New naphthoquinone derivatives against glioma cells

Article abstract of DOI:10.1016/j.ejmech.2015.04.039

This work was aimed to the development of a set of new naphtoquinone derivatives that can act against glioma. The compounds were tested in order to find out their ability to inhibit the growth of glioma cells, and the results of these assays were correlated with electrochemical analysis and NMR-based reoxidation kinetic studies, suggesting that a redox mechanism underlies and may explain the observed biological behavior. In addition to a full description of the synthetic pathways, electrochemistry, NMR and single crystal X-ray diffraction data are provided.

Full text of DOI:10.1016/j.ejmech.2015.04.039

Accepted Manuscript
New naphthoquinone derivatives against glioma cells
Marco Redaelli, Carla Mucignat-Caretta, Abdirisak Ahmed Isse, Armando Gennaro,
Raffaele Pezzani, Riccardo Pasquale, Valeria Pavan, Marco Crisma, Giovanni
Ribaudo, Giuseppe Zagotto
PII:
S0223-5234(15)30002-7
10.1016/j.ejmech.2015.04.039
EJMECH 7854
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 October 2014
Revised Date: 14 April 2015
Accepted Date: 17 April 2015
Please cite this article as: M. Redaelli, C. Mucignat-Caretta, A.A. Isse, A. Gennaro, R. Pezzani, R.
Pasquale, V. Pavan, M. Crisma, G. Ribaudo, G. Zagotto, New naphthoquinone derivatives against
glioma cells, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.04.039.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New naphthoquinone derivatives against glioma
cells
Marco Redaelli‡, Carla Mucignat-Caretta‡, Abdirisak Ahmed Isse§, Armando Gennaro§, Raffaele Pezzani°,
Riccardo Pasquale†, Valeria Pavan†, Marco Crisma††, Giovanni Ribaudo† and Giuseppe Zagotto†*
† Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131
Padova, Italy
‡ Department of Molecular Medicine, University of Padova, Via Marzolo 3, 35131 Padova, Italy
° Department of Medical and Surgical Sciences-Operative Unit of Endocrinology, University of Padova, Via
Ospedale 105, 35128 Padova, Italy,
§ Department of Chemical Sciences, University of Padova, Via Marzolo 1, 35131 Padova, Italy
†† Institute of Biomolecular Chemistry, Padova Unit, CNR, Via Marzolo 1, 35131 Padova, Italy
KEYWORDS: glioma, naphtoquinones, redox, temozolomide
ABSTRACT
This work was aimed to the development of a set of new naphtoquinone derivatives that can act against glioma. The
compounds were tested in order to find out their ability to inhibit the growth of glioma cells, and the results of these
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assays were correlated with electrochemical analysis and NMR-based reoxidation kinetic studies, suggesting that a
redox mechanism underlies and may explain the observed biological behavior. In addition to a full description of the
synthetic pathways, electrochemistry, NMR and single crystal X-ray diffraction data are provided.
1. INTRODUCTION
Gliomas are tumors most frequently affecting the central nervous system (CNS). According to the gliomas
classification provided by the World Health Organization [1], the most malignant grade is glioblastoma (Grade IV,
GB). The current therapeutic approach that FDA approved consists in neurosurgical resection, followed by
chemotherapy with temozolomide (TMZ) in combination with radiotherapy, though only 26% of treated patients
reached 2 years of survival [2]. Despite all the recent advances in neurosurgery [3] and chemotherapy [4],
glioblastoma presents highly invasive pattern and recurs in 90% of cases [5]. Many attempts to define the specific
biomolecular characteristics of these complex and variable tumors have been carried out during the last decades [6],
though no promising discoveries successfully migrated from the bench to the clinic [7]. At present, investigation on
modulation of specific targets and consequent effects on glioma cells is one of the most concrete approaches towards
a better understanding of the mechanism involved in gliomas malignancy. Alternative therapeutic approaches based
on novel compounds and/or discovery of interesting biological activities of known compounds are therefore
necessary.
Recently, indeed, the interest towards natural derivatives developed and naphthoquinones, among the others,
continuously captivated biological interest. In this connection, the applications of both natural (as in the case of 3-
hydroxy-5-methoxy-2-methylbenzoquinone and heliquinone, from Sterculiaceae family) [8] and semi-synthetic
derivatives have been widely investigated. For instance it has been reported that water-soluble naphthoquinone
derivatives – i.e. their conjugated with carbohydrates – show cytotoxic activity in JB6 P+ Cl41 cells [9], while other
1,4-naphthoquinones have been synthesized and evaluated for many other purpouses, such as their trypanocidal
activity [10].
Juglone is a natural compound deriving from the Juglandaceae family, particularly Juglans nigra, whose toxic and
growth-stunting effects are well known [11]. While Juglone was reported to induce generic oxidative stress in both
healthy cells and cancer cell lines [12], we previously highlited the cytotoxic effect of its derivative 1 in inducing
apoptotic cell death on human glioma cell lines [13] as a result of a screening on our in-house database. Starting
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from the discovery of this promising lead, the synthesis of novel naphthoquinone derivatives stems out with the aim
of describing and comparing structure-activity relationship (SAR) of Juglone and the obtained compounds by
evaluating their effects on glioma cell death, allowing the enlightenment of the mechanism of action and, eventually,
the optimization of the lead.
