H2S-Donating Doxorubicins May Overcome Cardiotoxicity and Multidrug Resistance

Article abstract of DOI:10.1021/acs.jmedchem.6b00184

Doxorubicin (DOXO) is one of the most effective antineoplastic agents in clinical practice. Its use is limited by acute and chronic side effects, in particular by its cardiotoxicity and by the rapid development of resistance to it. As part of a program ai

Full text of DOI:10.1021/acs.jmedchem.6b00184

Article
pubs.acs.org/jmc
H2S‑Donating Doxorubicins May Overcome Cardiotoxicity and
Multidrug Resistance
Konstantin Chegaev,† Barbara Rolando,† Daniela Cortese,† Elena Gazzano,‡ Ilaria Buondonno,‡
Loretta Lazzarato,† Marilu Fanelli,§ Claudia M. Hattinger,§ Massimo Serra,§ Chiara Riganti,*,‡
̀
Roberta Fruttero,*,† Dario Ghigo,‡ and Alberto Gasco†
†Department of Science and Drug Technology, University of Torino, via Pietro Giuria 9, 10125 Torino, Italy
‡Department of Oncology and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, via Santena,
5/bis, 10126 Torino, Italy
§Orthopaedic Rizzoli Institute, Laboratory of Experimental Oncology, Pharmacogenomics and Pharmacogenetics Research Unit, via
G. C. Pupilli 1, 40136 Bologna, Italy
S
* Supporting Information
ABSTRACT: Doxorubicin (DOXO) is one of the most effective
antineoplastic agents in clinical practice. Its use is limited by acute and
chronic side effects, in particular by its cardiotoxicity and by the rapid
development of resistance to it. As part of a program aimed at
developing new DOXO derivatives endowed with reduced cardiotox-
icity, and active against DOXO-resistant tumor cells, a series of H2S-
releasing DOXOs (H2S-DOXOs) were obtained by combining DOXO
with appropriate H2S donor substructures. The resulting compounds
were studied on H9c2 cardiomyocytes and in DOXO-sensitive U-2OS
osteosarcoma cells, as well as in related cell variants with increasing
degrees of DOXO-resistance. Differently from DOXO, most of the
products were not toxic at 5 μM concentration on H9c2 cells. A few of
them triggered high activity on the cancer cells. H2S-DOXOs 10 and 11
emerged as the most interesting members of the series. The capacity of 10 to impair Pgp transporter is also discussed.
of antibiotic, and a chronic, cumulative, dose-related form.2,3
The former is characterized by abnormal electrocardiographic
INTRODUCTION
Doxorubicin (DOXO) 1 (Chart 1), known also as Adriamicin,
is a potent broad-spectrum antineoplastic antibiotic, isolated
■
changes, and is rarely a serious problem; the latter can lead to
congestive heart failure that is unresponsive to digitalis. The
mortality rate in patients with congestive heart failure is close to
50%. The production of high levels of reactive oxygen species
Chart 1. Doxorubicin (DOXO)
−
(ROS), including peroxide anion (O2 ), hydrogen peroxide
(H2O2), and hydroxyl radical (OH•), by induction of the
antibiotic’s redox cycle at complex 1 of the mitochondrial
electron transport chain, is the most likely of the mechanisms
proposed to explain this cardiotoxicity.4,5 The heart is very
sensitive to oxidative stress because of its poor antioxidant
defenses compared to other organs.6,7 The cardiomyocyte
damage appears to be principally due to impairment of
mitochondrial functioning.4,7,8
Several synthetic DOXOs characterized by lower cardiotox-
icity have been described. For instance, the DOXO’s analogs N-
trifluoroacetyladriamycin-14-valerate (AD32), N-trifluoroacety-
ladriamycin-14-O-hemiadipate (AD143) and N-benzyladriamy-
cin-14-valerate (AD198) induce lower toxicity in rat hearts
thanks to their different effects on mitochondrial energy
from Streptomyces species, widely used as single agent or in
combination with other anticancer drugs in treating of
hematological cancers and solid tumors, lymphomas, and
sarcomas.1 Its use is accompanied by a number of clinical
toxicities, of which cardiomyopathy is the most important.
