sources can be used as starting points to generate novel structures with varied biological
activities.4 As discussed by Newman and Cragg,5 natural products still have a major role
in the design of new antitumor drugs.
The search for antitumor drugs is a big challenge with multiple questions.6 Some
of the key questions are associated to the mechanism of action of cytotoxic drugs and
the subcellular organelles where drugs preferentially accumulate. These questions can
be generally answered by bioimaging experiments7 as well as in vitro biological assays.8
Lapachones are naturally occurring naphthoquinones and among the most
studied quinones due their potent antitumor activity.9 Lately, diverse lapachone
derivatives have been reported as potent cytotoxic drugs against different cancer cell
lines.10 In this regard, advances in the synthesis of lapachones with potent antitumor
activity have been accomplished via modification of the A- and C-rings,11 with recent
progress being achieved by da Silva Júnior,12 Pinto,13 Hong,14 Ferreira,15 Bonifazi,16
among others17 (Scheme 1A).
4

As studied by the Boothman group, the mechanism of action of most β-
lapachones is related to the destruction of cancer cells with elevated levels of
NAD(P)H:quinone oxidoreductase 1 (NQO1).18,19,20 Recently, Ohayon and coworkers21
have shed some light on the possibility that β-lapachones might act non-reversibly as
inhibitors of deubiquitinases. The therapeutic effect of β-lapachones could be also
related to the oxidation of ubiquitin specific peptidase 2 (USP2), as a likely downstream
effect of reactive oxygen species (ROS) generation.
In this context, unraveling the mechanism of action of antitumor lapachone
derivatives is a recurring challenge for the scientific community. In the last few years,
our group has dedicated great efforts to prepare fluorescent lapachones by hybridization
of the quinoidal moiety with fluorescent compounds, such as the benzothiadiazole
structure (Scheme 1B).22 Fluorescent lapachones allow us to answer critical questions
related to their mechanism of pharmacological action, including subcellular localization
studies in live cells.23 Recently, our group has described the first fluorescent lapachone-
BODIPY hybrid as well as biological studies, including cytotoxic activity in different
cancer cell lines and cell imaging experiments (Scheme 1B).24
Herein, we have developed a new chemical platform to prepare new lapachone
derivatives coupled to the fluorescent BODIPY core, which has excellent photophysical
properties.24b Lapachones exhibit potent antitumor activity due to their ability to act on
multiple targets, as we have recently demonstrated.25 This family of quinoidal
derivatives can be divided into α-lapachones (para-quinones) and β-lapachones (ortho-
quinones). We have demonstrated that both α- and β-lapachones display cytotoxic
activity in cancer cells.26 In the present work, we have synthesized a small collection of
α- and β-lapachone derivatives (Scheme 1C) and evaluated their cytotoxicity in human
5

cancer and non-cancerous cell lines. Furthermore, we have also prepared fluorescent
BODIPY-lapachones to examine the subcellular localization of lapachone-based
compounds in cancer cells and any derived effects in their cytotoxic activity.
(A) Biologically significant lapachone derivatives:
O
O
O
O
O
O
O
O2N
O
O
O
O
A
O
H
N
N
N
A
I
N
N
C
C
N
C
O
O
O
Br
OH
I
(B) Recent attempts to prepare quinone-based luminescent hybrid compounds:
O
O
Cl
Cl
N
Benzothiadiazole
N
N
N3
F
F
(BTD) and BODIPY
fluorophores with
recognized
B
NH
or
O
N
N
N
H
luminescent properties
S
Click Reaction
O
Neto and
da Silva Júnior (ref. 22)
N
O
Emery and
NH
N
N
Non-Fluorescent
Lapachone-Based BTD
Strategy has failed
da Silva Júnior (ref. 24a)
N
H
N
O
N
O
O
Cl
N
S
F
N
B
F
N
N
N
N
Fluorescent
Lapachone-Based BODIPY
O
Cl
(C) This Work: New quinone-based BODIPY hybrids and their antitumor, mechanism of action and subcellular localization studies
O
O
O
O
N
N
O
N
N
N
N
H
N
O
O
O
O
N
15
F
O
O
N
B
Cl
N
11
B
n
Cl
F
F
F
N
O
O
N
F
N
F
F
N
B
N
H
N
Cl
F
N
B
8, n = 1
9, n = 2
N
N
H
N
N
N
N
10
Scheme 1. Overview of lapachone derivatives and the design of quinone-based BODIPY
hybrids.
6

2. Results and discussion
The synthesis of the quinone-based BODIPY hybrids 8-11 was accomplished by
a convergent synthetic route,24 using a classical copper(I)-catalyzed alkyne-azide
cycloaddition (CuAAC) reaction.27 We used an alkyne-containing BODIPY and azide-
containing quinones to assemble fluorescent, hybrid quinoidal-BODIPY molecules. We
started the preparation of quinone-containing BODIPYs by the synthesis of boron-
dipyrromethene with a terminal alkyne for subsequent CuAAC reaction. There are
numerous reports on synthetic approaches to generate BODIPY scaffold with bespoke
spectral properties.28 Among them, we used the methodology from the Dehaen group
with minor modifications30 for preparing compound 3. With regards to the azides 4-7,
we initially prepared 3-azido-nor-α-lapachone (4) and 4-azido-α-lapachone (5) from
lapachol. The methodology for the synthesis of 4 and 5 is well described in the
literature.31,32 Cyclic lapachones were obtained by insertion of bromine atoms and
nucleophilic substitution with sodium azide to afford the respective clickable analogues
4 and 5.
The synthesis of azides 6 and 7 was conducted following methodologies
described by our group.25,26,33 Compound 6 was prepared from C-allyl lawsone upon
reaction with iodine to obtain the cyclized product, which underwent nucleophilic
substitution with sodium azide. Finally, the last clickable derivative 7 was obtained
from nor-lapachol, previously synthesized from lapachol, by the well-established
Hooker oxidation method.34 Nor-lapachol was transformed into the key intermediate 3-
bromo-nor-β-lapachone, which was reacted with sodium azide to render 3-azide-nor-β-
lapachone (7). Once the azido-quinones 4-7 and the alkyne BODIPY 3 were obtained,
7

