N-Hydroxyphthalimide catalysts as bioactive pro-oxidants

Article abstract of DOI:10.1039/c5ra26556h

The catalytic role of N-hydroxyphthalimide (NHPI) in promoting free-radical hydrogen atom transfer (HAT) reactions, well-documented for processes of industrial and synthetic interest, is here investigated for the first time in a biological environment. While NHPI by itself did not show any bioactivity, selected NHPI-derivatives (NHPIDs) revealed the ability to activate the intracellular formation of reactive oxygen species (ROS), causing the depletion of glutathione (GSH) levels and an increase in oxidative stress (OS). The evident bioactivity of some of these derivatives resulted in a significant reduction of the viability in osteosarcoma MG-63, suggesting a new, potential role of NHPIDs as pro-oxidant drugs. The key role of the N-OH group in promoting oxidative stress is demonstrated.

Full text of DOI:10.1039/c5ra26556h

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N-Hydroxyphthalimide catalysts as bioactive pro-
oxidants†
Cite this: RSC Adv., 2016, 6, 21749
ab
a
a
a
L. Melone, P. Tarsini, G. Candiani* and C. Punta*
The catalytic role of N-hydroxyphthalimide (NHPI) in promoting free-radical hydrogen atom transfer (HAT)
reactions, well-documented for processes of industrial and synthetic interest, is here investigated for the
first time in a biological environment. While NHPI by itself did not show any bioactivity, selected
NHPI-derivatives (NHPIDs) revealed the ability to activate the intracellular formation of reactive oxygen
species (ROS), causing the depletion of glutathione (GSH) levels and an increase in oxidative stress (OS).
The evident bioactivity of some of these derivatives resulted in a significant reduction of the viability in
osteosarcoma MG-63, suggesting a new, potential role of NHPIDs as pro-oxidant drugs. The key role of
the N–OH group in promoting oxidative stress is demonstrated.
Received 12th December 2015
Accepted 17th February 2016
DOI: 10.1039/c5ra26556h
www.rsc.org/advances
Due to its unique catalytic role, in the last decade this
derivative has attracted increasing attention, and its use has
NHPI is a cheap molecule, which is emerging as a valuable been also suggested for processes of industrial relevance.
1
. Introduction
5,6
organocatalyst capable of promoting free-radical aerobic oxida-
tion by activating hydrogen atom transfer (HAT) processes.
We also showed how NHPI and related derivatives (NHPIDs)
1
are effective in catalysing the peroxidation of polyunsaturated
7,8
The efficiency and selectivity of this catalyst have been fatty acids (PUFAs). In that case the nal purpose was the
related to the favourable combination of enthalpic, polar, and selective synthesis of PUFAs' corresponding hydroperoxides.
2
entropic effects.
However, by considering that PUFAs are important components
As depicted in Scheme 1, the catalytic cycle implies the in situ of the cellular membrane and environment, the manifested
generation of phthalimide-N-oxyl (PINO) radical by several efficiency of this process prompted us to further investigate the
1
3
metal and metal-free initiators.
possible catalytic action of NHPIDs in promoting oxidative
PINO, whose formation was proved by Electron Spin Reso- stress (OS) in a biological environment in vitro.
4
nance (ESR) spectroscopy, is the real responsible of the cata-
The reasons of this interest rely on the still controversial role
lytic activity of NHPI in oxidative processes, being directly played by OS in regulating cancer development and apoptosis.
involved in the free-radical propagation step by hydrogen
atom abstraction from different activated organic substrates
(Scheme 1, path i). The resulting carbon centred radicals
undergo molecular oxygen addition, leading to the formation of
the corresponding peroxyl radicals (Scheme 1, path ii).
The catalytic cycle is completed by the HAT reaction from
a new NHPI molecule to the peroxyl radical (Scheme 1, path iii),
favouring the concomitant formation of hydroperoxides and
a new PINO unit, so that the radical chain is maintained.
Moreover, the possible presence of transition metal ions
would favour the reduction of the hydroperoxide, leading to the
formation of alkoxyl radicals which in turn are even faster in
abstracting hydrogen from C–H bonds and from NHPI itself
(Scheme 1, path iv).
a
Department of Chemistry, Materials, and Chemical Engineering “Giulio Natta”.