Clinically, there are two kinds of cardiomyopathy: an acute
form, with rapid onset after the administration of a single dose
Received: February 10, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1
a
)Reagents and conditions: (a) KF, DMF, rt; (b) dry THF, HCl in dry dioxane.
metabolism.9 Semisynthetic DOXOs conjugated with the
antioxidant ferulic or caffeic acids were less cardiotoxic but
still retain their antitumor efficacy.10 Similarly, a synthetic
doxorubicin targeting mitochondria, which is effective against
ovarian cancer cells, induces at the same time a lower
mitochondria-dependent cardiac damage.11 A critical issue in
the production of DOXO analogs with lower cardiotoxicity is to
prevent the loss of their cytotoxicity against tumors with low
sensitivity to the drug. Indeed, DOXO’s efficacy in cancer
therapy is also hampered by the ease with which resistance to it
develops. This occurs through a number of mechanisms,
principally the increased capacity of the cancer cells to efflux
the drug, thus limiting its cellular accumulation and reducing its
toxicity.12 Interestingly, DOXO resistance is decreased by nitric
oxide (NO) donors, namely compounds able to increase the
intracellular NO concentration.13−17
(HNO).28,29 H2S exhibits strong cytoprotective effects through
a combination of antioxidant and antiapoptotic signals.30−33 It
is able to scavenge ROS and reactive nitrogen species (RNS).33
In particular, it is able to suppress peroxinitrite (HOONO)
induced cell damage, owing to its ability to react with
HOONO, giving rise to sulfinyl nitrite (HSNO2), a new NO
donor.34 A number of recent reports show that sodium
hydrosulfide (NaSH), which at physiological pH is in
equilibrium with H2S, attenuates DOXO-induced cardiotoxicity
in H9c2 embryonic rat cardiac cells, by inhibiting endoplas-
matic reticulum (ER) stress and oxidative stress.35 Inhibition of
the p38 MAPK pathway, activated by DOXO, seems to be an
important mechanism underlying this protection.36 Exogenous
H2S has also recently been shown to attenuate DOXO-induced
cardiotoxicity in H9c2 cells, by inhibiting calreticulin (CRT)
expression.37 On the basis of this rationale, DOXO linked to
H2S donors (H2S-DOXO), namely compounds capable of
releasing H2S under physiological conditions,38−40 might be
expected to give rise to chimeras endowed with improved
biochemical profiles compared to the antibiotic lead.
Hydrogen sulfide (H2S) is a colorless gas with a characteristic
smell of rotten eggs that, as a biochemical agent, has long only
been considered as a poisonous and toxic pollutant. It is a weak
acid (pKa1 ≈ 6.9, pKa2 > 12 at 25 °C), and at physiological pH
(7.4) the two species present in equilibrium are HS− and H2S,
in a ratio of about 3:1. At 37 °C its water solubility is about 80
mM.18 It is able to cross lipid membranes by simple diffusion,
This paper describes a number of such compounds (Scheme
1) obtained by combining DOXO with H2S donors through an
ester linkage at C-14. They are shown to be less cardiotoxic
than the lead, and they maintain high levels of activity against
DOXO-resistant cancer cell lines.