we readily prepared a small collection of quinone-based BODIPY hybrids (8-11) in
good yields (Scheme 2).
Scheme 2. Synthesis of quinone-based BODIPY hybrids 8-11.
Lapachone-based BODIPYs 8-11 were evaluated for in vitro cytotoxicity using
an MTT assay in five human cancer cell lines (i.e. PC3, SF295, MDA-MB435, SW620
and HCT-116) and two murine non-cancer (L929 and V79) cells. As previously
reported,35 compounds 8-11 were classified according to their cytotoxicity against
cancer cells as highly active (IC50 < 2 µM), moderately active (2 µM < IC50 < 10 µM),
or inactive (IC50 > 10 µM). As shown in Table 1, no active para-quinones (8-10) were
8

identified. We have reported α-lapachone derivatives with potent cytotoxicity against
cancer cells prepared by C-ring modification and insertion of arylamino groups at
positions C-3 (nor-α-lap) and C-4 (α-lap).36 In fact, molecular hybridization37 between
α-lapachones and 1,2,3-triazole groups has been reported as an efficient strategy for
antitumour compounds.22 Herein, the same approach rendered hybrids compounds (8-
10) that were not active against any the tested cancer cell lines, with IC50 values round 8
µM for all compounds.
On the other hand, compound 11 exhibited significant cytotoxicity against all
human cancer cell lines with IC50 values in the low micromolar range (i.e. 0.3 to 0.9
µM, Table 1). This observation is in line with previous reports of ortho-quinones (i.e. β-
lapachone) being more active than the corresponding para-quinones, such as α-
lapachone.38 Moreover, Abreu and collaborators39 have shown that the radicals
generated during the reduction of β-lapachones reduction confer more stability to
reduced ortho-quinone derivatives when compared to their reduced para-isomers. As a
result, ortho-quinones, such as the hybrid derivative 11, might present better redox
cycling ability, which is the ability of a molecule to undergo repeated reduction and
oxidation, producing more free radicals than compounds 8-10 (para-quinones), which is
in agreement with the absence of cytotoxicity determined for 8-10.
Table 1. Cytotoxic activity expressed by IC50 (µM, 95% CI) of compounds 8-11 in
cancera and normal cellsb after 72 h. Values obtained by nonlinear regression from three
independent experiments and compared to the antitumor drug doxorubicin (DOXO).
Compd PC3
> 8.10
SF295
> 8.10
MDA-MB435
> 8.10
8
9

> 7.83
> 8.19
0.38
> 7.83
> 8.19
0.28
> 7.83
> 8.19
0.81
9
10
11
(0.35-0.41) (0.17-0.40) (0.67-0.91)
0.05 0.45 0.94
(0.02-0.07) (0.41-0.49) (0.89-0.98)
DOXO
Compd
SW620
HCT-116
L929
V79
> 8.10
> 7.83
> 8.19
0.68
> 8.10
> 7.83
> 8.19
0.32
> 8.10
> 7.83
> 8.19
0.92
> 8.10
> 7.83
> 8.19
0.65
8
9
10
11
(0.60-0.75) (0.25-0.38) (0.75-1.00) (0.51-0.72)
0.29 0.21 0.22 0.25
(0.26-0.37) (0.18-0.23) (0.19-0.24) (0.21-0.28)
DOXO
aPC3 prostate carcinoma, SF295 glioblastoma, MDA-MB435 melanoma, and SW620
and HCT-116 colon carcinomas cell lines. bChinese hamster V79 and mouse L929 cells.
The lapachone derivative 11 presented high activity in all the cancer cell lines
evaluated, showing the strongest effect in human glioblastoma cells (SF295) with an
IC50 value around 300 nM. This result represents an improvement in the potency of
previously described lapachone-based BODIPY derivatives.24 The results against non-
tumor cells showed that 11 presented IC50 values in V79 and L929 cells in the range
0.92-0.65 µM. These results indicate that 11 was slightly less cytotoxic against non-
tumor cells, with selectivity indexes (i.e. ratio of cytotoxicities between cancer and
normal cells) around 3. Whereas further optimization of the lapachone structure would
be needed to enhance these selectivity indexes, these values are in line with those of
doxorubicin, a clinically used antitumor drug and one of the most potent and important
antitumor quinones.
10

