In vitro inhibition of Hsp90 protein by benzothiazoloquinazolinequinones is enhanced in the presence of ascorbate. a preliminary in vivo antiproliferative study

Article abstract of DOI:10.3390/molecules25040953

A series of benzo[g]benzothiazolo[2,3-b]quinazoline-7,12-quinones were prepared from 2-acylnaphthohydroquinones and 2-aminobenzothiazoles and were evaluated for their in vitro antiproliferative activity. After screening using the MTT reduction assay, their IC₅₀ values were calculated on a panel of cancer cells (T24, DU-145, MCF-7). Current standard anticancer drugs were included as control, and their calculated IC₅₀ values were 7.8 and 23.5 μM for 5-fluorouracil and tamoxifen, respectively. Non-cancer cells (AG1523) were included to assess cancer cell sensitivity and drug selectivity. Four members of the series, with IC₅₀ values from 0.11 to 2.98 μM, were chosen for further assays. The selected quinones were evaluated regarding their effects on cancer cell proliferation (clonogenic assay) and on Hsp90 and poly(ADPribose)polymerase (PARP) protein integrity. The most active compound (i.e., 15) substantially inhibited colony forming unit (CFU) formation at 0.25 μM. In the presence of ascorbate, it induced an oxidative cleavage of Hsp90 but had no effect on PARP protein integrity. In an in vivo animal model, it discreetly increased the mean survival time (m.s.t.) of tumor-bearing mice. In light of these results, compound 15 represents a potential lead-molecule to be further developed.

Full text of DOI:10.3390/molecules25040953

molecules
Article
In Vitro Inhibition of Hsp90 Protein by
Benzothiazoloquinazolinequinones Is Enhanced in
The Presence of Ascorbate. A Preliminary In Vivo
Antiproliferative Study
1
,2,
1
3
1,4
Jaime A. Valderrama
*, David Ríos , Giulio G. Muccioli , Pedro Buc Calderon
and
Julio Benites 1
,2,
*
1
Química y Farmacia, Facultad de Ciencias de la Salud, Universidad Arturo Prat, Casilla 121,
Iquique 1100000, Chile; darios@unap.cl (D.R.); pedro.buccalderon@uclouvain.be (P.B.C.)
2
3
Instituto de Ciencias Exactas y Naturales, Universidad Arturo Prat, Casilla 121, Iquique 1100000, Chile
Bioanalysis and Pharmacology of Bioactive Lipids (BPBL), Louvain Drug Research Institute, Université
catholique de Louvain, 72 Avenue E. Mounier, BPBL 7201, 1200 Brussels, Belgium;
giulio.muccioli@uclouvain.be
Research Group in Metabolism and Nutrition, Louvain Drug Research Institute, Université catholique de
Louvain, 73 Avenue E. Mounier, 1200 Brussels, Belgium
4
*
Correspondence: jaimeadolfov@gmail.com (J.A.V.); julio.benites@unap.cl (J.B.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 9 January 2020; Accepted: 18 February 2020; Published: 20 February 2020
Abstract: A series of benzo[g]benzothiazolo[2,3-b]quinazoline-7,12-quinones were prepared from
-acylnaphthohydroquinones and 2-aminobenzothiazoles and were evaluated for their in vitro
2
antiproliferative activity. After screening using the MTT reduction assay, their IC50 values were
calculated on a panel of cancer cells (T24, DU-145, MCF-7). Current standard anticancer drugs were
included as control, and their calculated IC50 values were 7.8 and 23.5
tamoxifen, respectively. Non-cancer cells (AG1523) were included to assess cancer cell sensitivity
and drug selectivity. Four members of the series, with IC50 values from 0.11 to 2.98 M, were
µM for 5-fluorouracil and
µ
chosen for further assays. The selected quinones were evaluated regarding their effects on cancer
cell proliferation (clonogenic assay) and on Hsp90 and poly(ADPribose)polymerase (PARP) protein
integrity. The most active compound (i.e., 15) substantially inhibited colony forming unit (CFU)
formation at 0.25 µM. In the presence of ascorbate, it induced an oxidative cleavage of Hsp90 but
had no effect on PARP protein integrity. In an in vivo animal model, it discreetly increased the mean
survival time (m.s.t.) of tumor-bearing mice. In light of these results, compound 15 represents a
potential lead-molecule to be further developed.