Politecnico di Milano, Piazza L. Da Vinci 32 and INSTM local unit, 20133 Milano,
Italy. E-mail: gabriele.candiani@polimi.it; carlo.punta@polimi.it
b
Universit a` degli Studi e-Campus, Via Isimbardi 10, Como, 22060 Novedrate, Italy
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c5ra26556h
Scheme 1 General free-radical cycle for the NHPI-catalysed oxidation
of organic substrates.
1
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9
OS occurs when redox homeostasis within the cell is altered.
solutions suggested to overcome the industrial limit of NHPI
20
While experimental evidences show that an increase in the related to its solubility.
cellular concentrations of reactive oxygen species (ROS), like
Compounds 1-OH and 2-OH were obtained starting from
alkoxyl and peroxyl radicals, can promote DNA damage, angio- trimellitic anhydride chloride, aer optimization of the experi-
1
0
21
genesis, and metastasis, more recently researchers recognized mental procedure reported by Ishii for the synthesis of 2-OH.
the possible role of ROS in cancer therapy, leading to the Similarly, compound 3-OH was synthesized in the presence
conclusion that, to consider OS as outright toxic would be of the purposely prepared (R)-2-(1-hydroxybutan-2-yl)-1H-benzo
11
a mistake. In fact, it was reported that an increase of intracel- [de]isoquinoline-1,3(2H)-dione, following the approach recently
lular ROS content to cytotoxic levels would selectively induce reported by our group. Interestingly, specically functional-
apoptotic cell death in cancer. Thereby, besides the investiga- ized naphthalimides have already shown good anticancer
tion of natural and synthetic agents with antioxidant activity,
the development of pro-oxidant agents capable of inducing OS is intercalation affinity towards DNA. The cytotoxic properties of
nowadays emerging as an intriguing anticancer strategy.
22
9
12,13
activity upon a variety of human cancer cells due to their high
23
14–16
these derivatives were usually ascribed to the side chains
Herein, for the rst time we demonstrate that the pro- present on the naphthalene moiety. However, data reported in
oxidant catalytic activity of NHPIDs, originally developed for the literature suggested that this target group would favor
industrial applications, can be exerted also in vitro to address cellular internalization, rendering derivative 3-OH attractive for
relevant biological issues. Striking outcomes of the study our purpose. Moreover, we recently reported how catalyst 3-OH
include (i) the key role that lipophilic tails on the aromatic ring could be photo-activated by UV radiation, further promoting
22
play in favouring the interaction of NHPI with cells; (ii) the aerobic oxidation.
association between the cytotoxic action of NHPIDs and their In addition, we also synthesized compound 4-OH, a bis-
catalytic activity as pro-oxidants, evidenced by the increase of NHPI lipophilic catalyst, by reacting hydroxylamine hydro-
0
0
OS, followed by the depletion of intracellular glutathione chloride with 4,4 -(4,4 -isopropylidenediphenoxy)bis (phthalic
L-g-glutamyl-L-cysteinyl-glycine, GSH) levels and consequently anhydride), a cheap and commercially available di-anhydride
(
apoptotic cell death; (iii) the importance of having free N–OH obtained from bisphenol A. The catalytic efficiency and the
moieties in order to exhibit a pro-oxidant activity, suggesting high lipophilic character of compound 4-OH have been already
that the catalytic action in vitro could still follow the free-radical documented by our group, for the solvent-free selective aerobic
20
HAT mechanism described in Scheme 1.
oxidation of alkyl aromatics.
NHPIDs were tested in MG-63 cancer cell line to check their
potential, concentration-dependent, cytotoxicity. The results
are reported in Fig. 1. Compounds 2-OH, 3-OH, and 4-OH dis-
played EC50 at 100, 200 and 110 mM, respectively (Fig. 1C–E, full
2. Results and discussion
Human osteosarcoma MG-63 cells were chosen as target recip- black cycles), well below the values for the poorly cytotoxic
17,18
ients because being responsive to OS insult.
compound 1-OH (Fig. 1B).