Quinones are highly redox active molecules leading to formation of reactive
oxygen species (ROS), which cause oxidative stress by oxidising lipids, proteins and
DNA.40 Lipid peroxidation is a well-established mechanism of cellular damage, and it
can be used as an intracellular indicator of oxidative stress. The measurement of
malondialdehyde (MDA), which is one of the most abundant products from lipid
peroxidation, is a highly sensitive assay to quantitatively evaluate the formation of lipid
peroxides in cells. We determined the levels of MDA in three cancer cell lines (prostate,
glioma and colon) and one non-tumor cell line (murine fibroblast) 24 h after exposure
of compound 11, and observed that the levels of MDA in all cancer cells as well as
normal fibroblasts evaluated were significantly higher than in the controls (Figure 1). In
addition, when comparing the equal concentrations of compound 11 between cell lines
used in this current study, there is no more sensitive strain than another in relation to the
extent of byproduct of lipid peroxidation, thiobarbituric acid reactive substances
(TBARS) formation. Moreover, the pretreatment of the cells with N-acetylcysteine
(NAC) showed a strong protective effect by preventing lipid peroxidation mainly by
promoting GSH synthesis by NAC, and by its ROS scavenger ability41 highlighting the
prooxidant mechanism linked to cancer and non-cancer cells death. The role of ROS as
an important colaborative factor in cell death was well correlated by variation of thiols
content after tested compound exposure. Oxidative stress burden usually correlates with
cellular thiol levels or vice versa cellular thiol/disulfide ratio is a well-accepted indicator
of the redox state of a cell.42 Naphthoquinones with higher redox potential showed
stronger cytotoxicity, presumably because of stronger electrophilicity against thiols and
because their GSH conjugates are more readily reduced to semiquinones which activate
oxygen.43 To determine whether oxidative stress induced by compound 11 is
accompanied by changes in total glutathione, the reduced (GSH), oxidized glutathione
11

(GSSG) and GSH/GSSG ratio were measured. After 24 h exposure, compound 11
significantly reduced (p<0.05) the total content of glutathione of all cell cultures (Table
2) The decrease in total amount of glutathione after compound 11 exposure was
accompanied by an increase in GSSG levels (p<0.05) in relation to non-treated cultures.
Due to observed compound 11 exposure effects on both reduced and oxidized
glutathione the ratio of GSH/GSSG was decreased. On the other hand, in all cultures
pre-exposed to NAC before quinone treatment the level of total glutathione was kept
nearly or slightly above at the control value.
Figure 1. Determination of MDA levels (TBARS assay) in human cancer cells (PC3,
SF295 and HCT-116) and in non-cancer mouse fibroblasts (L929) after 24 h-incubation
with compound 11 or 10 µM H2O2 (positive control) in the presence or absence of NAC
12

(5 mM). *p < 0.05 as compared to control by ANOVA followed by Tukey's test. Values
as means s.e.m. for three independent experiments in triplicate.
However, the neutralization of ROS by pretreatment with NAC suggests that an
alternative mechanism different from ROS generation might be involved in the
cytotoxic effect of compound 11, as stated in Table 3, in co-treated (GSH-OEt +
naphthoquinones) cancer and non-cancer cells, GSH-OEt only reduced the sensitivity of
compound 11 exposure but not abrogated it, which pointed that another mechanism
differently of ROS generation capacity may be involved in its cytotoxic effects.
Quinonoid compounds can potentially damage multiple macromolecules in cells, such
as DNA, resulting in single or double strand breaks or the formation of oxidative
nucleobases such as 8-oxo-7,8-dihydroguanine, thymine and uracil glycol.44 In order to
assess this possibility, we performed comet assays, which are consistent with the
reported DNA damage potential attributed to quinonoid molecules.45 Compound 11
caused DNA strand breaks after 24 h exposure in all three cancer cell lines as well as in
murine fibroblasts. In relation to the antioxidant potential of NAC, it is known that
NAC can regulate of oxidative stress related gene expression, having an antagonistic
effect on oxidative injuries.47 However, as shown in Figure 2, NAC pretreatment did not
prevent DNA damage but only resulted in slightly lower levels of fragmented DNA.
Altogether, these results suggest that the high cytotoxicity of compound 11 is not
exclusively limited to the generation of ROS generation. The results of ongoing studies
in our laboratories will be reported in due course.
13

Table 2. Effects of compound 11 on intracellular thiols after 24 h with or without NAC
pretreatment.
Intracellular thiols (µg/mg protein)
GSH
Cell
line
Treatment
Concentration Total
GSSG
GSH/GSSG
PC3
Controla
NAC
11
0.1% DMSO
5 mM
4.23 0.75 3.04 0.51 1.11 0.12 2.69 0.21
4.74 0.50 3.53 0.33 1.18 0.21 2.87 0.55
3.53 0.81* 1.35 0.10* 2.16 0.15* 0.37 0.11*
4.26 0.55 3.13 0.22 1.07 0.15 2.88 0.33
5 µM
11 plus NACb 5 µM
SF295 Control
0.1% DMSO
5 mM
5 µM
4.51 0.83 3.16 0.21 1.31 0.55 2.34 0.56
NAC
11
4.86 0.71 3.81 0.55
1.02 0.10 3.61 0.81
3.44 0.10* 1.31 0.16* 2.09 0.21* 0.39 0.20*
4.72 0.22 3.47 0.55 1.21 0.10 2.82 0.10
11 plus NAC
5 µM
HCT- Control
0.1% DMSO
5.02 0.91 3.74 0.55 1.19 0.37 3.09 0.33
116
NAC
11
5 mM
5 µM
5 µM
5.38 0.45 4.09 0.61 1.23 0.33 3.26 0.21
3.81 0.56* 1.78 0.25* 2.01 0.22* 0.56 0.10*
5.12 0.11 3.71 0.25 1.38 0.33 2.63 0.22
11 plus NAC
L929
Control
NAC
11
0.1% DMSO
5 mM
5 µM
4.87 0.25 3.76 0.51 1.05 0.33 3.55 0.51
5.43 0.81 3.89 0.75 1.52 0.10 2.52 0.33
3.50 0.15* 1.15 0.21* 2.27 0.33* 0.33 0.10*
5.21 0.33 3.89 0.21 1.24 0.25 3.08 0.50
11 plus NAC
5 µM
a Negative controls treated with vehicle (0.1% DMSO); b cells were pretreated for 24 h
with NAC (5 mM); *p < 0.05 as compared to control by ANOVA followed by Tukey's
test. Values as means s.e.m. for three independent experiments in triplicate.
14