Keywords: benzothiazoloquinazoline; quinones; antiproliferative activity; clonogenic assay; Hsp90;
PARP protein
1
. Introduction
Many natural and synthetic compounds sharing the 1,4-naphthoquinone scaffold display a wide
variety of biological activities [1]. A key mechanism explaining the activities of such molecules is the
intercalation of DNA due to the high ability of their large planar polycycles to bind the base pairs
through hydrogen bonds and “
occurs with compounds containing three to four coplanar rings, such as mitoxantrone and doxorubicin.
This interferes with normal DNA functioning and leads to cell death [ ]. Interestingly, depending
on the molecules, such DNA damage can be caused by the parent form or following its metabolic
π-stacking” interactions [2,3]. Hence, an optimal DNA intercalation
4
Molecules 2020, 25, 953; doi:10.3390/molecules25040953
www.mdpi.com/journal/molecules

Molecules 2020, 25, 953
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conversion to electrophilic or radical species [
5
]. In this context, quinones having redox-cycling
properties are endowed with potential anticancer activities [
on a particular ambivalence of cancer cells: they produce a large amount of reactive oxygen species
ROS), while they are generally deficient in antioxidant enzymes [10 13]. Such a dichotomy represents
1–9]. The rationale behind this is based
(
–
a vulnerability of tumor cells to an oxidative stress, which can be therapeutically exploited. Indeed,
an ROS-generating system (i.e., quinone redox cycling) yields a huge amount of ROS that exceeds
the antioxidant defense capacity, thus, compromising their fine redox equilibrium. In this context,
we have induced the alteration of intracellular redox homeostasis of cancer cells by using redox-cycler
quinones as a new strategy in the research and development of new antitumor drugs. To this end,
numerous quinone derivatives have been synthesized and assessed for their biological activity in order
to optimize this redox-cycling approach [11,14–16].
Recently, we have reported results on the synthesis and preliminary in vitro cytotoxic evaluation of
a coplanar heteroaromatic scaffold namely benzo[g]benzothiazolo[2,3-b]quinazoline-7,12-quinones [16].
The synthetic accessibility to these heterocycles as well as the encouraging cytotoxic activities of some
members of the series on cancer cells suggest that these heterocyclic quinones might be good antitumor
compound candidates. Based on these preliminary results, we assessed the anticancer potential
of the previously reported benzothiazoloquinazolinequinones [16] together with new members of
the series using a double in vitro/in vivo approach. The in vitro effect of quinones was compared
to two well-known anticancer drugs, namely tamoxifen (TAM) and 5-fluorouracil (5-FU). TAM has
been utilized against prostate and mammary cancer cells [17], while 5-FU has been used against
bladder tumors [18,19]. Moreover, to bring some mechanistic insight to the mechanism of action,
we have also evaluated the effect of quinones on important intracellular cancer cell targets, namely
poly(ADPribose)polymerase (PARP) and the chaperone Hsp90 protein. Previous studies have shown
that his latter protein is a good target for oxidant-based antitumor treatments [20].
2
. Results
2
.1. Synthesis of Benzo[G]Benzothiazolo[2,3-B]Quinazoline-7,12-Quinones
The quinones employed in this study were prepared by heteroannulation between
acylnaphthoquinones generated in situ from their respective naphthohydroquinones
-aminobenzothiazoles 10 according to our previously reported procedure [16], (Supplementary data).
Table 1 summarizes the results for the preparation of the members of the series 11–26. The structures
1–6, and
2
7–
1
13
of quinones 11
–
16
,
18,
19,
21
,
22 were confirmed by comparing their spectral properties ( H,
C
NMR) to those reported in the literature [16]. According to the proposed mechanism for heterocycle
heteroannulation [16], the reaction is initiated by attacking the NH group of the aminobenzothiazoles
2
at the 3-position of the acylnaphthoquinones. This attack generates Michael intermediate adducts,
which by a further 6-exo trig ring closure followed by aerobic oxidation yields the heterocycles
containing a chiral carbon atom at the 13-position of the heteropentacyclic scaffold.