Disappointingly, rst experiments carried out in the pres-
These observations prompted us to further investigate the
ence of increasing amounts of NHPI for 24 h showed that it did reasons for such a bioactivity. If our original assumption was
induce signicant cytotoxicity only at extremely high concen- correct, the decrease in cell viability following challenging with
trations (EC50 ¼ 7 mM), as determined by AlamarBlue® uori- compounds 2-OH, 3-OH, and 4-OH had to be due to a signi-
metric assay (Fig. 1A). We thus hypothesized that this behavior cant elevation of the OS. In fact, being NHPIDs 2-OH, 3-OH, and
would be associated to the strong polar character of NHPI, a key 4-OH more lipophilic than, and as pro-oxidant as 1-OH, they
19,20
factor already investigated for industrial applications.
In would be expected to interact with cells and exert potential pro-
biological systems, high polarity could drastically reduce the oxidant effects to a greater extent than the latter molecule.
reactivity of NHPI towards the plasma membrane, preventing Moreover, onto consideration of the HAT mechanism according
its effective interaction with the cell.
to which the NHPIDs promote oxidation (Scheme 1), we wanted
In order to ascertain this, we synthesized NHPIDs bearing to be sure that the eventual pro-oxidant action was specic to
different lipophilic tails (Fig. 2), reproducing the same synthetic these compounds and relied on the presence of the N–OH
Fig. 1 Cytotoxicity of (A) NHPI, (B) 1-OH, (C) 2-OH vs. 2-Me (full black cycles vs. empty squares, respectively), (D) 3-OH vs. 3-Me (full black cycles
vs. empty squares, respectively) and (E) 4-OH vs. 4-Me (full black cycles vs. empty squares, respectively) after 24 h of exposure as a function of
concentration in MG-63 human osteosarcoma cells.
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Fig. 2 NHPIDs and corresponding N-methyl derivatives.
group, rather than to the introduction of the lipophilic tails. To related to such OS insult, as conrmed by the signicantly
do so, we challenged osteosarcoma cells with compounds 2-OH, greater GSH depletion with the parent molecule 4-OH, if
3
-OH, and 4-OH and the purposely synthesized N-methyl compared to 4-Me compound (71 ꢀ 4% vs. 92 ꢀ 7%, respec-
molecules 2-Me, 3-Me, and 4-Me (Fig. 1C–E, empty squares vs. tively, p < 0.05) (Fig. SI9†).
full black cycles, respectively). In this way we could evaluate and
We would like to point out that these results cannot be taken
compare the eventual cytotoxicity exclusively associated to the for granted. From one point of view, the aqueous environment
graing of the phthalimide aromatic ring (Fig. SI8†). In line could inhibit NHPIDs' catalytic properties by means of the
with our expectations, 2-Me to 4-Me derivatives were far less hydrogen bonding involving N–OH moiety. On the other hand,
cytotoxic than parent N-OHs.
we operated for the rst time at really lower concentrations of
Following these observations, we moved to consider the the catalyst with respect to classical synthetic applications. It is
possible occurrence of OS both directly, by evaluating the in fact well known that peroxidation kinetics promoted by NHPI
1
increase of ROS, and indirectly, by measuring GSH depletion. In are signicantly dependent on catalyst concentration.
fact, chemotherapy agents (e.g. cisplatin) are detoxied by
The difference noticed among the EC50 of 2-OH (100 mM),
antioxidant defense mechanisms, such as enzymatic conjuga- 3-OH (200 mM) and 4-OH (110 mM) highlights one more the key
24
tion to the tripeptide GSH. Abrogation of such drug-resistant role played by the lipophilic tails, suggesting that a proper
mechanisms by redox modulation would thus have signicant design of the nitroxide pro-oxidant would allow to improve its
therapeutic implications.
biological performance. Such a difference is emphasized by the
Interestingly, we herein show the generation of large quan- inherent behavior of the resulting 2-OH to 4-OH but not 1-OH,
tities of ROS induced by the treatment with compounds 2-OH, to aggregate. In our experimental conditions, 2-OH, 3-OH, and
3
ꢀ
-OH, and 4-OH used each at its respective EC50 (130 ꢀ 5%, 294 4-OH compounds did form monodisperse aggregates with
20%, 234 ꢀ 23%, all p < 0.05 vs. CTRL cells). Furthermore, our hydrodynamic diameters comprised between 136 and 628 nm
results about OS induced by derivatives 2-Me, 3-Me, and 4-Me (Fig. SI10–SI12†).