Figure 2. Effects of compound 11 on DNA strand breaks index after 24 h of exposure
using alkaline comet assay exposure in the presence or absence of NAC (5 mM). *p <
0.05 as compared to control by ANOVA followed by Tukey's test. Data are presented as
mean values SEMs for three independent experiments in triplicate.
Table 3. IC50 values (µM) for compounds 8-11 after 24 h of exposure in different cells
in the presence or absence of reduced GSH.
Compound GSH-OEt PC3
SF295
> 40
> 40
> 40
> 40
> 40
> 40
> 40
HCT-116
> 40
> 40
> 40
> 40
> 40
> 40
> 40
L929
> 40
> 40
> 40
> 40
> 40
> 40
> 40
+
-
> 40
> 40
> 40
> 40
> 40
> 40
> 40
8
+
-
9
+
-
10
+
15

-
4.47 0.73
1.96 0.21 5.28 0.55
5.91 0.10
11
+
15.83 1.15 9.81 0.10 14.25 2.10 12.41 1.75
a Cell-permeable ethyl ester analogue of reduced GSH (15 mM).
Finally, in order to analyse the subcellular localization studies of the lapachone-
BODIPY hybrid 11, we measured its photophysical properties. However, compound 11
presents a very low fluorescence quantum yield, which limits its capabilities for
fluorescence cell imaging (Table 4 and Figures S16 and S17 in ESI). As reported for
other BODIPY dyes,47b the amino group in the position 5 of the BODIPY core can
cause significant quenching of the fluorophore via photoinduced electron transfer (PeT),
making it unsuitable for imaging studies. Whereas the quenching effect is highly
pronounced in compound 11, the extent of PeT quenching is dependent on the structure
of the quinone-based BODIPY core and the amino substituents. In view of these results,
we synthesized the BODIPY-alkyne 14 as described in the literature and conjugated it
to the quinone 7 by CuAAC reaction to render the hybrid BODIPY-quinone 15.48,49 We
measured the photophysical properties of compound 15, and as expected, determined its
excitation and emission maxima in the green visible range, with high extinction
coefficients and fluorescence quantum yields (Table 4 and Figure S1 in ESI), being a
suitable fluorescent probe for live-cell imaging.
16

Scheme 3. Synthesis of the quinone-based BODIPY derivative 15.
With compound 15 in hands, we tested its cytotoxicity in the same five cancer
cell and non-cancer cell lines. Compound 15 displayed antiproliferative effects only in
PC3 prostate and HCT-116 colon cell lines with IC50 values equal to 2.39 (2.14-2.65)
and 2.85 (2.65-3.07) µM, respectively. For cancer cells (SF295, MDA-MB435 and
SW620) and normal cells (L929 and V79) IC50 values were determined to be > 7.72
µM. Given the differences in cytotoxicity, we assessed the subcellular localization of
compound 15 in PC3 cancer cells. Fluorescence confocal microscopy experiments
revealed the preferential accumulation of 15 in the subcellular lysosomal compartments,
showing strong co-localization with commercially available LysoTracker Red but not
with MitoTracker Red dyes (Figure 3). Under these conditions, compound 11 was not
17

detectable under the fluorescence microscope due to its low quantum yield (Figure S18
in ESI).
Table 4. Photophysical data for compounds 11 and 15.
Compound λabs.(nm) λabs.(nm) ε (M-1cm-1) QYa
495
498
530
511
30,300
68,800
0.03
0.82
11
15
a Determined as relative to fluorescein in basic EtOH (QY: 0.97).49b
Mefloquine, a known antimalarial and antitumoral molecule, has been reported
to accumulate in the subcellular lysosomes of acute myeloid leukemia cells, where it
exhibits high cytotoxicity due to lysosomal disruption.50 This observation led us to
evaluate the compound 15 in 2 leukemia cancer cell lines (HL-60 and Jurkat cells).
Notably, 15 presented high activity in these cell lines with IC50 values in the low
micromolar range [for HL-60 cells, IC50 = 0.92 (0.90-1.05) and for Jurkat cells, IC50 =
0.94 (0.86-0.98) µM]. Furthermore, because compound 15 showed very low
cytotoxicity in non-cancer cells (i.e. L929 and V79), its selectivity index was over 8.5
(i.e. ~7-fold higher than compound 11). Finally, we also assessed the effect of
compound 15 on intracellular thiols in HL-60 and Jurkat cancer cell lines (Table 5).
Remarkably, a weaker protective effect was observed for cells pretreated with NAC,
suggesting a different mechanism of action between both lapachone-BODIPY hybrids.
The ROS-independent cytotoxic effect of compound 15 is in line with the subcellular
localization studies and its potential activity in lysosomal disruption of leukemia cancer
cells.
18