2. RESULTS AND DISCUSSION
2.1 Chemistry. Juglone (JUG) was obtained from radical oxidation of 1,5-dihydroxynaphthalene in the presence of
CuCl according to a literature procedure and to the indication previously reported by Zonta et al [14-16]. Starting
from Juglone a series of derivatives containing various functional groups and structural variations was prepared to
test for the existence of a structure-activity relationship. A selection of the synthesized compounds is summarized in
Figure 1 (6, 14, and 16 were known compounds). A first set of naphthoquinone derivatives was synthesized starting
from Juglone via a modified Micheal’s addition. Unfortunately, these reactions gave poly-oxidized species that were
difficult to eliminate. Then the reaction was tried under inert nitrogen atmosphere. The reactions showed moderate
to good yields and the raw products had to be purified by flash chromatography while recrystallization from n-
hexane gave satisfactory results only for few derivatives. Hetero- and homo-nuclear 2D NMR studies proved that
substitution occurred at position 3 (see Supporting Information). Another class of derivatives containing sulfur (6, 8)
was prepared in a similar manner by direct addition of the appropriate nucleophilic thiophenol precursor to the
position 3 of Juglone, but ethanol was used as solvent instead of acetic acid. Preparation of a 3-bromo Juglone
precursor (7) via chemoselective bromination of Juglone and subsequent reaction of the bromo-juglone with amines
gave few more derivatives (9, 10) that, with the sulfur derivative, showed different redox properties potentially
useful to establish a SAR. NMR studies proved that substitution occurred at position 3. More in detail, 7 was used as
the model compound and deeply investigated through 2D homo- and heteronuclear 2D NMR experiments.
Correlation Spectroscopy (CoSY), Heteronuclear Single Quantum Coherence (HSQC) and Heteronuclear Multiple
Bond Correlation (HMBC) spectra (see Supporting Information) confirmed the reactivity of the 3- position of the
naphthoquinone scaffold.
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Figure 1 here
Derivatives 6, 7 and 8 were also unambiguously identified through single crystal X-ray diffraction analysis (see
Figure 2 for 7 and 8, whereas for 6, already reported in the literature [17], see Supporting Information). In these
structures, C-O(H) bond distances are within 1.339(2) – 1.356(6) Å, slightly shorter than those commonly observed
for phenolic hydroxyls [1.362(15) Å] [18]. The C=O bond lengths, comparing with the average value reported for
benzoquinones [1.222(13) Å] [18], show a bimodal distribution where those observed for the C=O group proximal
to the hydroxyl are longer [1.223(5) – 1.227(2) Å] than those of the distal C=O group [1.206(5) – 1.218(2)Å]. In all
structures the hydroxyl group is intramolecularly H-bonded to the proximal C=O group to form a six-membered
pseudo-cycle. Such features are common among juglone-related compounds [17, 19].
Figure 2 here
O-Acetylated derivatives (3, 5) and 7-methyl-8-chloro naphthoquinones derivatives (14, 15) were also
synthesized to explore the influence of these groups on structure-activity relationships.
2.2 Cytotoxicity. Synthesized compounds were tested at different concentrations (50 µM, 5 µM, 0.5 µM or 0.05
µM) using GLI36 human glioma cell line. Cells were incubated with the compounds (1-16), Juglone (JUG),
Temozolomide (TMZ) and Paclitaxel (PTX) for 24 or 72 hours. After treatment cells were tested for cell viability
with MTT assay and cell death (apoptosis and necrosis) with morphological analysis (see Supporting Information).
Cell cultures treated with 1 for 24 and 72 hours showed a significant decrease in cell viability in comparison with
JUG, TMZ, PTX and most of the other tested compounds (see Supporting Information).
Unraveling the status of the treated cells with morphological analysis, apoptosis and necrosis were evaluated in all
the conditions tested. Data showed a strongly significant increase in percentage of apoptotic cells and a decrease of
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necrosis level in comparison with reference compounds (JUG, TMZ and PTX) and the other synthesized compounds
(figure 2). Analysis carried out at 72h provided similar results (see Supporting Information).
According to what previous research works suggest [20], we postulated, as working hypothesis, that the preliminary
results of biological assays could be explained by a redox-based mechanism promoted by the examined compounds.
Figure 3 here
In this connection we synthesized 1,4,5-trihydroxy naphthalenes as reduction products of selected
naphthoquinones of a particular biological interest (JUG and 1): compounds JUG-R and 1-R were prepared via
reduction of Juglone and 1, respectively, with Na2S2O4 (Figure 4).
Figure 4 here
This reaction involving 1 was found to produce the quite unstable and easily reoxidable 1-R, as indicated by
color changes and confirmed by an NMR-based kinetic study (see Supporting Information), where the signals in the
proton spectra of JUG-R and 1-R were compared to those of JUG and 1, respectively. In general, a shift towards
lower δ was observed for the reduced derivatives, especially for what concerning the singlet attributed to the proton
in the 2- position. In order to prevent reoxidation of the reduced compounds to the naphthoquinone form, NMR
spectra for characterization and biological assays were carried out in the presence of Na2S2O4.
The synthesized compounds were subsequently used for cytoxicity assays. Glioma cells were incubated with
JUG, JUG-R, JUG + Na2S2O4, JUG-R + Na2S2O4, 1 + Na2S2O4, 1-R + Na2S2O4 and Na2S2O4 as control, at different
concentrations (50 µM, 5 µM, 0.5 µM or 0.05 µM). After 24 hours apoptosis and necrosis were evaluated by
morphological analysis. MTT test could be otherwise affected by the presence of Na2S2O4.