all not statistically signicant vs. CTRL cells) (Fig. 3) pointed It is worth noting that, although also 2-Me, 3-Me, and 4-Me
out that the mechanism whereby NHPIDs 2-OH, 3-OH, and do form aggregates in water in our experimental conditions
-OH induce cytotoxicity is associated to the N–OH moiety. (Fig. SI13–SI15†), sometimes (2-Me) with lower, sometimes
(
4
Indeed, injury to cells occurs only when ROS overwhelm the (3-Me, 4-Me) with higher hydrodynamic diameters than the
9
biochemical defense of the cell. Moreover, GSH consumption corresponding N-hydroxy derivatives, they are in any case much
25
that mirrors the depletion of cellular antioxidant defenses, is less cytotoxic (Fig. 1).
Fig. 3 Oxidative stress (OS) levels expressed in terms of reactive
oxygen species (ROS) production in untreated MG-63 cells (CTRL Fig. 4 Caspase-3 and -7 activities in untreated MG-63 cells (CTRL
group) as compared to 1-OH (200 mM), 2-OH and 2-Me (100 mM, group) as compared to 1-OH (200 mM), 2-OH and 2-Me (100 mM,
both), 3-OH and 3-Me (200 mM, both), and 4-OH and 4-Me-treated both), 3-OH and 3-Me (200 mM, both), and 4-OH and 4-Me-treated
human osteosarcoma cells (110 mM, both).
human osteosarcoma cells (110 mM, both).
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This conrms once again the key function of any N–OH possibility of the development of novel anticancer chemicals
moiety. Overall, the inherent tendency of pro-oxidants 2-OH to and strategies based on NHPI catalysis.
4-OH to aggregate do have a twofold advantage over less lipo-
philic molecules as 1-OH. First, submicrometric aggregates can
be easily endocytosed by cells. Moreover, apart from being pro-
4. Experimental
oxidant drugs per se, –OHs do form aggregates in water that 4.1. General
might offer place to accommodate hydrophilic, lipophilic as
All reagents used in this work are commercially available
well as amphiphilic drugs. This colloidal behavior would extend
their use as chemotherapeutic carriers or drug delivery
(
Sigma-Aldrich, Italy) and were used as received without further
1
13
purication unless otherwise stated. H and C-NMR of the
products were recorded at room temperature (r.t.) with a Bruker
Avance-400 NMR spectrometer. Compounds 1–4 were stored
26
systems.
Excess OS kills or damages cells mostly by apoptosis, and
27–29
eventually by other mechanisms.
In the former case, alter-
ꢁ
dried at 4 C and solubilized in dimethyl sulfoxide (DMSO)
ations in the redox status of the cell to a more oxidizing envi-
ronment occurs prior to activation of the nal phase of cysteine
aspartic acid-specic protease known as caspases that orches-
trate the execution phase of apoptosis by cleaving multiple
before use.
4.2. Synthetic procedures
9
structural and repair proteins. Between the fourteen caspases
4.2.1. Synthesis of NHPIDs 1-OH, 2-OH, and 3-OH.
identied in mammals, a subset of downstream, or executioner Compound 3-OH was obtained according to the procedure
22
caspases (Caspase-3, and -7) is actually responsible for the previously described, by adding dropwise and under stirring at
30
ꢁ
demolition phase of apoptosis. We thereby focused our 0 C for ꢂ30 min, 20 mL of an anhydrous THF solution of (R)-2-
attention on the primary effector caspases necessary for trig- (1-hydroxybutan-2-yl)-1H-benzo[de]isoquinoline-1,3 (2H)-dione
gering the cleavage of the vast majority of subcellular proteins. (10 mmol) into 20 mL of an anhydrous solution of THF con-
As shown in Fig. 4, Caspase-3 and -7 activity was signicantly taining trimellitic anhydride chloride (10 mmol, 2.106 g) and
ꢁ
higher in cells treated with any of the NHPIDs as compared to 4 mL of anhydrous pyridine. The mixture was kept at 0 C for 6 h
each respective –Me derivative (597 ꢀ 47% vs. 126 ꢀ 7% for and then overnight at r.t. Aer ltrating the white precipitate,
2
6
-OH and 2-Me, 679 ꢀ 25% vs. 104 ꢀ 6% for 3-OH and 3-Me, the clear liquid was vacuum evaporated obtaining a white solid
.664 ꢀ 223% vs. 126 ꢀ 7% for 4-OH and 4-Me, all p < 0.05), that was dissolved in pyridine (ꢂ40 mL) and reacted with
strengthening the idea that overt cytotoxicity specically NH OH$HCl (50 mmol) in microwave for 1 h under reux. The
2
induced by 2-OH, 3-OH, and 4-OH was linked to OS insult. This, evaporation of the solvent and the washing of the mixture with
ꢁ
in turn, led to some GSH depletion and engages cells in caspase- 0.1 M HCl at 0 C resulted in a white solid that was puried by
mediated apoptotic cell death.