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Table 1. Synthesized dibenzothiazoloquinazoline-7,12-quinones 11–26.
N
O
R1
2
H N
O
HO R1
N
OH
O
S
R2
R2
7
-10
R1
Ag2O, MgSO4
DCM, rt
O
DCM, rt
S
N
O
O
OH
1-6
11-26
Acylhydroquinone
Aminobenzothiazol Product
R1
R2
Yield (%) a
67 b
1
2
3
4
1
2
3
4
1
2
3
4
5
5
6
6
7
7
7
7
8
8
8
8
9
9
9
9
7
9
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Me
H
H
H
b
1-Propyl
1-Pentyl
1-Heptyl
Me
1-Propyl
1-Pentyl
1-Heptyl
Me
1-Propyl
1-Pentyl
1-Heptyl
2-Furyl
78
47
41
61
b
b
H
b
Me
Me
Me
Me
OMe
OMe
OMe
OMe
H
b
68
87
b
84
43
b
61
b
75
60
b
55
63
63
63
2-Furyl
2- Thiophen
2- Thiophen
OMe
H
F
a
Isolated yield after column chromatography with reference to their precursors 1–6; b Reference [20].
2
.2. In Vitro Antitumor Activity of Heterocyclic Quinones
As a first approach, an MTT reduction test was performed using a panel of human cancer (MCF-7,
T24, and DU-145), non-tumor (AG1523) cell lines in the presence of increasing concentrations of the
test compounds. As positive control, two well-known anticancer drugs, namely TAM and 5-FU,
were also tested on these cell lines. TAM, a non-steroidal anti-estrogen drug, is of interest because
it has been used against both MCF-7 and DU-145 cells [17]. Regarding 5-FU, it is a broad-spectrum
chemotherapeutic drug used to treat a variety of malignancies. Particularly, it has a long history of use
against bladder tumors [18,19].
Table 2 shows that the most active quinones were compounds 15
, 16, 19, and 20 with IC50 values
ranging from 0.11 to 1.77 M. Excluding these compounds, no particular sensitivity of cancer cell lines
µ
was observed with regard to quinones although DU-145 cells seemed a little more resistant compared
to both MCF-7 and T24 cells. Otherwise, a more dispersed range of sensitivity was noticed within the
quinone series in healthy fibroblasts.
Table 2. In vitro inhibitory effect of compounds 11–26 on the proliferation of the human-derived tumor
cell lines: T24 (bladder), DU-145 (prostate), and MCF7 (breast) and the non-tumor fibroblasts (AG 1523).
IC50 ± SEM (Standard Error of the Mean) a (µM)
MSIb
Structure
No.
T24
DU-145
MCF-7
AG 1523
1
1
1
2
4.65 ± 0.43
9.59 ± 0.32
6.83 ± 1.29
1.52
10.71 ± 0.57
4.86 ± 0.50c
11.58 ± 2.05c
4.25 ± 0.50c
1.28
8.82 ± 0.27

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Table 2. Cont.
IC50 ± SEM (Standard Error of the Mean) a (µM)
MSIb
Structure
No.
T24
DU-145
MCF-7
AG 1523
13
14
15
16
17
6.85 ± 0.54
7.99 ± 0.17
5.83 ± 0.25
1.24
8.55 ± 0.35
8.42 ± 1.27
0.22 ± 0.06 c
1.36 ± 0.15c
6.86 ± 0.33
9.04 ± 0.69
0.11 ± 0.03 c
0.85 ± 0.10c
8.46 ± 0.31
6.84 ± 0.79
2.98 ± 0.52 c
1.77 ± 0.26c
5.26 ± 0.22
0.90
29.4
2.62
1.09
7.28 ± 0.39
32.31 ± 3.31
3.46 ± 0.08
7.50 ± 0.69
18
19
20
21
22
5.60 ± 0.29
1.32 ± 0.11c
1.89 ± 0.15
7.09 ± 0.14
5.95 ± 0.45
7.55 ± 0.69
0.79 ± 0.11c
1.13 ± 0.05
7.43 ± 0.28
6.62 ± 0.19
7.12 ± 0.65
2.72 ± 0.18c
1.67 ± 0.12
4.63 ± 0.30
3.52 ± 0.32
0.89
3.97
3.17
1.78
1.11
6.02 ± 0.29
6.40 ± 0.46
4.99 ± 0.59
11.32 ± 1.10
5.97 ± 0.24
2
3
4
9.29 ± 0.77
10.41 ± 1.00
8.72 ± 0.41
19.95 ± 0.50
4.69 ± 0.33
7.85 ± 0.60
8.62 ± 0.72
11.69 ± 0.65
7.32 ± 0.36
1.72
10.1
1.60
21.70 ± 0.27
90.12 ± 1.21
12.77 ± 0.31
2
S
N
O HO
N
25
S
O

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Table 2. Cont.