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The cytotoxic effect (apoptosis and necrosis) induced by JUG and 1 was not influenced by the presence of Na2S2O4
while the reduced trihydroxy naphthalene derivatives JUG-R and 1-R showed a strong decrease in cytotoxicity in
comparison with the original hydroxynaphthoquinones (Figure 5). The significantly lower level of apoptosis and
necrosis induced in human glioma cells by the reduced derivatives (JUG-R and 1-R) could represent a first
important indication supporting the hypothesis that a redox mechanism is involved. This is hypothesized to be
related to a marked difference in their reoxidation tendency showing that 1-R is more promptly reconverted to 1 as
compared to JUG-R to JUG.
Figure 5 here
The potential capability of the compounds of reaching and interacting with the target cells in a more complex system
was investigated by evaluating in silico the logP values, ranging from -0.5 to 2.7, in line with the one calculated for
TMZ. Nevertheless the major limitation of this study is related to this point: the biological evaluation was carried
out at in vitro level. Further insights in complex systems are needed to assess this issue.
2.3 Electrochemistry. To account for the different biological activity of the reduced and non-reduced
naphthoquinone derivatives cyclic voltammetry (CV) analysis of naphthoquinones was performed to better
understand if and how much the redox activity may be involved in inducing cell death. Since most of the
investigated compounds are sparingly soluble in water, all voltammetric investigations were carried out in aqueous
solutions containing 20% (v/v) CH3CN and the data for the derivatives reaching an adequate concentration are
repoted below. The redox chemistry of quinones is well documented in the literature and it is known that both the
voltammetric pattern and the redox potential are strongly influenced by pH [21-23]. Therefore all experiments were
conducted in buffered solutions, using 0.1 M phosphate buffer at pH 7; the bath temperature was set at 25°C.
Examples of cyclic voltammograms are reported in Fig. 6 for juglone and some other representative naphthoquinone
derivatives. It is well known that reduction of quinones in protic media occurs by a 2e−, 2H+ reaction leading to the
formation of the corresponding hydroquinones. The redox reaction in the case of juglone is
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(1)
A single peak couple similar to those shown in Fig. 6 was observed for all investigated compounds and therefore the
voltammetric pattern was assigned to the quinone/hydroquinone redox couple according to the redox reaction of eq.
(1). Table 1 summarizes the cathodic and anodic peak potentials, Epc and Epa respectively, as well as the peak
separation, ∆Ep = Epa – Epc, and half-wave potential E1/2, measured for each naphthoquinone at the scan rate v = 0.05
Vs-1. The separation between the cathodic and anodic peaks is significantly greater than 30 mV, predicted for a
reversible 2e- process and increases with increasing scan rate, indicating that these compounds undergo quasi-
reversible electron transfer processes [24]. The standard reduction potential, Eo, can be measured from cyclic
voltammetry by assuming it to be approximately equal to the half-wave potential:
Epa + Epc
Eo ≈ E1/ 2
=
(2)
2
Figure 6 here
Values of E1/2 are included in Table 1 (last column). As can be observed, although an extensive series of
naphthoquinones bearing both electron withdrawing and electron donating groups was chosen for this study, the
measured redox potentials lie in a rather narrow range. In fact, if 9 is excluded, the maximum difference between
E1/2 values for the whole series is only 65 mV, indicating that the ability of these molecules to act as oxidizing
agents is little affected by molecular structure. Therefore if the biological activity of the substrates is strongly related
to their redox power as oxidizing agents, the activity is not expected to vary significantly throughout the whole
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series of naphthoquinones. This is in part true, but as we shall discuss in the next section there are important
exceptions, underscoring the importance of structural factors and/or presence of functional groups not affecting E1/2.
Table 1 here
Figure 7 here
Figure 8 here
Figure 7 enlightens a definite linear correlation between the half-wave potential and the citotoxicty expressed in
terms of apoptosis. According to the reported data, the fit to the straight correlation line is better at 5µM
concentration of naphtoquinones but compounds show a similar behavior also at different concentrations. On the
other hand Figure 8 shows a scattered correlation pattern between the E1/2 values and the cell necrosis data.
3. CONCLUSIONS
A set of naphthoquinone derivatives have been synthesized and tested for their ability to inhibit the growth of
glioma cells. Preliminary data [13] on the peculiar properties of 2-(2',4'-dihydroxyphenyl)-8-hydroxy-1,4-
naphthoquinone (1) as strong cytotoxic agent for glioma cells were confirmed within this small focused library of
1,4-naphthoquinone derivatives. The results obtained from biological studies were found to be in good agreement
with preliminary kinetic data, supported by NMR results, and strongly suggests that a redox mechanism underlies. In
addition to the redox pathway, which has often been reported in connection with the biochemical properties of
hydroxynaphthoquinone derivatives [25, 26], kinetic aspect of the reaction was demonstrated to be strongly
involved, since only 1-R showed fast reoxidation to 1. In conclusion, besides the similar behaviour described by the
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electrochemical results we assume, as working hypothesis, that the reaction kinetic is influenced by the effects of the
substituents, leading to different reoxidation tendency and, hence, different degrees of cytotoxicity in human glioma
and normal cells.
4. EXPERIMENTAL SECTION
4.1 Chemistry
4.4.1 Materials and instruments. Commercially available chemicals were purchased from Aldrich, and used as
received, unless otherwise stated. All air-sensitive manipulations were conducted according to Schlenk line
techniques, using dry nitrogen and glassware. If required, solvents were dried prior to use. For work-up and
chromatographic purification, commercial grade solvents were used; chromatographic separations were carried out
using silica gel 60 (230-400 mesh, Grace Davisil). In addition, semi-preparative and preparative purification of the
derivatives were carried out on Isolera One, an automated flash chromatography system provided by Biotage
(Upsala, Sweden); the chromatography was carried out using disposable cartridges made of silica gel as stationary
phase and bench solvents as mobile phase.