chromatography on silica-gel using 9 : 1 (v : v) CHCl : MeOH as
3
f 3
eluent (R : 0.60 – orange color spot if TLC is exposed to NH ).
ꢁ
1
Yield: 90%. Mp: 115.0 C. H-NMR (400 MHz, DMSO-d6)
d (ppm): 10.91 (s, 1H), 8.49 (d, 2H), 8.42 (d, 2H), 8.18 (d, 1H),
3
. Conclusions
8
.01 (s, 1H), 7.84 (m, 1H + 2H), 5.43 (s, 1H), 4.95 (dd, 1H), 4.76
1
In summary, lipophilic NHPIDs 2-OH, 3-OH, and 4-OH were (dd, 1H), 2.21 (m, 1H), 2.018 (m, 1H), 0.97 (t, 3H). H-NMR and
1
3
synthesized and their activity as pro-oxidant catalysts was
substantiated in vitro in osteosarcoma MG-63 cells.
C-NMR spectra of this derivative is reported in Fig. SI4.†
Compounds 1-OH and 2-OH were synthetised similarly to
20
The overt cytotoxicity of these derivatives was associated to 3-OH, following the procedures previously described, rst
an increase of OS levels, and to a consequent depletion of preparing the ester from MeOH and dodecyl alcohol respec-
intracellular GSH content, leading to caspase activation and tively and trimellitic anhydride chloride. The corresponding
apoptotic cell death. Interestingly, the markedly lower bioac- NHPIDs 1-OH and 2-OH were obtained using NH
2
OH$HCl and
tivity of the N-methyl derivatives 2-Me, 3-Me, and 4-Me shed puried by chromatography on silica-gel using 9 : 1 (v : v)
light on the key role played by N–OH group in promoting CHCl
3
: MeOH as eluent (R
f
: 0.60 (3), 0.76 (4) – orange color spot
ꢁ
oxidation even in vitro, suggesting the occurrence of the clas- if TLC is exposed to NH
). Yield: 94%. Mp: 172.0 C (1-OH),
3
ꢁ
1
sical HAT process.
85.0 C (2-OH). H NMR of 1-OH (400 MHz, acetone-d6) d (ppm):
Taken together, these observations provide unambiguous 9.95 (broad s, 1H), 8.43 (d, 1H), 8.28 (s, 1H), 7.96 (d, 1H), 3.98
1
evidence that NHPI, an oxidation catalyst widely used in (s, 3H). H-NMR of 2-OH (400 MHz, DMSO-d6) d (ppm): 8.44 (d,
a plethora of abiotic industrial applications, once tethered to 1H), 8.31 (s, 1H), 7.96 (s, 1H), 4.40 (t, 2H), 1.83 (q, 2H), 1.6–1.2
1
13
suitable lipophilic moieties is a promising pro-oxidant in vitro. (m, 18H), 0.88 (t, 3H). H-NMR and C-NMR spectra of these
It is worth noting that, in this preliminary investigation, our derivatives are reported in Fig. SI1 and 2,† respectively.
aim was to verify an hypothesis, that is the potential bioactivity
of NHPIDs, rather than to design the best drug solutions for in prepared according to the procedure previously described.
vitro and in vivo applications.