and does not require facilitation by membrane channels.19
A
huge amount of experimental evidence has accumulated in the
past 15 years to show that hydrogen sulfide is an endogenous
gasotransmitter produced from L-cysteine by the action of two
pyridoxal-5′-phosphate (PLP)-dependent enzymes: cystathio-
nine β-synthase (CBS) and cystathionine γ-lyase, also called
cystathionase (CSE).20
Like the other two gasotransmitters, NO and carbon
monoxide (CO), H2S performs a variety of homeostatic
functions, especially in the nervous and cardiovascular
systems.20−25 Several reports indicate that there is cross-talk
between H2S and NO. This relation is complex, and it requires
further investigation. It has been suggested that these two
species may interact, producing an unidentified nitrosothiol,
which triggers a weak vasorelaxing effect, if any.26,27 In addition,
H2S plays roles in modulating the cellular S-nitrosothiol profile,
probably through the formation of thionitrous acid (HSNO),
which is a source of nitrosonium ion (NO+), NO, and nitroxyl
RESULTS AND DISCUSSION
■
Synthesis. The H2S-DOXOs 10−16 were obtained by the
reaction of 14-bromo/chloro daunomicin hydrobromide (2)
with the appropriate H2S donor acids 3−9 (Scheme 1). The
reaction was performed in dry DMF in the presence of KF at
room temperature. The resulting products were purified by
flash chromatography, successively suspended/dissolved in dry
THF, and treated with 1 equiv of HCl in dry dioxane to obtain
the corresponding hydrochlorides. The purity of the com-
pounds was evaluated by RP-HPLC techniques.
Stability of the H2S-DOXOs. The stability of all the new
DOXO derivatives was evaluated by high-performance liquid
chromatography (HPLC) in Dulbecco’s Modified Eagle
Medium (DMEM), in Iscove’s Modified Dulbecco’s Medium
(IMDM), as well as in human serum. The products were
B
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
hydrolyzed following pseudo-first-order kinetics. The half-lives
(t1/2), determined by fitting the data to a one-phase exponential
decay equation, are reported in Table 1.
manipulation of the ester bridge (13), but also transformation
of 2-thioxo-1,3-dithiole-4-carboxylic acid into the ester (12),
decreased H2S release. Compounds 14 and 15, two aliphatic
esters, were markedly less potent H2S donors than 11. Very
similar results were obtained when the experiments were
performed in IMDM (data not shown). In human serum, H2S
release was increased, except in the case of compound 14
(Supporting Information Figure 1).
Table 1. Stability (t1/2) of H2S-DOXOs in Cell Culture
a
Medium (DMEM) and in Human Serum
DMEM
Human serum
Compound
t1/2
t1/2
BIOLOGICAL ASSAYS
■
10
11
12
13
14
15
16
>24 h
21.5 h
17 min
16 min
<1 min
5.6 h
2.8 h
Accumulation of H2S-DOXOs and their cytotoxic
effects in H9c2 cardiomyocytes. We compared the effect
of DOXO and H2S-DOXOs at 5 μM concentration, that we
previously found to be cytotoxic in H9c2 cardiomyocytes10,16,17
and in DOXO-sensitive cancer cells, but not in DOXO-resistant
ones.13,16,42,43 The accumulation of H2S-DOXOs within H9c2-
cardiomyocytes was measured by a fluorimetric assay, and their
cytotoxic effects were assayed by detecting the activity of lactic
dehydrogenase (LDH) in the extracellular medium, considered
as an index of doxorubicin-induced damage.13,15 As shown in
Figure 2A, H2S DOXOs were retained within H9c2 cells at
4.6 h
17 min
3 min
11 min
3.8 h
3.4 h
38 min
a
Data derived from 3−5 independent experiments.
In DMEM, the compounds may be separated into four
clusters: compounds possessing high (10, 11, t1/2 > 20 h),
medium (15, 16, t1/2 = 3−6 h), low (12, 13, t1/2 = 16−17 min),
and very low (14, t1/2 < 1 min) stability (Table 1).
Similar results (data not shown) were obtained in IMDM. In
human serum, the products 12−14 and 16 showed t1/2 < 38
min, while the more stable 10, 11, and 15 showed t1/2 values in
the range 2.8−4.6 h. The t1/2 values measured in the different
media principally reflect the presence of two vulnerable
moieties in the H2S-DOXOs: the ester group, and the H2S
donor substructure. HPLC analysis (see Supporting Informa-
tion) showed that both these moieties contribute to
determining this parameter, in different degrees depending on
the specific product.