Figure 3. Fluorescence confocal microscopy of PC3 cells upon incubation with
compound 15 as well as Mitotracker Red (top panel) and LysoTracker Red (bottom
panel). Images arranged from left to right as: green fluorescence (15), red fluorescence
(MitoTracker/LysoTracker), merged green/red channels and brightfield images. Scale
bar: 10 µm.
Table 5. Effects of compound 15 on intracellular thiols after 24 h of cell exposure with
or without NAC pretreatment.
Intracelular thiols (µg/mg protein)
GSH
Cell line Treatment
Concentration Total
0.1% DMSO
5 mM
GSSG
GSH/GSSG
HL-60
Controla
NACb
15
3.61 0.22 3.01 0.11 0.66 0.17 4.53 0.55
3.85 0.81 3.03 0.25 0.75 0.90 4.02 0.61
5 µM
3.01
0.44*
2.83
0.71*
0.72
0.20*
1.19
0.10*
2.18
0.10*
1.47
0.33*
0.31 0.05*
0.76 0.10*
15 plus
5 µM
NACc
Jurkat
Controla
NACb
15
0.1% DMSO
5 mM
5 µM
3.87 0.55 3.02 0.21 0.78 0.33 3.81 0.22
4.13 0.25 3.25 0.17 0.81 0.55 3.97 1.17
3.28
0.60*
3.11
0.05*
0.60
0.33*
1.58
0.55*
2.57
0.10*
1.44
0.51*
0.21 0.11*
1.08 0.33*
15 plus
5 µM
NACc
19

a Negative controls treated with vehicle (0.1% DMSO); b cells were pretreated for 24 h
with NAC (5 mM); *p < 0.05 as compared to control by ANOVA followed by Tukey's
test. Values as means s.e.m. for three independent experiments in triplicate.
3. Conclusions
We have synthesized and characterized a small collection of novel quinone-
based BODIPY hybrids of the natural products lapachol and lawsone. All compounds
were evaluated in cancerous and non-cancerous cell lines, and we identified two nor-β-
lapachone hybrids (11 and 15) with potent cytotoxic activity. Mechanistic studies for
both compounds suggest that the action of compound 11 may be related to the
generation of reactive oxygen species whereas the fluorescent lapachone 15 may exert
its cytotoxic action in subcellular lysosomal organelles. This study provides new
structure-activity relationships in the preparation of biologically active lapachone
derivatives as well as new insights in the potential mechanism of action for their
cytotoxic activity.
4. Experimental Section
4.1. Chemistry
Melting points were obtained on Thomas Hoover and are uncorrected. Analytical
grade solvents were used. Column chromatography was performed on silica gel
(SilicaFlash G60 UltraPure 60-200 µm, 60 Å). 1H and 13C NMR were recorded at room
temperature using a Bruker AVANCE DRX400, in the solvents indicated, with TMS as
20

internal reference. Chemical shifts (δ) are given in ppm. Electron-impact mass spectra
(70 eV) were obtained using a VG Autospec apparatus (Micromass, Manchester, UK).
Absorption spectra were obtained on a Varian Cary 100 spectrophotometer at room
temperature in the solvents described above. Fluorescence spectra were obtained on a
Varian Cary Eclipse spectrofluorimeter with a xenon arc lamp as the light source while
using an excitation wavelength (λexc) corresponding to a higher absorption band. In all
experiments, a quartz cuvette was employed with a 1 cm optical path length. Azide
derivatives 4-7 and BODIPY 14 were synthesized as previously reported in the
literature.25,26,30-33 Structures of the novel compounds 3, 8-11 and 15 were determined by
1H and 13C NMR. Electrospray ionization mass spectra were also obtained to confirm
compound identities.
Procedure for the synthesis of BODIPY 3. To a solution of dichlorinated BODIPY
(215 mg, 0.639 mmol) in acetonitrile (15 mL) under stirring at room temperature,
propargylamine (102 µL, 1.6 mmol, 2.5 eq) was added. After 1 hour under stirring at
room temperature TLC control showed full conversion of the starting material. Solvent
was evaporated under reduced pressure and, after purification via silica column
chromatography (C6H14/DCM, 3:2-1:3), the desired product 3 (201 mg, 0.56 mmol,
89% yield) was obtained. 1H NMR (400 MHz, CDCl3) δ: 7.49-7.43 (m, 5H), 6.93 (d,
1H, J = 4 Hz), 6.49 (br, 1H), 6.39 (d, 1H, J = 4 Hz), 6.28 (d, 1H, J = 4 Hz), 6.21 (d, 1H,
J = 4 Hz), 4.17 (dd, 2H, J = 2.4 and 3.6 Hz), 2.38 (t, 1H, J = 2.4 Hz). 13C NMR (100
MHz, CDCl3) δ: 169.3, 135.9, 133.7, 133.2, 132.0, 131.2, 130.3, 129.5, 128.3, 121.6,
113.2, 110.9, 78.8, 34.1. EI/MS (m/z) [M+H]+: 356.0. Calcd for [C18H14BClF2N3]+:
356.0.
21