IC50 ± SEM (Standard Error of the Mean) a (µM)
MSIb
Structure
No.
T24
DU-145
MCF-7
AG 1523
26
13.37 ± 2.37
10.75 ± 1.01
7.46 ± 0.53
1.88
19.79 ± 0.83
5
-Fluorouracil
Tamoxifen
-
-
3.61 ± 0.77
27.9 ± 4.8
7.10 ± 1.45
23.0 ± 4.1
12.68 ± 1.9
19.6 ± 2.6
b
0.84
0.95
6.59 ± 1.04
22.5 ± 2.3
a
Data represent IC50 mean values
values of fibroblasts/IC50 values of tumor cells; Data were got from Valderrama et al. [16].
±
SEM of at least three different experiments; MSI: mean selective index = IC50
c
By taking a mean IC50 value calculated from the individual IC50 values calculated for the three
cancer cell lines, the quinones may be ranked in three groups with different activities. The first one,
encompassing the most active quinones, included compounds 15
from 1.10 to 1.61 M). A second group is shown by the less active quinones (23 and 26) with IC50 mean
values ranging from 10.52 to 12.61 M. The third one included quinones with IC50 values ranging from
.36 to 8.92 M. It should be noted that 12 out of 16 quinones were more active than 5-fluorouracil.
, 16, 19, and 20 (mean IC50 values
µ
µ
5
µ
Finally, all quinones had IC50 values lower than those obtained with TAM.
Table 2 also shows the mean selectivity index (MSI) of quinones and the standard drugs. Such a
selective index was calculated as the ratio between IC50 values of healthy cells/IC50 values in cancer cells.
Interestingly, the most active quinones, namely 15, 16, 19, and 20, as well as a less active compound 24
show high values of MSI.
The data in Table 2 can be used to establish a preliminary structure–activity relationship (SAR)
analysis. Such SAR study reveals that for quinones 11 14 and 23 26, the nature and size of the carbon
–
–
ligands bonded to the chiral carbon atom of the pentacyclic scaffold did not significantly influence
either their activity or their selectivity. The substitution effects are more significant in enhancing the
antiproliferative activity for the members containing the methyl or propyl groups in the 13-position.
Thus, the insertion of methyl or methoxy groups in the 3-position of the compounds 11 and 12, as in
1
5
,
16
their quinone precursors, reaching in some cases nanomolar IC50 values.
Based on these results, the most active quinones, namely 15 16 19, and 20, were chosen for a
, 19, and 20, induces a dramatic increase of the antiproliferative activity compared to those of
,
,
clonogenic assay. Table 3 shows the effects of four selected quinones on the growth capacity of T24
cells as shown by the number of colony forming units (CFUs). Compound 15 appeared as the most
active member of the series. Indeed, at a dose of 0.25 µM, it decreased from 195 to 72 the number of
CFUs. Otherwise, it inhibits the proliferation of T24 cells by 63% as compared to the control untreated
cells. At such concentration, the other compounds showed a residual activity with the exception of
19, which is able to inhibit cell proliferation by 28%, however, it is still two times less active than 15.
The percentages of the observed cell growth compared to control conditions (100%) are indicated
in brackets.
Table 3. Effect by quinones on antiproliferative capacity (CFUs—colony forming units) of T24 cells.