Hydrogen sulfide release from the H2S-DOXOs. H2S
release was assessed using a fluorometric assay based upon the
reaction of H2S with dansyl azide (DNS-Az) to give the
fluorescent related amide, which was detected with a HPLC
system equipped with a fluorimetric detector.41 The results of
H2S release in DMEM are reported in Figure 1. The most
potent H2S donor was 11, a derivative of benzoic acid bearing,
in para-position, the 3-thioxo-3H-1,2-dithiol-5-yl group. In-
troduction into 11 of a phenyl substituent at the 4-position of
the thione ring (10) reduced this capacity. Substitution of the
thione ring with a thiocarbamoyl group (16), and in particular
Figure 2. Intracellular accumulation and cytotoxicity of H2S-DOXOs
in cardiomyocytes. H9c2 cardiomyocytes were incubated for 24 h in
fresh medium (CTRL) or in medium containing 5 μM DOXO or H2S-
DOXOs (10−16). (A) The amount of intracellular DOXOs was
measured fluorimetrically in cell lysates in duplicate (n = 3). Data are
presented as means SD. Vs DOXO: * p < 0.002. (B) The release of
extracellular LDH in the culture medium was measured spectrophoto-
metrically in duplicate (n = 3). Vs CTRL: * p < 0.001; vs DOXO: ° p
< 0.05.
similar concentrations as DOXO was. However, while DOXO
induced significant cytotoxicity, the hybrid antibiotics 10−14
were significantly less toxic than the lead, and did not produce
significantly higher toxicity compared to the untreated cells
(Figure 2B).
Figure 1. H2S release assessment from the H2S-DOXOs at 1, 4, and 24
h incubation time in DMEM. Data are presented as mean
SEM
(SEM ≤ 3; number of determinations = 3−5).
C
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Compound 3, the H2S-releasing moiety present in 10, did
not modify the intracellular retention of DOXO within H9c2
cells when co-incubated at an equimolar concentration
(Supporting Information Figure 2A), and it was not toxic
when used alone. Conversely, co-incubation significantly
reduced DOXO’s toxicity, suggesting that the H2S-DOXOs’
lack of cardiotoxicity is due to the presence of the H2S-releasing
moieties (Supporting Information Figure 2B). The case is
different with compounds 15 and 16, which are significantly
more cardiotoxic than the lead (Figure 2B). To shed light on
this difference, the ethyl ester of acid 8 and the methyl ester of
acid 9 (17 and 18 respectively; see Supporting Information),
the H2S donor substructures present in 15 and 16, were tested
for their toxicity on H9c2 cells. The results clearly indicate that
these two products are more toxic than the lead (Supporting
Information Figure 3). It may thus reasonably be supposed that
the cardiotoxicity of H2S-DOXOs 15 and 16 is due to the
presence in their structure of these two moieties.
Effect of H2S-DOXOs on ROS intracellular levels. The
intracellular levels of ROS, which are critical mediators of
DOXO-induced cardiotoxicity,7 were measured by a fluori-
metric assay. DOXO significantly increased the ROS levels in
H9c2 cardiomyocytes; by contrast, the intracellular ROS levels
measured in the same cells treated with H2S-DOXO were
significantly lower than those produced by the parent antibiotic
and did not differ from untreated cells (Figure 3A). To
determine whether the reduced ROS levels were caused by the
release of H2S, ROS levels were again measured in the presence
of hydroxocobalamin (OHCbl), a well-known H2S scavenger.44
The product did not affect the ROS levels in cells treated with
DOXO, but markedly increased them in those exposed to H2S-
DOXOs, producing the same range of ROS measured in
DOXO-treated cells (Figure 3A). In line with this data, OHCbl
was not toxic in untreated cardiomyocytes, and did not modify
the cytotoxicity of DOXO; conversely, it significantly increased
the cytotoxic effects induced by all H2S-DOXOs except for 15
and 16, which showed the opposing trend (Figure 3B).
Compound 3 alone was sufficient to reduce basal ROS levels in
H9c2 cells, an event that was reversed by OHCbl (Supporting
Information Figure 4A). Similarly, the presence of OHCbl
increased the cytotoxicity of 3, alone or co-incubated with
DOXO (Supporting Information Figure 4B). Similarly, the
antioxidant N-acetyl-L-cysteine (NAC), used at a concentration
that significantly reduced the amount of ROS produced by
DOXO (Supporting Information Figure 5A), strongly reduced
the release of LDH (Supporting Information Figure 5B), as did
compounds 10−14. By contrast, the co-incubation of DOXO
with dexrazoxane, which achieves significant cardioprotection
acting through an ROS-independent mechanism,45 failed to
reduce DOXO’s cytotoxicity in H9c2 cells (Supporting
Information Figure 6).