1H and 13C{1H} NMR spectra were recorded on a Bruker Avance III 400 MHz and a Bruker AMX 300 MHz
spectrometers. All spectra were recorded at room temperature, the solvent for each spectrum is given in parentheses.
Chemical shifts are reported in ppm and are relative to TMS internally referenced to the residual solvent peak. The
multiplicity of signals is reported as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broad (br) or a
combination of any of these.
High resolution mass-spectra were recorded on a ESI-TOF Mariner from Perseptive Biosystem (Stratford, Texas,
USA), using electrospray (ES) ionization.
The degree of purity of the compounds synthesised throughout this investigation was assayed by HPLC, using a
Varian Pro-Star system equipped with a Biorad 1706 UV-VIS detector (254 nm) and an Agilent C-18 column (5ꢀm,
4.6 x 150 mm). An appropriate ratio of water (A) and acetonitrile (B) was used as mobile phase with an overall flow
rate of 1 mL min-1; the general method for the analyses is reported here: 0 minutes (90% A-10% B), 15 minutes
(10% A-90% B), 20 minutes (10% A-90% B), 21 minutes (90% A-10% B), 25 minutes (90% A-10% B).
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Cyclic voltammetry was performed on a PC controlled Autolab PGSTAT30 potentiostat (Eco-Chimie, Utrecht,
Netherlands), with a positive feedback for ohmic drop compensation. All experiments were carried out at 25 °C in a
three-electrode cell with a glassy carbon (GC) working electrode, a Pt counter-electrode and an aqueous saturated
calomel reference electrode. All solutions were prepared in a mixture of phosphate buffer (pH 7)/acetonitrile (80:20
v/v) and were carefully deaerated with Ar. The GC electrode was a 3 mm diameter disc embedded in glass, which
was fabricated from a GC rod (Tokai, GC-20) and polished to a mirror finish with silicon carbide papers and
diamond paste. Before every experiment it was cleaned by polishing with a 0.25 ꢀm diamond paste followed by
ultrasonic rinsing in ethanol for about 5 minutes.
Chemical names and calculated logP values were obtained with ChemDraw Ultra 8.0, CambridgeSoft, USA.
4.1.2 Synthesis of Juglone JUG
Juglone was synthesised according to the literature procedure [15, 16] and to the indication previously reported by
Zonta et al [14].
4.1.3 General Procedure for the Synthesis of 3-Substituted-5-Hydroxy Naphthoquinones
A round bottom flask was charged with 5-hydroxynaphthalene-1,4-dione (juglone, 2 eq) in acetic acid, followed by
addition of the appropriate phenol (1 eq) in acetic acid (2 ml) and 0.5 ml of 2 M H2SO4. The mixture was stirred at
room temperature for 2 hours under nitrogen at atmospheric pressure, followed by addition of water (20 ml) and
neutralization with 5% sodium bicarbonate. The mixture was extracted with ethyl acetate (3 × 15 ml), and the
combined organic layers were dried (Na2SO4) and evaporated to dryness. Purification was carried out by
recrystallization or by flash chromatography (n-hexane / ethyl acetate = 7:3 v / v) if necessary.
4.1.3.1 Synthesis of 8-hydroxy-2-(2,4-dihydroxyphenyl)naphthalene-1,4-dione (1)
Yield 66 %; δH (400 MHz, DMSO-d6) 11.93 (1H, s, OH ), 9.71 (1H, s, OH ), 9.69 (1H, s, OH ), 7.8-7.7 (1H, m,
ArH), 7.54 (1H, dd J 1.1 Hz, J 6.4 Hz, ArH) 7.37 (1H, dd J 1.1 Hz, J 7.3 Hz, ArH,), 7.10 (1H, d J 8.4 Hz, ArH), 6.98
(1H, s, CH), 6.39 (1H, d J 2.3 Hz, ArH), 6.32 (1H, dd J 2.3 Hz, J 8.4 Hz, ArH); δC (100 MHz, DMSO-d6) 189.6,
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184.6, 160.9, 160.4, 157.2, 147.6, 137.1, 136.3, 132.7, 132.4, 124.3, 118.3, 115.8, 111.7, 107.7, 103.1; HRMS (ESI)
[M+1]+ found 283.0646 [C16H11O5]+, calcd. 283.0601. HPLC purity 98%.
4.1.3.2 Synthesis of 8-hydroxy-2-(2,4-dihydroxy-3-methylphenyl)naphthalene-1,4-dione (2)
Yield 55%; δH (400 MHz, DMSO-d6) 11.91 (1H, s, OH), 9.54 (1H, s, OH), 8.61 (1H, s, OH), 7.80-7.70 (1H, m,
ArH), 7.52 (1H, dd J 1.0 Hz J 7.2 Hz, ArH), 7.33 (1H, dd J 1.0 Hz J 7.3 Hz, ArH), 6.85 (1H, s, CH), 6.81 (1H, d J
8.3 Hz, ArH), 6.39 (1H, d J 8.3 Hz, ArH), 1.97 (3H, s, Me); δC (100 MHz, DMSO-d6) 184.8, 183.7, 160.9, 158.1,
154.3, 149.1, 137.0, 132.6, 128.5, 124.4, 118.2, 115.9, 113.0, 111.7, 106.9, 106.2, 9.3; HRMS (ESI) [M+1]+ found
297.0804 [C17H13O5]+, calcd. 297.0762. HPLC purity 97%.