Dose (µM)
.00
15
16
19
20
0
195 ± 16 (100)
180 ± 11 (92)
162 ± 18 (83)
72 ± 5 * (37)
22 ± 9 * (11)
16 ± 3 * (8)
202 ± 28 (100)
204 ± 19 (101)
216 ± 29 (107)
189 ± 12 (94)
79 ± 9 * (39)
191 ± 24 (100)
195 ± 16 (102)
183 ± 21 (96)
138 ± 18 * (72)
68 ± 12 * (36)
30 ± 16 * (16)
35 ± 11 * (18)
213 ± 23 (100)
204 ± 17 (96)
214 ± 25 (100)
225 ± 19 (106)
118 ± 22 * (55)
89 ± 10 * (42)
46 ± 7 * (22)
0
0
.025
.125
0
.25
1
.25
2
.5
58 ± 12 * (29)
23 ± 15 * (11)
5
.0
2 ± 1 * (1)
T24 cells were incubated for 24 h in the absence or in the presence of varied concentrations (from 0 to 5 µM) of
quinones, and the proliferative capacity of cells was evaluated as shown in the Materials and Methods section.
p < 0.05 as compared to control values.
*

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2
.3. Effect of Selected Quinones on Intracellular Targets
Next, we wanted to assess how these quinone derivatives affect cancer-related intracellular targets.
Here, we selected Hsp90 and PARP. Indeed, Hsp90 is highly overexpressed in cancer cells, where it
plays a critical role in stabilizing proteins that are essential for carcinogenesis such as Akt, RIP, and
others [20]. PARP, and more specifically PARP cleavage, is a classical marker of apoptosis making it a
relevant target to assess here.
Figure 1A shows the effect of four selected quinones on a chaperone Hsp90 protein. When T24
cells were incubated with quinones (5
of the Hsp90 protein. Nevertheless, when incubation was done in the presence of 1 mM ascorbate
vitamin C) plus quinones (including menadione), a new peptide fragment of about 70 kD was detected
by the antibody against Hsp90. In previous studies, it was demonstrated that ascorbate/menadione
asc/men) provoked an oxidative proteolytic cleavage at the N-terminal domain of Hsp90, and the
new peptide fragment did not come from a new protein synthesis [17 21 22]. The results depicted in
µM), no changes were observed with regard to the integrity
(
(
,
,
Figure 1A show that all quinones (19 to a lesser extent) are engaged within a redox cycle induced by
ascorbate generating ROS, which ultimately leads to Hsp90 protein cleavage and likely to a loss of its
chaperone function. Notably, the incubation of ascorbate with menadione induced a strong Hsp90
cleavage as compared to synthesized quinones.
(
A)
(
B)
Figure 1. Effects of selected quinones of Hsp90 (
integrity in T24 cells. Cancer cells were incubated for 4 h with 5
either in the absence or in the presence of 1 mM ascorbate (Asc). Ctl represents control untreated cells.
p < 0.05 as compared to control conditions.
A
) and poly(ADPribose)polymerase (PARP) (
B) protein
µM menadione (K3), 15, 16, 19, or 20
*

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Regarding PARP cleavage, neither the quinone derivatives nor menadione induced a detectable
PARP cleavage (Figure 1B). This was the case regardless of the presence of ascorbate (1 mM). The obtained
results make a potential role of apoptosis unlikely to be the underlying mechanism of cell impairment
induced by quinones, including menadione. Indeed, PARP is a substrate of caspase-3, therefore, when
an apoptosis process is triggered, it is expected that PARP protein will be cleaved yielding a second
protein band also recognized by the anti-PARP antibody.
Figure 2 shows the effects of quinone 15 on Hsp90 (Figure 2A) and PARP protein (Figure 2B) by
using the hepatocarcinoma TLT cells instead of T24 cells. As already observed by using the T24 cell
line, quinones in the presence of ascorbate were able to produce the same effect on TLT cells: oxidative
cleavage of Hsp90 but not PARP degradation.
(
A)
(
B)
Figure 2. Effects of selected quinones of hsp90 (A) and PARP (B) protein integrity in transplantable
primary liver tumor (TLT) cells. Cancer cells were incubated for 4 h with 5 M menadione (K3) or 15
either in the absence or in the presence of 1 mM ascorbate (Asc). Ctl represents control untreated cells.
p < 0.05 as compared to control conditions.