Figure 3. Intracellular ROS and cytotoxicity of H2S-DOXOs in
cardiomyocytes treated with the H2S scavenger hydroxocobalamin.
H9c2 cardiomyocytes were incubated for 24 h in fresh medium
(CTRL) or in medium containing 5 μM DOXO or H2S-DOXOs (10−
16), in the absence (−) or in the presence (+) of 100 μM OHCbl,
chosen as H2S scavenger. (A) The amount of intracellular ROS was
measured fluorimetrically in cell lysates in duplicate (n = 3). Data are
presented as means SD. Vs respective CTRL: * p < 0.01; vs DOXO:
° p < 0.002; − OHCbl vs + OHCbl: p < 0.01 for all compounds (not
shown). (B) The release of extracellular LDH in the culture medium
was measured spectrophotometrically in duplicate (n = 3). Vs
respective CTRL: * p < 0.05; vs DOXO: ° p < 0.05; − OHCbl vs
+ OHCbl: p < 0.05 for all compounds (not shown).
since DOXO is the first-line drug in this kind of tumor;46
however, its efficacy is limited in osteosarcoma expressing P-
glycoprotein (Pgp/ABCB1),47 a membrane transporter that
effluxes DOXO and limits its anticancer efficacy.12 The effects
of H2S-DOXOs in DOXO-sensitive U-2OS osteosarcoma cells,
and in the related cell variants with increasing degrees of
DOXO-resistance, namely U-2OS/DX30, U-2OS/DX100, and
U-2OS/DX580, were investigated. The DOXO IC50 values of
these variants show a progressive increase (Supporting
Information Table 1), according to the increasing expression
of Pgp.48 As expected, DOXO was accumulated in progressively
smaller amounts in the resistant cells (Figure 4A), where it
progressively lost its toxicity (Figure 4B). By screening the
intracellular retention of the H2S-DOXOs, 10, 11, 15, and 16
were identified as being accumulated to a significantly greater
extent than DOXO itself, in both sensitive and resistant cells
(Figure 4A).
In line with their higher intracellular retention, these
compounds were significantly more cytotoxic than DOXO in
both drug-sensitive and drug-resistant cells (Figure 4B). Co-
incubation of 3 with DOXO increased drug retention and
cytotoxicity in U-2OS/DX30 and U-2OS/DX100 cells;
however, this combination did not show superior efficacy
than DOXO in U-2OS/DX580 cells (Supporting Information
Figure 7A, B). These data suggest that the sole presence of a
H2S-releasing group may be sufficient to improve DOXO’s
As demonstrated by the experiments with OHCbl and NAC,
the presence of appropriate H2S-releasing moieties linked to
DOXO or the presence of an excess of an antioxidant molecule
plays a pivotal role in lowering ROS levels and in preventing
toxicity in cardiomyocytes. These results strongly support the
hypothesis that the reduced cardiotoxicity of compounds 10−
14 was due to the decreased intracellular level of ROS.
H2S-DOXOs as effective anticancer agents. Since
oxidative stress is one of the main mechanisms involved in
DOXO’s antitumor activity,7 the next point to clarify was
whether the H2S-DOXOs still retained their activity on cancer
cells. The attention was focused on human osteosarcoma cells,
D
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Intracellular accumulation and cytotoxicity of H2S-DOXOs
in osteosarcoma cells. DOXO-sensitive U-2OS cells and the DOXO-
resistant variants (U-2OS/DX30, U-2OS/DX100, U-2OS/DX580)
were incubated for 24 h in fresh medium (CTRL) or in medium
containing 5 μM DOXO or H2S-DOXOs (10−16). (A) The amount
of intracellular DOXOs was measured fluorimetrically in cell lysates in
duplicate (n = 3). Data are presented as means SD. For DOXO,
resistant cells vs U-2OS cells: * p < 0.05; for all compounds, vs
DOXO: * p < 0.05. (B) Release of extracellular LDH into the culture
medium was measured spectrophotometrically in duplicate (n = 3).