4.1.3.3 Synthesis of 8-hydroxy-2-(2,4-dimethoxyphenyl)naphthalene-1,4-dione (4)
Yield 20%; δH (400 MHz, DMSO-d6) δ 11.95 (1H, s, OH), 7.7-7.6 (1H, m, ArH), 7.48 (1H, d J 7.4 Hz, ArH), 7.20
(1H, d J 7.4 Hz, ArH), 7.16 (1H, d J 8.6 Hz, ArH), 6.83 (1H, s, CH), 6.51 (1H, d J 2.3 Hz, ArH), 6.56 (1H, dd J 8.6
Hz J 2.3 Hz, ArH), 3.82 (3H, s, OMe), 3.73 (3H, s, OMe); δC (100 MHz, DMSO-d6) 187.8, 183.3, 160.4, 159.4,
154.4, 145.4, 137.0, 133.4, 129.5, 122.5, 120.0, 116.0, 113.4, 111.7, 107.3, 105.9, 55.2, 45.1; HRMS (ESI) [M+1]+
found 311.0925 [C18H14O5]+ calcd.311.1113. HPLC purity 98%
4.1.3.4 Synthesis of 8-hydroxy-2-(2,3,4-trihydroxyphenyl)naphthalene-1,4-dione (11)
Yield 20 %; δH (400 MHz, Acetone-d6) 11.97 (1H, s, OH), 9.47 (1H, s, OH), 8.76 (1H, s, OH), 8.49 (1H, s, OH),
7.8-7.7 (1H, m, ArH), 7.55 (1H, d J 6.6 Hz, ArH), 7.37 (1H, d J 8.4 Hz, ArH), 6.94 (1H, s, CH), 6.60 (1H, d J 8.4
Hz, ArH), 6.39 (1H, d J 8.4 Hz, ArH,); δC (100 MHz, Acetone-d6); 189.9, 184.0, 156.5, 155.8, 149.3, 147.4, 142.5,
136.8, 136.5, 123.7, 121.6, 117.9, 112.8, 107.0, 94.5, 88.4; HRMS (ESI) [M-1]- found 297.0803 [C16H9O6]-,
calcd.297.0800. HPLC purity 94%.
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4.1.3.5 Synthesis of 8-hydroxy-2-(2,4,6-trihydroxyphenyl)naphthalene-1,4-dione (12)
Yield 93%; δH (400 MHz, Acetone-d6) 11.92 (1H, s, OH), 8.76 (1H, s, OH), 8.57 (1H, s, OH), 8.49 (1H, s, OH),
7.9-7.8 (1H, m, ArH), 7.60 (1H, d J 6.6 Hz, ArH), 7.40 (1H, d J 8.4 Hz, ArH), 6.76 (1H, s, CH), 6.43 (2H, d J 8.4
Hz, 2 x ArH,); δC (100 MHz, Acetone-d6) 174.3, 173.7, 162.5, 159.4, 159.2, 158.2, 151.8, 132.2, 131.9, 111.0,
102.2, 94.5, 90.4, 79.7; HRMS (ESI) [M-1]- found 297.0435 [C16H9O6]-, calcd.297.0405. HPLC purity 97%.
4.1.3.6 Synthesis of 2-(p-tolylthio)-8-hydroxynaphthalene-1,4-dione (6) [17]
Yield 47%; δH (300 MHz, Acetone-d6) 11.66 (1H, s, OH), 7.8-7.7 (1H, m, ArH), 7.6-7.4 (5H, m, ArH), 7.32 (1H,
dd J 1.0 Hz J 7.4 Hz, ArH,), 5.86 (1H, s, CH), 2.45 (3H, s, Me); δC (75 MHz, Acetone-d6) 186.1, 181.0, 161.0,
142.6, 138.6, 136.8, 132.5, 128.3, 124.9, 124.7, 124.6, 124.7, 124.6, 119.2, 21.7; HRMS (ESI) [M+1]+ found
297.0596 [C17H13O3S]+ calcd.297.0580. HPLC purity 96%.
4.1.3.7 Synthesis of 2-(3-methoxyphenylthio)-8-hydroxynaphthalene-1,4-dione (8)
Yield 43%; δH (300 MHz, Acetone-d6) 11.48 (1H, s, OH), 7.60 (1H, dd J 8.2 Hz J 8.3, ArH), 7.4-7.3 (2H, m, 2 x
ArH), 7.15 (1H, dd J 1.0 Hz J 8.3 Hz, ArH), 7.1-7.0 (3H, m, ArH), 5.87 (1H, s, CH), 3.72 (3H, s, Me); δC (75 MHz,
Acetone-d6) 188.4, 181.7, 162.9, 162.3, 157.0, 138.7, 133.6, 132.7, 130.1, 129.5, 128.9, 124.7, 121.8, 120.1, 117.9,
116.1, 56.4; HRMS (ESI) [M+1]+ found 313.0588 [C17H13O4S]+, calcd.313.0531. HPLC purity 94%.
4.1.4 Synthesis of 3-N-Substituted Juglone Derivatives
3-Bromo juglone (7) was synthesised according to the literature [15]. Then, the precursor 7 (1 eq) was dissolved in
acetic acid, followed by the addition of the appropriate amine in large excess (7 eq). The work-up followed the
procedure previously reported.