µ
*

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2
.4. In Vivo Antitumor Activity by 15
To go one-step further, we assessed the effect of the quinone derivative 15 in the TLT-bearing
mice model. The dose of 15 used in this study (i.e., 10 mg/kg) was calculated according to doses
of menadione, previously used by Verrax et al. [23]. In mice treated with 15, the calculated mean
survival time was 24.0 days as compared to 21.9 days in control untreated-animals, representing an
increase in life span of 9.6 % (Figure 3). Although this ILS represents a marginal effect as compared
to a minimum optimal effect (<25%), a small trend can be inferred supporting a potential protective
activity by 15, that requires to be further confirmed. When considering how long every individual
mouse survived, a mean of 22.8
control group had a mean of 21.2
no significant difference between treated and control group (t(38) = 1.13, p = 0.262). To go further,
studies are intended in the near future using a larger range of doses of 15.
±
6.1 days was obtained for the experimental treated group while the
1.6 days. A two-sample t-test on survival time (in days) revealed
±
Figure 3. Kaplan–Meier diagram in groups of TLT-bearing NMRI mice receiving intraperitoneal (i.p.)
saline (control untreated animals). Compound 15 was also i.p. administered as 10 mg/kg body weight,
2
4 h after i.p. transplantation of TLT cells.
3
. Materials and Methods
3
.1. Synthesis Of Benzo[G]Benzothiazolo[2,3-B]Quinazoline-7,12-Quinones
The new members of the series 17
acylnaphthoquinones in situ generated from their respective naphthohydroquinones
-aminobenzothiazoles 10 according to our previously reported procedure [16]. NMR data for
,
20
,
23
–
26 were prepared by heteroannulation between
2, 3, 5, 6, and
2
7–
compound 23 is not reported due to its extremely low solubility in the common solvents used in NMR
spectroscopy (the experimental procedure and the characterization of the compounds is reported in
the Supplementary Materials).
3
.2. Cell Lines and Cell Cultures
Human cancer cell lines T24 (bladder), DU-145 (prostate), MCF-7 (breast) and non-tumor fibroblasts
AG 1523 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell
culture conditions were the same as reported elsewhere [18]. Briefly, cell lines were kept in DMEM
containing 10% fetal calf serum. In addition, 100 U/mL of penicillin and 100
from Gibco (Grand Island, NY, USA) were further added. Cell cultures were kept at 37 C under an
µ
g/mL streptomycin
◦
atmosphere of 95% air/5% CO and 100% humidity. Sodium L-ascorbate, menadione (vitamin K3),
2
TAM, and 5-FU were purchased from Sigma (St. Louis, MO, USA).

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3
.3. Cell Survival Assays
The cytotoxicity of the quinones was assessed by following the reduction of MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan blue [24] and the capability
of cancer cells to proliferate, namely a clonogenic assay [25].
(
3
.3.1. MTT Reduction Assay
Cells were seeded into 96-well plates at a density of 10,000 cells/well for 24 h and then incubated
for 48 h with or without the quinone derivatives. Tamoxifen and 5-fluorouracil were used as standard
chemotherapeutic agents (positive controls). The IC50 values were determined using the GraphPad
Prism software (San Diego, CA, USA). Further incubation procedures and optical density reading of
colored solutions were performed according to Benites et al. [26].
3
.3.2. Clonogenic Assays
They were performed by seeding T24 cells (500) in six-well plates at a single-cell density. T24 cells
were chosen because of their facility to enumerate when they form colonies while the other cell lines
form cellular aggregates during their proliferation making the counting process difficult. Cells were
allowed to adhere overnight, and then treated with quinones for 24 h. Afterward, they were washed
with warm PBS, given fresh medium, and allowed to grow for 10 days. Clonogenic survival was
determined by fixing and staining colonies using crystal violet and further counting their number. The
number of colonies calculated under control conditions was set as 100%.