General procedure to prepare quinone-based BODIPY hybrids 8-11 and 15. In a
round bottom flask, the respective azide derivatives (0.5 mmol), alkyne BODIPYs (0.6
mmol), and 10 mL of CH3CN were added. The reaction mixture was stirred until
complete solubilization of the reagents after which, CuI (10% per mole) was added. The
system was kept under inert atmosphere (Ar) until complete consumption of BODIPY
and monitored by TLC until its completion. The solvent from the crude was evaporated
under reduced pressure and it was purified by column chromatography on silica-gel,
using eluents with an increasing polarity gradient mixture of hexane and ethyl acetate.
Nor-α-lapachone-based BODIPY (8). Compound 8 was obtained as a brown solid
after 18 h of reaction (258 mg, 83% yield); mp 173-175 °C. 1H NMR (400 MHz,
CDCl3) δ: 8.16-8.14 (m, 1H), 8.05-8.03 (m, 1H), 7.75-7.73 (m, 2H), 7.59 (s, 1H), 7.48-
7.44 (m, 5H), 6.86 (d, 1H, J = 4.8 Hz), 6.82 (sl, 1H), 6.35 (d, 1H, J = 3.8 Hz), 6.29 (d,
1H, J = 4.8 Hz), 6.18 (d, 1H, J = 3.8 Hz), 6.00 (s, 1H), 4.73 (d, 2H, J = 6.1 Hz), 1.74 (s,
3H), 1.16 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 180.9, 179.8, 177.8, 144.6, 135.9,
134.8, 135.9, 134.8, 133.6, 133.5, 131.9, 131.7, 130.5, 130.3, 129.3, 128.2, 127.2,
126.8, 126.5, 121.9, 121.1, 118.1, 94.3, 67.8, 40.4, 27.5, 21.0. EI/HRMS (m/z)
[M+Na]+: 647.1551. Calcd for [C32H24BClF2N6O3Na]+: 647.1557.
α-Lapachone-based BODIPY (9). Compound 9 was obtained as a brown solid after 18
h of reaction (229 mg, 72% yield); mp 171-173 °C. 1H NMR (400 MHz, CDCl3) δ:
8.12-8.10 (m, 1H), 7.95-7.93 (m, 1H), 7.69-7.67 (m, 3H), 7.47-7.42 (m, 5H), 6.87 (d,
22

1H, J = 4.4 Hz), 6.81 (sl, 1H), 6.35-6.32 (m, 2H), 6.17 (d, 1H, J = 3.6 Hz), 5.78 (t, 1H, J
= 6.6 Hz), 4.74 (d, 2H, J = 6 Hz), 2.72 (dd, 1H, J = 14.6 and 6.8 Hz), 2.40 (dd, 1H, J =
14.6 and 6.8 Hz), 1.50 (s, 3H), 1.35 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 182.8,
179.3, 161.9, 156.2, 143.8, 135.9, 134.5, 133.7, 133.5, 132.7, 131.9, 131.8, 131.1,
131.3, 129.4, 128.3, 126.7, 126.4, 122.3, 120.9, 115.1, 112.9, 111.2, 79.3, 50.2, 40.5,
39.3, 26.9, 26.2. EI/HRMS (m/z) [M+Na]+: 661.1704. Calcd for
[C33H26BClF2N6O3Na]+: 661.1714.
Nor-α-lapachone-based BODIPY derivative (10). Compound 10 was obtained as a
brown solid after 18 h of reaction (268 mg, 88% yield); mp 165-168 °C. 1H NMR (400
MHz, CDCl3) δ: 7.94 (d, 1H, J = 7.6 Hz), 7.91 (d, 1H, J = 7.6 Hz), 7.84 (s, 1H), 7.61 (t,
1H, J = 7.6 Hz), 7.55 (t, 1H, J = 7.6 Hz), 7.48-7.45 (m, 5H), 7.09 (sl, 1H), 6.85 (d, 1H, J
= 4,8 Hz), 6.41 (d, 1H, J = 4.8 Hz), 6.33 (d, 1H, J = 4 Hz), 6.16 (d, 1H, J = 4.0 Hz),
5.40 (m, 1H), 4.82 (d, 2H, J = 5.2 Hz), 4.78 (d, 2H, J = 6.4 Hz), 3.41-3.34 (m, 1H),
3.04-2.97 (m, 1H). 13C NMR (100 MHz, CDCl3) δ: 181.7, 177.3, 161.9, 159.1, 144.7,
135.9, 134.2, 133.7, 133.4, 133.1, 132.6, 132.2, 131.9, 131.3, 130.3, 129.8, 129.3,
128.3, 126.2, 125.2, 124.2, 123.7, 120.4, 112.8, 111.4, 83.2, 53.0, 40.0, 30.2. EI/HRMS
(m/z) [M+Na]+: 633.1386. Calcd for [C31H22BClF2N6O3Na]+: 633.1401.
Nor-β-lapachone-based BODIPY (11). Compound 11 was obtained as a brown solid
after 48 h of reaction (227 mg, 73% yield); mp 190-193 °C. 1H NMR (400 MHz,
CDCl3) δ: 7.96 (d, 1H, J = 7.2 Hz), 7.83-7.79 (m, 2H), 7.73 (t, 1H, J = 8.9 Hz), 7.65 (t,
1H, J = 7.8 Hz), 7.46-7.42 (m, 5H), 6.89 (sl, 1H), 6.84 (d, 1H, J = 4 Hz), 6.34 (sl, 2H),
23