4.2.2. Synthesis of NHPID 4-OH. Compound 4-OH was
20
0 0
4,4 -(4,4 -Isopropylidenediphenoxy)bis(phthalic anhydride) was
Even though further investigation is necessary to make these puried by recrystallization from acetic anhydride. 5 g of dia-
catalysts more effective and selective towards target cells, we nhydride (commercially provided as yellowish akes) were
ꢁ
believe that these preliminary results may open up the dispersed in 40 mL of acetic anhydride and reuxed at 139 C
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ꢃ2
for 1 h in a microwave reactor with the automatic control of a fresh aliquot (300 mL cm ), supplemented with 1.5% (v : v)
power (Micro-SYNTH Labstation – Milestone Inc., USA). Aer DMSO-containing compounds at the desired concentration and
cooling at r.t. pure crystals were recovered by ltration on lter kept in contact with cells for 24 h before analysis. Cells grown in
paper and extensively washed with cold diethyl ether in order to complete medium containing 1.5% (v : v) DMSO were consid-
remove the acetic anhydride. The white crystals were dried ered as untreated controls (CTRL).
under vacuum and stored in desiccator until next use.
4.3.3. Cell viability test. Cytotoxicity was evaluated using
0
In a 50 mL two-necked ask, 2.60 g (5 mmol) of puried 4,4 - AlamarBlue® cell viability assay (Thermosher Scientic, Italy)
0
(
4,4 -isopropylidenediphenoxy)bis(phthalic anhydride) were according to manufacturer's guidelines. Briey, at the end of
added to 30 mL of anhydrous pyridine. Aer adding an excess of treatment, the cell culture medium was replaced with fresh
hydroxylamine hydrochloride (30 mmol, 2.85 g), the mixture medium containing 10% of AlamarBlue® reagent and aer 2 h
ꢁ
ꢁ
was heated under reux (119 C) for 1 h in a microwave reactor. of incubation at 37 C, uorescence (lex ¼ 540 nm; l
¼
em
Aer cooling the pyridine was evaporated under vacuum, 595 nm) was measured by a multimethod plate reader (GENios
obtaining a viscous orange product which was dissolved in Plus, TECAN, Italy). Experiments were performed at least
ethylacetate (150 mL). The organic phase was extensively 3 times in triplicate and cell viabilities were expressed as
washed with 0.5 M HCl (100 mL, 3 times) and deionized water percentage of CTRL cells (assigned as 100%). Cytotoxicity (%)
(
dH
2
O, 100 mL, 3 times) in order to remove the residual pyri- was calculated as follows: 100 (%) – viability (%).
4.3.4. Evaluation of oxidative stress (OS) levels. OS was
dine and nally dried with anhydrous sodium sulphate. The
evaporation of the solvent gave a pale yellow foam, which was monitored by evaluating the uorescence of 2 ,7 -dichloro-
puried by chromatography on silica-gel using 9 : 1 (v : v) uorescein (DCF) produced by intracellular deesterication and
CHCl : MeOH as eluent (R : 0.54 – orange color spot if TLC is oxidation of 2 ,7 -dichlorodihydrouorescein diacetate (DCFH-
exposed to NH
0
0
0
0
3
f
1
3
). Yield: 93%. H-NMR of 4-OH (400 MHz, DA). Briey, at the end of treatment, cells were washed with
ꢁ
DMSO-d6): d ¼ 10.77 (s, 2H), 7.80 (d, 2H), 7.34 (d, 4H), 7.29 (d, PBS, incubated for 15 min at 37 C with 10 mM DCFH-DA in
1
13
2
H), 7.24 (s, 2H), 7.08 (d, 4H), 1.69 (s, 6H). H-NMR and C- Phosphate Buffered Saline (PBS), washed twice with the same
NMR spectra of this derivative is reported in Fig. SI6.† buffer and lysed with 0.5% (v : v) Tween in 50 mM Tris–HCl
.2.3. Synthesis of methylated derivatives. Compounds buffer, pH 7.5 for at least 10 min at r.t. Cells were then scraped,
4
2
4
-Me, 3Me and 4-Me were prepared similarly to 2-OH, 3-OH and collected and subjected to 3 consecutive freezing/thawing cycles
ꢁ
-OH using MeNH $HCl in place of hydroxylamine hydrochlo- (ꢃ80 C/r.t.) and the uorescence in cell lysates measured by
2
ride. The NMR spectra of all these derivatives are reported means of a microplate reader (l ¼ 485 nm; l ¼ 530 nm),
ex
em
in ESI.†
-Me. Yield: 95%; H-NMR (400 MHz, CDCl
then normalized over the total protein content of each sample
): d (ppm): 8.44 as determined by bicinchoninic acid (BCA) protein assay kit
1
2
3
(
(
d, 1H), 8.37 (s, 1H), 7.88 (s, 1H), 4.44 (t, 2H), 3.19 (s, 1H), 1.77 (Pierce, USA). Experiments were performed at least 3 times in
q, 2H), 1.45–1.10 (m, 18H), 0.85 (t, 3H).