3
.4. Immunoblotting Procedures
Cancer cells were incubated for 4 h in the absence and in the presence of compounds. Afterward,
cells were washed twice with ice-cold PBS and then resuspended in RIPA lysis buffer supplemented
with 1% protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA) and 3% phosphatase inhibitor
cocktail (Calbiochem, Darmstadt, Germany). Treatment of samples and electrophoresis conditions
were conducted as previously reported [24]. Mouse monoclonal primary antibodies against hsp90
C-terminus (F8), and against -actin (clone AC-15), were purchased from Santa Cruz Biotechnology
Santa Cruz, CA, USA) and from Abcam (Cambridge, UK), respectively. Rabbit polyclonal antibodies
α/β
β
(
against PARP were purchased from BD Biosciences (San Diego, CA, USA). Goat anti-rabbit antibody
and rabbit anti-mouse polyclonal antibody were purchased respectively from Chemicon International
(Temecula, CA, USA) and DakoCytomation (Glostrup, Denmark).
3
.5. Animals and Diet
Young adult male NMRI mice (25–30 g) were obtained from Janvier Labs (Saint-Berthevin, France).
After their placement at the University Animal Facilities, animals were randomized according to their
3
body weight. They were housed in groups of five mice per cage (40
×
25
×
15 cm ) in a constant
◦
temperature environment (22 C) with alternating 12 h day/night cycles receiving standard food (UAR,
Villemoisson-sur-Orge, France) and tap water ad libitum.
3
.6. Experimental Tumor Model
Taper et al. [27] first observed the transplantable primary liver tumor (TLT) in 1966 in a two-month
old female Swiss Webster mouse. It had a rapid growth in both solid and ascetic forms and it was called
hepatoma indicating the organ of its origin. Fritzler et al. [28] further characterized the ultrastructure
of TLT hepatoma ascites cells. Regarding our experimental conditions, the TLT tumor was maintained
4
6
by weekly intraperitoneal (i.p.) transplantation (10 cells/mice). For antitumor assays, 1
were i.p. implanted in NMRI mice, and after 24 h, animals were i.p. receiving a solution of either saline
control group) or 15 (10 mg/kg body weight). After intraperitoneal tumor transplantation, animal
health conditions were daily verified. To avoid unnecessary animal suffering, mice were killed by
×
10 TLT cells
(

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cervical dislocation when posterior leg paralysis was noted. Then, mice mortality was daily recorded.
Such a protocol was repeated twice, with a final number of 20 mice per experimental group. Procedures
were conducted in accordance with legal requirements and with the approval of the local bioethics
committee (2014/UCL/MD/010).
The antitumor activity was calculated according to Geran et al. [29] using the mean survival time
(
1
m.s.t.) at day 30 and the increase in life span (ILS) calculated as ILS = [(m.s.t. control/m.s.t. 15)
00. This murine hepatoma-derived cell line was selected for the in vivo experiments because of its
previous use for oncologic studies [30,31] and due to its deficiency in antioxidant enzymes [32].
−
1]
×
3
.7. Statistical Analysis
All in vitro experiments were done at least three times. The experimental data were examined
using either a one-way ANOVA or an unpaired t-test, using GraphPad Prism software (GraphPad
Software, San Diego, CA, USA). A value of P < 0.05 was set as the level of significance. The statistical
analyses of the treatment of mice were done according to procedures reported by Geran et al. [29].
4
. Discussion
Severe adverse effects frequently restrain the successful achievement of antitumor chemotherapy.
Due to such current difficulties, the search for new drugs and alternative therapies has become a
crucial issue. In this context, we have developed a new and original series of quinones endowed with
in vitro antiproliferative activity in the range of 0.1–20 µM, as shown by the MTT reduction test and a
clonogenic assay. The molecular mechanisms by which quinones display their anticancer activities
remain elusive, but they are likely different from those of both TAM and 5-FU. Indeed, in vitro studies
indicate that TAM is a potent inhibitor of PKC [33,34], while the mechanism of cytotoxicity of 5-FU has
been attributed to fluoronucleotide misincorporation into DNA inhibiting DNA synthesis thus leading
to cell death [35]. Despite these differences in the mechanism of action, the range of in vitro activities
by quinones was in the same order of magnitude of standard anticancer drugs [17]. Interestingly,
when some of these quinones were compared with free-radical-generating anticancer drugs such as
doxorubicin and mitomycin C, they were less active than doxorubicin but fairly more active than
mitomycin C [16].