6.14 (sl, 1H), 6.00 (sl, 1H), 4.74 (d, 2H, J = 4.4 Hz), 1.77 (s, 3H), 1.17 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ: 180.2, 174.9, 171.4, 161.8, 144.3, 136.0, 134.6, 133.7,
133.2, 132.4, 131.9, 131.6, 130.3, 120.8, 129.3, 128.3, 126.8, 125.6, 122.3, 120.5,
112.8, 111.4, 111.4, 95.9, 67.1, 40.4, 27.7, 21.2. EI/HRMS (m/z) [M+Na]+: 647.1521.
Calcd for [C32H24BClF2N6O3Na]+: 647.1557.
Fluorescent nor-β-lapachone-based BODIPY (15). Compound 15 was obtained as a
red solid after 48 h of reaction (226 mg, 70% yield); mp 176-179 °C. 1H NMR (400
MHz, CDCl3) δ: 8.21 (d, 1H, J = 7.2 Hz), 7.83 (t, 1H, J = 7.2 Hz), 7.79-7.73 (m, 2H),
7.64 (s, 1H), 7.18 (d, 2H, J = 8.0 Hz), 7.09 (d, 2H, J = 8.0 Hz), 6.03 (s, 1H), 5.98 (s,
2H), 5.24 (d, 2H, J = 4.0 Hz), 2.56 (s, 6H), 1.80 (s, 3H), 1.41 (s, 6H), 1.22 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ: 180.1, 174.6, 171.4, 158.9, 155.5, 143.9, 143.2, 141.7,
135.0, 133.6, 131.9, 131.7, 130.2, 129.4, 127.9, 126.7, 125.7, 122.5, 121.3, 115.6,
111.2, 96.0, 67.2, 62.3, 27.8, 21.3, 14.6. EI/HRMS (m/z) [M+Na]+: 670.2401. Calcd for
[C36H32BF2N5O4Na]+: 670.2413.
4.2 Biological Data
Inhibition of cell proliferation – MTT tests. Cell growth was quantified by the ability
of living cells to reduce the yellow dye MTT to a purple formazan product. Cytotoxicity
was checked on different human cancer cell lines (HL-60 and Jurkat leukemias, PC3
prostate carcinoma, SF295 glioblastoma, MDA-MB435 melanoma, SW620 and HCT-
116 colon carcinomas cell lines), and two non-cancer murine fibroblasts (Chinese
hamster V79 and mouse L929 cells). All cancer cells were obtained from the National
24

Cancer Institute in Bethesda, MD, USA. The V79 cells were kindly provided by Dr.
Henriques JAP (Federal University of Rio Grande do Sul, Porto Alegre, Brazil), and
L929 cells were purchased from Rio de Janeiro Cell Bank (BCRJ, Rio de Janeiro,
Brazil). For the experiments, cells were plated in 96-well plates (0.7 x 105 to 0.3 x 106
cells/ well for cancer cells and 0.5 x 105 cells/well for murine fibroblasts), and tested
compounds, dissolved in DMSO (0.1%), was then added to each well, followed by
incubation for 24 h at concentrantions ranging from 0.04 to 40.92 µM or 72 h at
concentrantions ranging from 0.04 to 8.19 µM. In some experiments, the contribution of
ROS to the cytotoxicity of tested quinones was assessed by cells co-treated with GSH-
OEt (15 mM). Our preliminary experiments showed that the ethyl ester of GSH was not
cytotoxic and provided a more efficient protection than GSH at the same concentration
(15 mM). In fact, GSH is not readily transported into most cells. Thus, in the MTT
experiments, we used GSH ethyl ester, which is more lipophilic, readily taken up by
cells and hydrolyzed to GSH by cellular nonspecific esterases.51 Afterward, the plates
were centrifuged and the medium replaced by fresh medium (150 µL) containing 0.5
mg/mL MTT. Three hours later, the MTT formazan product was dissolved in 150 µL
DMSO, and absorbance was measured using a multiplate reader (Spectra Count,
Packard, Ontario, Canada). Drug effect was quantified as the percentage of control
absorbance of the reduced dye at 595 nm. Doxorubicin (0.001-1.06 µM) was used as
positive control. Experiments were carried out in triplicate and repeated at least three
times.
Lipid peroxidation (TBARS assay). The extent of tested compound-induced lipid
peroxidation was determined by the reaction of thiobarbituric acid (TBA) with
25

malondialdehyde (MDA), a product formed by lipid peroxidation.52 The assays were
performed according to Salgo and Pryor,53 with minor modifications. Cells were
incubated with compound for 24 h, and after lysis with Tris-HCl (15 mM for 1 h). Two
milliliters 0.4 mg/mL trichloroacetic acid, 0.25 M HCl were added to the lysate, which
was then incubated with 6.7 mg/mL TBA for 15 min at 100 ºC. The mixture was
centrifuged at 750 x g for 10 min. As TBA reacts with other products of lipid
peroxidation in addition to MDA, results are expressed in terms of thiobarbituric
reactive species (TBARS), which are determined by absorbance at 532 nm. Hydrolyzed
1,1,3,3-tetramethoxypropan was used as the standard. The results were normalized by
protein content.54 In order to evaluate the contribution of ROS on lipid peroxidation
extent, cells were pre-treated for 24 h with NAC (5 mM), and after they were exposed to
tested compound during 24 h. H2O2 (10 µM) was used as positive control. All
experiments were performed in triplicate in three independent experiments.
Determination of reduced (GSH), oxidized (GSSG) glutathione, and GSG/GSSG
ratios. Total glutathione (GSH + GSSG) was determined by spectrophotometer
determination of 5-thio-2-nitrobenzoate (TNB), which was produced from DTNB,
according to Akerboom and Sies55 with minor modification. Briefly, cells were exposed
with tested compound (5 µM) during 24 h. Then, cells were washed with ice-cold PBS,
and resuspended in 0.1 sodium phosphate (5 mM EDTA, pH 8.0), and sonicated to
obtain the cell homogenate. An equal volume of 2 M HClO4-4 mM EDTA was added to
the cell extract, and the precipitated proteins were sedimented by centrifugation at 8000
x g for 15 min at 4 ºC. The supernatant was neutralized with 2 M KOH, and the
insoluble residue was removed by centrifugation under the same conditions. For the
26