quadruplicate and values expressed as percentage of CTRL cells.
1
3-Me. Yield: 94%; H-NMR (400 MHz, CDCl
3
) d (ppm): 8.59 (d,
4.3.5. Quantication of intracellular GSH content. For
2
2
H), 8.33 (s, 1H), 8.26 (d, 1H), 8.21 (d, 2H), 7.79 (d, 1H), 7.75 (t, evaluating the total intracellular glutathione (GSH) content
H), 5.55 (m, 1H), 5.05 (dd, 1H), 4.85 (dd, 1H), 2.34 (m, 1H), 2.09 aer treatment, cells were trypsinized, harvested, centrifuged at
3
(m, 1H), 1.01 (t, 3H).
5 ꢄ 10 g for 5 min and washed once with PBS. Pellets were
1
4
-Me. Yield: 94%; H-NMR (400 MHz, CDCl
3
) d (ppm): 7.77 lysed in 5% (w : v) sulfosalicylic acid (SSA) in dH
2
O and sub-
ꢁ
(
(
d, 2H), 7.36 (d, 4H), 7.29 (d, 2H), 7.24 (s, 2H), 7.00 (d, 4H), 3.15 jected to 2 freezing/thawing cycles (ꢃ80 C/r.t.). Aer an incu-
ꢁ
s, 6H), 1.73 (s, 6H).
bation of 10 min at 4 C, lysates were nally deproteinized by
1
13
3
H-NMR and C-NMR spectra of these derivatives are re- centrifugation for 10 min at 6 ꢄ 10 g and GSH levels deter-
ported in Fig. SI3, SI5 and SI7,† respectively.
mined in supernatants with Glutathione assay kit according to
the manufacturer's instructions. Experiments were performed
at least twice in triplicate and values, normalized for protein
content, were expressed as percentage of CTRL cells.
4
.3. Biological procedures
4.3.1. Cell cultures. MG-63 cells (human osteosarcoma cell
4.3.6. Biophysical characterization of aggregates. The
line, CRL-1427) were purchased from the American Type existence and the size (hydrodynamic diameter) of aggregates
ꢁ
Culture Collection (ATCC, USA) and cultured at 37 C, 99% were determined by Dynamic Light Scattering (DLS) utilizing
2
humidity with regular supply of 5% CO , in complete medium Zetasizer Nano ZS (ZEN3500, Malvern Instruments Ltd., UK).
consisting in Dulbecco's Modied Eagle Medium (D-MEM) Briey, all compounds were solubilized in DMSO, diluted
added with 10% (v : v) of Fetal Bovine Serum (FBS), 1 mM 66 times in 10 mM Hepes buffer to the nal concentration of
ꢃ1
sodium pyruvate, 10 mM Hepes buffer, 100 U mL penicillin, 200, 100, 200 mM, and 110 mM for compounds 1, 2, 3, and 4,
ꢃ1
ꢁ
0
.1 mg mL streptomycin, and 2 mM glutamine.
4
respectively, and incubated 30 min at 37 C before analysis. All
ꢁ
.3.2. Cell treatment. Cultures were continuously kept in measurements were performed at 37 C in a nal volume of 500
exponential-phase growth before experiments. The day before mL. Single measurements were repeated at least twice in inde-
the beginning of the treatments, cells were trypsinized, plated at pendent experiments. Representative intensity distributions are
4
2
the density of 1.5 ꢄ 10 cells per cm and allowed to adhere shown in Fig. SI10–SI15.†
overnight. 24 h post-seeding, the medium was replaced with
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