Currently, the best and most definitive therapeutic outcome for a potential anticancer drug is
done by an in vivo assay showing tumor inhibition or at least, a delay in tumor growth. However,
several issues, such as the nature of the tumor, the administration route, the doses employed, the
applied schedule (single or multiple doses of the anticancer compound), etc., restrict the in vivo
efficacy of a given drug. We would like to stress the point that our experimental approach implies
stringent conditions, because the compound was administered as a single dose. In addition, the TLT
hepatocarcinoma is extremely aggressive and no standard drugs have been capable of inhibiting
in vivo TLT growth [30]. For all these reasons, it should be kept in mind that although the in vivo effect
of 15 was rather small, such a low activity may represent a promising in vivo anticancer effect.
Since menadione (vitamin K3) also has the 1,4-naphthoquinone scaffold, we inferred that the
selected quinones would be have a redox behavior similar to that of menadione. That means the
occurrence of an ascorbate-driven quinone redox cycling leading to ROS formation that ultimately
causes an oxidative Hsp90 cleavage. Indeed, we have recently shown the in situ formation of hydroxyl
free radicals by a Fenton-type reaction at the N-terminal nucleotide binding pocket of Hsp90 forming a
protein radical, which, by rearrangement, causes the rupture of the peptide backbone [21]. It is worth
recalling that Hsp90 is a chaperone protein that stabilizes numerous proteins considered crucial for
cancer cell survival. Such proteins have been called “client” proteins and the list includes Akt, RIP,
Bcr-Abl (specifically in K562 cells), etc. Therefore, when Hsp90 is either destabilized due to the action
of geldanamycin [4,36,37] or cleaved by an oxidative attack [20,21], its chaperone function is lost, client
proteins are then degraded in the proteasome disturbing cellular equilibrium and ultimately causing
cancer cell death.

Molecules 2020, 25, 953
11 of 13
Regarding PARP protein, the obtained results are in agreement with previous reports acquired
with other types of cancer cells (i.e., K562, TLT). Under such conditions, the cell demise induced
by ascorbate/menadione was not caspase-3 dependent but close to a necrotic-like cell death [23,32].
Therefore, since PARP is a substrate of caspase-3, it would be expected that PARP protein should
be cleaved when apoptosis is activated, which was not the case here. Although our results lead to
the conclusion that apoptosis is unlikely to be playing a role, additional assays are required to better
characterize the type of cell death induced by quinones. Indeed, we are working to identify which
intracellular targets are impaired in order to explain the antiproliferative activity of quinones.
In conclusion, original quinones were prepared by in situ heteroannulation between
acyl-naphthoquinones and substituted 2-aminobenzothiazoles. Most quinone derivatives have
shown anticancer activities at lower doses than 5-FU and TAM. Four molecules (15, 16, 19, 20) were
extremely active against the three cancer cell lines. Among them, compound 15 displays a strong
in vitro antiproliferative effect but a tiny in vivo activity.
Supplementary Materials:
Description of the general procedure for the preparation of
20 23 26, and spectral data of nuclear magnetic
benzo[g]benzothiazolo[2,3-b]quinazoline-7,12-quinones 17
,
,
–
1
13
resonance ( H NMR and C NMR) and high-resolution mass spectrometry (HRMS).
Author Contributions: D.R. proposed the subject and designed the study. J.B. performed the synthesis of quinone
compounds. P.B.C. performed the biological evaluation. G.G.M. carried out the HRMS analysis. J.A.V., P.B.C., and
J.B. contributed to experimental design, results discussion, and manuscript writing. All authors have read and
agreed to the published version of the manuscript.
Funding: The present study was supported by grants no. 1100376 (D.R.) and no. 1190577 (P.B.C.) of the Fondo
Nacional de Ciencia y Tecnología, Chile
Acknowledgments: We thank Pilar Díaz from the Universidad Arturo Prat, Chile, for technical support.
Conflicts of Interest: The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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Sample Availability: Samples of the synthesized compounds 17, 20, 24, 25, 26 are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(