4.1.4.1 Synthesis of 2-bromo-8-hydroxynaphthalene-1,4-dione (7)
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Yield 59%; δH (400 MHz, DMSO-d6) 11.48 (1H, s, OH), 7.78 (1H, dd J 8.3 Hz J 7.8 Hz, ArH), 7.72 (1H, s, CH),
7.58 (1H, dd J 7.8 J 1.1 Hz, ArH), 7.38 (1H, dd J 8.3 J 1.1, ArH); δC (100 MHz, DMSO-d6) 182.4, 182.3, 160.9,
141.1, 139.5, 137.5, 132.3, 124.6, 119.4, 114.9; HRMS (ESI) found 250.9301 (C10H4O3Br79, M79-H -), 252.9370
(C10H4O3Br81, M81-H -), requires 250.9349 and 252.9330. HPLC purity 95%.
4.1.4.2 Synthesis of 8-hydroxy-2-morpholinonaphthalene-1,4-dione (9)
Yield 39%; δH (400 MHz, Acetone-d6) 12.89 (1H, s, OH), 7.7-7.6 (1H, m, ArH), 7.6-7.5 (1H, m, ArH), 7.24 (1H,
dd J 1.0 Hz J 7.4 Hz, ArH), 6.00 (1H, s, CH), 3.71 (4H, m, 2 x O(CH2)), 3.59 (4H, m, 2 x N(CH2)); δC (100 MHz,
Acetone-d6) 190.1, 187.6, 161.6, 153.2, 137.7, 125.1, 124.9, 120.0, 109.8, 109.6, 67.4, 50.4; HRMS (ESI) [M+1]+
found 260.0912 [C14H14O4N]+ calcd. 260.0911. HPLC purity 96%.
4.1.4.3 Synthesis of 8-hydroxy-2-(piperidin-1-yl)naphthalene-1,4-dione (10)
Yield 39%; δH (400 MHz, Acetone-d6) 13,13 (1H, s, OH), 7.7-7.5 (1H, m, ArH), 7.44 (1H, dd J 6.4 Hz J 1.3 Hz,
ArH), 7.26 (1H, dd J 7.4 Hz J 1.3, ArH), 5.97 (1H, s, CH), 3.6-3.5 (4H, m, 2xCH2), 2.12 (6H, m, 3xCH2); δC (100
MHz, Acetone-d6) 189.8, 189.1. 161.9, 156.4, 135.4, 134.6, 124.9, 119.8, 115.8, 108.2, 51.4, 27.8, 25.3; HRMS
(ESI) [M+1]+ found 258.1123 [C15H16O3N]+ calcd. 258.1120. HPLC purity 99%.
4.1.5 Synthesis of O-Acetyl Juglone Derivatives
4.1.5.1 Synthesis of 1,4-dihydro-1,4-dioxonaphthalen-8-yl acetate (3)
A round bottom flask was charged with 5-hydroxynaphthalene-1,4-dione (juglone, 1 eq), acetic anhydride (5 eq) and
sodium acetate. The mixture was refluxed at 120 °C and stirred overnight. The mixture was then added to a
phosphate buffer (20 mL, pH: 7,4) and extracted with chloroform (3 × 15 ml) and the combined organic layers were
dried (Na2SO4) and evaporated to dryness. Purification was carried out by recrystallization.
13

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Yield 51%; δH (300 MHz, Acetone-d6) 8.06 (1H, dd J 6.6 Hz J 1.3 Hz, ArH), 8.0-7.9 (1H, m, ArH), 7.59 (1H, dd J
6.7 Hz J 1.3 Hz, ArH), 7.07 (1H, d J 10.4 Hz, CH3), 6.97 (1H, d 10.4 Hz, CH2), 2.40 (3H, s, CH3); δC (75 MHz,
Acetone-d6) 185.2, 184.9, 169.9, 150.9, 141.1, 138.7, 136.3, 134.9, 131.1, 125.6, 124.6, 21.4; HRMS (ESI) found
239.2309 (C12H8O4Na, M+H)+ requires 239.0315. HPLC purity 94%.
4.1.5.2 Synthesis of 1,4-dihydro-2-(2,4-dihydroxyphenyl)-1,4-dioxonaphthalen-8-yl acetate (5)
Compound 5 was synthesized according to the general procedure for the synthesis of 3-substituted-5-hydroxy
naphthoquinones previously reported, starting from 5-acetoxy-1,4-naphthoquinone and resorcinol.
Yield 17%; δH (400 MHz, DMSO-d6) 11.87 (1H, s, OH), 10.21 (1H, s, OH), 7.9-7.8 (1H, m, ArH), 7.59 (1H, dd J
1.0 Hz, J 7.4 Hz, ArH), 7.42 (1H, dd J 1.0 Hz, J 7.4 Hz, ArH), 7.34 (1H, d J 8.5 Hz, ArH), 6.93 (1H, s, CH), 6.81
(1H, dd J 2.3 Hz, J 8.5 Hz, ArH), 6,71 (1H, d J 2.3 Hz, ArH), 2.11 (3H, s, CH3); δC (100 MHz, DMSO-d6) 182.2,
175.4, 169.0, 160.9, 158.7, 154.2, 145.8, 138.8, 135.5, 132.3, 128.4, 127.9, 127.2, 121.9, 112.3, 106.5, 100.3, 20.3;
HRMS (ESI) [M+1]+ found 325.0692 [C18H13O6]+calcd. 325.0707. HPLC purity 98%.