spectrophotometric determination 910 µL of the cell extract supernatant or of the
standard glutathione solution, in the same phosphate-EDTA buffer, were mixed with 50
µL of 4 mg/mL NADPH in 0.5% (w/v) NaHCO3, 20 µL of 6 U/mL glutathione
reductase in phosphate-EDTA buffer, and 20 µL of 1.5 mg/mL DTNB in 0.5%
NaHCO3. The increase in absorbance was measured at 412 nm. The results were
normalized by protein content.54 Total glutathione content was determined as µg/mg
protein. For GSSG determination, 4-vinypyridine was added to a final concentration of
0.1% (v/v), and then incubated for 1 h at room temperature. At this concentration, 4-
vinypyridine is able to react with all GSH without interfering with GSSG determination.
GSH was determined based on the total glutathione and GSSG concentration results. In
order to evaluate the contribution of ROS on glutathione modulation content, cells were
pre-treated for 24 h with NAC (5 mM), and after they were exposed to tested compound
during 24 h. All experiments were performed in triplicate in three independent
experiments.
Alkaline comet assay (Single cell gel electrophoresis). The alkaline comet assay was
performed as described by Singh et al.56 with minor modifications,57 and following the
recommendations of the International Workshop on Genotoxicity Test Procedures.58 At
the end of the treatment with 11 (2.5, 5, and 10 µM during 24 h) exposure in the
presence or absence of 5 mM NAC (pre-treated for 24 h), cells were washed with ice-
cold PBS, detached with 100 µL trypsin (0.15%) and resuspended in complete RPMI
medium. Next, 20 µL of cell suspension (~106 cells/mL) were mixed with 0.75% low
melting point agarose and immediately spread onto a glass microscope slide precoated
with a layer of 1% normal melting point agarose. The agarose was allowed to set at 4˚C
27

for 5 min. The slides were incubated in ice-cold lysis solution (2.5 M NaCl, 10mM Tris,
100mM EDTA, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4˚C for a minimum of 1
h to remove cellular proteins, leaving the DNA as “nucleoids.” After the lysis
procedure, the slides were placed on a horizontal electrophoresis unit. The unit was
filled with fresh buffer (300 mM NaOH and 1mM EDTA, pH>13.0) to cover the slides
for 20 min at 4˚C to allow DNA unwinding and expression of alkali-labile sites.
Electrophoresis was conducted for 20 min at 25 V and 300 mA (0.86 V/cm). After
electrophoresis, the slides were neutralized (0.4 M Tris, pH 7.5), stained with ethidium
bromide (20 µg/mL) and analyzed using a fluorescence microscope. All the above steps
were conducted under yellow light or in the dark to prevent additional DNA damage.
Images of 100 randomly selected cells (50 cells from each of two replicate slides) were
analyzed for each concentration of test substance. Cells were scored visually and
assigned to one of five classes, according to tail size (from undamaged-0, to maximally
damaged-4), and a DNA strand breaks index value was calculated for each sample of
cells. DNA strand breaks index thus ranged from 0 (completely undamaged: 100 cells x
0) to 400 (with maximum damage: 100 cells x 4).59
Spectral characterization. Spectral characterization of BODIPY-prodrug conjugates.
Spectroscopic and quantum yield data were recorded on a Synergy HT
spectrophotometer (Biotek). Compounds were dissolved at the indicated concentrations
and spectra were recorded at r.t. Spectra are represented as means from at least two
independent experiments with n = 3. Quantum yields were calculated by measuring the
integrated emission area of the fluorescence spectra and comparing it to the area
measured for fluorescein in basic EtOH.
28

Fluorescence confocal microscopy. PC3 cells were grown in DMEM cell culture
media supplemented with 10% FBS, antibiotics (100 U mL-1 penicillin and 100 mg mL-
1 streptomycin) and 2 mM L-glutamine in a humidified atmosphere at 37 °C with 5%
CO2. Cells were plated on glass chamber slides Lab-Tek™ II (Nunc), incubated with
compounds 11 or 15 (5 µM) with or without Tracker dyes at 37 °C for 10 min. Cells
were imaged in phenol red-free DMEM under a Zeiss LSM 510 META fluorescence
confocal microscope equipped with a live cell imaging stage. Fluorescence and
brightfield images were acquired using a 40X oil objective. Fluorophores were excited
with 488 nm (compound 15) or 543 nm (Lysotracker Red, Mititarcker Red) lasers. All
images were analysed and processed with ImageJ.
Acknowledgements
This research was funded by grants from the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq 305385/2014-3, PVE 401193/2014-4, 477346/2013-8
and Edital Universal MCTI/CNPq Nº 01/2016), FAPEMIG (APQ-02478-14) and
Programa Pesquisador Mineiro (PPM-00638-16), INCT-Catálise, CAPES/DAAD
99999.008126/2015-01 and CAPES. M. V. acknowledges the Medical Research
Council, the Marie Curie Integration Grant (333847), and the Biotechnology and
Biological Sciences Research Council (BB/M025160/1). The authors acknowledge Dr.
Bin-Zhi Qian (Univ. Edinburgh) for the supply of PC3 cancer cells for imaging studies.
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