4.1.6 Synthesis of 5-Hydroxy-7-Methyl-8-Chloro-1,4-Naphthoquinones
4.1.6.1 Synthesis of 5-chloro-8-hydroxy-6-methylnaphthalene-1,4-dione (14)
A round bottomed flask was equipped with AlCl3 (28 eq) and NaCl (13 eq) were introduced. The mixture was
heated to melt the salts at 180 °C under vigorous stirring and 4-chloro-3-methylphenol (1 eq) and maleic anhydride
(4 eq) were added. After 10 minutes at 180 °C the mixture was poured into ice/water made strongly acid with HCl
12 M, and was stirred at room temperature for half an hour. The precipitate was collected, dried and extracted with
n-hexane in a soxhelet. The solvent was evaporated to dryness.
Yield: 67%; %; δH (400 MHz, Acetone-d6) 12.61 (1H, s, OH), 7.39 (1H, s, ArH), 7.07 (1H, d J 10.3 Hz, ArH), 7.04
(1H, d J 10.3 Hz, ArH), 2.54 (3H, s, CH3); δC (100 MHz, Acetone-d6) 183.1, 182.2, 160.3, 143.7, 139.1, 138.7,
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130.3, 126.6, 122.4, 114.6, 15.5; HRMS (ESI) [M-1]- found 220.9937 [C11H6O3Cl35]- calcd. 221.0001; [M-1]- found
222.9985 [C11H6O3Cl37]- calcd.222.9999. HPLC purity 93%.
4.1.6.2 Synthesis of 5-chloro-8-hydroxy-2-(2,4-dihydroxyphenyl)-6-methylnaphthalene-1,4-dione (15)
Compound 15 was synthesized according to the general procedure for the synthesis of 3-substituted-5-hydroxy
naphthoquinones previously reported, starting from 5-hydroxy-7-methyl-8-chloro-1,4-naphthoquinone and
resorcinol.
Yield: 77%; %; δH (400 MHz, DMSO-d6) 12.54 (1H, s, OH), 9.75 (1H, s, OH), 9.73 (1H, s, OH), 7.47 (1H, s,
ArH), 7.12 (1H, d J 8.4 Hz, ArH), 6.42 (1H, d J 2.3 Hz, ArH), 6.35 (1H, dd J 2.3 Hz J 8.4 Hz, ArH), 2.49, (3H, s,
CH3); δC (100 MHz, DMSO-d6) 183.1, 182.2, 160.3, 159.6, 159.2, 143.7, 139.1, 138.7, 130.3, 129.9, 126.6, 122.4,
114.6, 110.4, 108.9, 103.4, 15.4; HRMS (ESI) [M+1]+ found 331.0372 [C17H10O5Cl35]+ calcd. 331.0368; (ESI)
[M+1]+ found 333.0407 [C17H10O5Cl37]+ calcd. 333.0345. HPLC purity 96%.
4.1.7 Synthesis of Other Naphthoquinones Derivatives
4.1.7.1 Synthesis of 2-(2,4-dihydroxyphenyl)naphthalene-1,4-dione (13)
3-(2,4.diidrossiphenyl)-1,4-naphthoquinone (13) was synthesized following the general procedure reported above
for the synthesis of 3-substituted-5-hydroxy naphtoquinone starting from 1,4-naphthoquinone and resorcinol.
Yield: 37%; δH (400 MHz, DMSO-d6) 9.70 (2H, sb, 2 x OH), 8.00 (2H, m, ArH), 7.89 (2H, m, ArH), 7.07 (1H, d J
8.4 Hz, ArH), 7.02 (1H, s, CH), 6.39 (1H, d J 2.3 Hz, ArH), 6.32 (1H, dd J 2.3 Hz J 8.4 Hz, ArH) ); δC (100 MHz,
DMSO-d6) 186.6, 183.4, 161.0, 160.2, 147.2, 137.1, 136.9, 134.2, 132.3, 132.0, 124.1, 116.2, 115.2, 111.0, 107.7,
103.2; HRMS found (ESI) 267.0667 C16H10O4H (M+1)+, requires 267.0579. HPLC purity 98%.
4.1.8 Naphthazarin naphthalene-1,4-dione (16)
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Naphthazarin (16) was used as received from Aldrich and used without any further purification.
4.1.9 General Procedure for the Synthesis of 1,4,5-Trihydroxy Naphthalenes
1,4,5-trihydroxy naphthalenes were obtained following a previously reported procedure. A round bottom flask was
charged with a solution of Na2S2O4 (5 eq) in water (5 mL), followed by the addition of a mixture of diethyl ether (15
mL) and a solution of the naphthoquinone (1 eq) in dichloromethane (3 mL). The mixture was vigorously stirred at
room temperature for 15 minutes and the organic layer was collected and washed with brine. The solvent was
evaporated to dryness. In order to prevent reoxidation of the compounds to the naphtoquinone form, NMR spectra
and biological assays were carried out in the presence of Na2S2O4. Each NMR spectrum was recorded using a
mixture of DMSO-d6 (800 µL) and D2O (200 µL containing 10 mg of Na2S2O4) as solvent.
4.1.9.1 Synthesis of naphthalene-1,4,5-triol (JUG-R)
Yield 68%; δH (400 MHz, DMSO-d6) 10.71 (1H, s, OH), 10.34 (1H, s, OH), 9.36 (1H, s, OH), 7.50 (1H, dd J 8.4
Hz J 1.1 Hz, ArH), 7.22 (1H, dd J 8.4 Hz J 7.8 Hz, ArH), 6.71 (1H, dd J 7.8 Hz J 1.1 Hz, ArH), 6.66 (1H, dd J 8.0
Hz, ArH), 6.54 (1H, d J 8.0, ArH); δC (75 MHz, DMSO-d6) 154.4, 146.2, 146.0, 127.4, 126.0, 115.4, 113.7, 109.2,
108.8, 108.3. HPLC purity 95%.
4.1.9.2 Synthesis of 3-(2,4-dihydroxyphenyl)naphthalene-1,4,5-triol (1-R)
Yield 59%; δH (400 MHz, DMSO-d6) 9.97 (1H, s, OH), 8.14 (1H, s, OH), 7.43 (1H, dd J 8.1 Hz J 1.0 Hz, ArH),
7.19 (1H, dd J 8.1 Hz J 7.6 Hz, ArH), 7.01 (1H, d J 8.8 Hz, ArH), 6.67 (1H, dd J 7.6 Hz J 1.0 Hz, ArH); 6.66 (1H, s;
ArH), 6.33 (2H, m, ArH); δC (75 MHz, DMSO-d6) 154.7, 154.3, 146.1, 146.0, 142.6 127.8, 125.0, 124.7, 118.4,
115.3, 114.1, 112.7, 110.5, 109.9, 108.6, 108.3. HPLC purity 94%.
16

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4.2 Biological assays.
4.2.1 Cells. GLI36 Human glioma cell line was established by Dr. Anthony Campagnoni (UCLA, Los Angeles, CA,
[28]). Cells were maintained in a monolayer using complete growth medium (CGM): 90% Dulbecco Modified
Eagle’s Medium (DMEM), 10% Foetal Bovine Serum, 100 I.U./mL penicillin, 10 I.U./mL streptomycin, 10 I.U./mL
tetracycline, 25 I.U./mL Plasmocin (InVivogen, Milan, Italy). Cells were incubated at 37 °C in a humidified
environment with 95% air and 5% CO2, up to 80–90% confluence (4–6 days).
4.2.2 MTT assay. Cells were plated in 96-well plates in CGM. After 48 h, addition to CGM of JUG, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, TMZ and paclitaxel (PTX) was carried out to reach the concentrations of 50 µM, 5
µM, 0.5 µM or 0.05 µM, in 10 µL DMSO. TMZ was used as the reference molecule, since it is used for human
glioma treatment. PTX was used as a different anti-tumor agent, not clinically approved for glioma treatment.
Control was performed with 10 µL DMSO. After 24h or 72h, MTT (2.5mg/mL in phosphate buffered saline, PBS)
was added for additional 3h and the percentage of cell viability was obtained. Each analysis was performed in 5
replicates and repeated in 3 independent experiments.
4.2.3 Assessment of apoptosis and necrosis by Wright’s staining. Cells were cultured in 24-well plates on 9 mm
coverslips for 24h in CGM, followed by incubation for 24h or 72h with JUG, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, TMZ and PTX and DMSO as control at different concentrations (50 µM, 5 µM, 0.5 µM or 0.05 µM).
Then, cells were washed in PBS, fixed in methanol for 5 min and Wright’s stained (250 µL/well) for 5 min. Cell
morphology was evaluated under light microscope at 400x magnification. At least 600 cells were counted for each
coverslip in 5 different fields. Each experiment was repeated 2 times.
4.2.4 Statistical Analysis. Statistical analysis was performed with GraphPad Prism5 (GraphPad Software Inc.,San
Diego, CA). The percentage of apoptotic and necrotic cells was calculated, t-test was used to estimate the difference
in apoptotic and necrotic cells between treated and control cells. Results are expressed as the mean ±SEM, p<0.05
was considered as statistically significant.
Supporting Information
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Experimental details and spectra (biological assays, NMR, HPLC, X-ray data) are reported as Supporting
Information. This material is available free of charge via the Internet
Corresponding Author
*Giuseppe Zagotto
giuseppe.zagotto@unipd.it
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via Marzolo 5, 35131 Padova,
Italy.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version
of the manuscript. All the authors contributed equally.
Acknowledgment
M. C. is grateful to Prof. A. Dolmella (Department of Pharmaceutical and Pharmacological Sciences, University of
Padova) for granting access to the Gemini diffractometer and for his help with X-ray data collection and processing.
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Tables and Figures
Figure 1
22

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Figure 2
23

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A
Apoptosis
Apoptosis 50uM
100
80
60
40
20
0
B
Necrosis
Necrosis 50uM
100
80
60
40
20
0
Figure 3
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OH
O
O
O
O
OH
OH
OH
OH OH
OH
OH
OH OH
JUG-R
OH
OH
JUG
1
1-R
Figure 4
25

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A
C
Necrosis 50uM
Apoptosis 50uM
100
100
***
***
***
80
60
40
20
0
80
60
40
20
0
***
***
***
B
D
Apoptosis 5uM
Necrosis 5uM
100
80
60
40
20
0
100
80
60
40
20
0
***
***
***
***
***
***
Figure 5
26

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Figure 6
27

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Epc
Epa
E1/2
∆Ep
Quinone
(V)
(V)
(V)
(mV)
JUG
1
-0.254
-0.256
-0.385
-0.313
-0.291
-0.299
-0.413
-0.292
-0.282
-0.347
-0.208
-0.199
-0.200
-0.263
-0.197
-0.247
-0.355
-0.177
-0.231
-0.195
46
-0.231
-0.228
-0.293
-0.288
-0.244
-0.273
-0.384
-0.235
-0.257
-0.271
57
2
185
50
4
5
94
8
52
9
58
11
14
15
115
51
152
Table 1
28