Data are presented as means SD. Vs respective CTRL: * p < 0.01; vs
DOXO: ° p < 0.05.
Figure 5. Intracellular ROS and cytotoxicity of H2S-DOXOs in
osteosarcoma cells treated with OHCbl. DOXO-sensitive U-2OS cells
and the DOXO-resistant variants (U-2OS/DX30, U-2OS/DX100, U-
2OS/DX580) were incubated for 24 h in fresh medium (CTRL) or in
medium containing 5 μM DOXO or H2S-DOXOs (10−16), in the
absence (−) or in the presence (+) of 100 μM OHCbl. (A and B) The
amount of intracellular ROS was measured fluorimetrically in cell
lysates in duplicate (n = 3). Data are presented as means SD. For
panel A: vs respective CTRL: * p < 0.001; vs DOXO: ° p < 0.05. For
panel B: vs respective CTRL: * p < 0.001; − OHCbl vs + OHCbl: p <
0.001.
resistant cells, the efflux kinetics of parental DOXO, DOXO
co-incubated with 3, and 10 were measured in the Pgp
overexpressing U-2OS/DX580 cells. As shown in Figure 6, the
presence of 3 reduced the Vmax of DOXO efflux, without
affecting the Km; by contrast, 10 had a lower Vmax and a higher
Km than DOXO.
These results suggest that the release of H2S by an
appropriate H2S donor impairs the catalytic efficacy of Pgp,
thus explaining the increased intracellular accumulation of
DOXO in the presence of 3. In the case of 10, the presence of
an H2S donor linked to the anthracycline moiety not only
impairs the catalytic activity Pgp, but also reduces the
compound’s affinity for the protein, producing maximal benefit
in terms of drug accumulation and toxicity.
efficacy in mildly chemoresistant cells, but not in strongly
resistant ones. Interestingly, while DOXO increased the ROS
levels in U-2OS cells but not in resistant cells, in sensitive cells
the H2S-DOXOs produced significantly less ROS than DOXO
did (Figure 5A). Of note, 10 and 11, which exerted high
cytotoxicity, also significantly reduced the ROS amount
compared with untreated osteosarcoma cells (Figure 5A).
This reduction of ROS levels is likely due to the presence of the
H2S-releasing groups, since ROS levels were decreased in
osteosarcoma cells exposed to DOXO plus 3 to a significantly
greater extent than they were in cells exposed to DOXO alone
(Supporting Information Figure 7C). The observed increase in
ROS production in cells treated with 10 in the presence of
OHCbl is in line with this result (Figure 5B).
Similar data were obtained with 11 (not shown). These
results indicate that 10 and 11 are cytotoxic in both drug-
sensitive and drug-resistant cells, notwithstanding the lower
ROS production, leading to the hypothesis that they may exert
their anticancer effects through an oxidative stress-independent
mechanism. Several works highlight that part of the anticancer
efficacy of anthracyclines is due to the inhibition of
topoisomerase II.49 By contrast, all H2S-DOXOs were unable
to inhibit this enzyme (Supporting Information Figure 8),
leading to exclusion of the possibility that their anticancer
effects were mediated by this mechanism.
CONCLUSION
■
A series of H2S-releasing moieties were linked through an ester
bridge to C-14 of DOXO to improve its pharmaceutical profile,
in view of their ability to release H2S. All products were tested
on cardiac H9c2 cells, and on U-2OS osteosarcoma cells and
related cell variants with increasing degrees of DOXO
resistance. The specific H2S-releasing moiety strongly influ-
enced the products’ biological behavior. All H2S-DOXOs were
able to reduce the oxidative stress induced by the antibiotic on
the cardiomyocytes, and most of them were significantly less
toxic on the cardiomyocytes than the lead. Compounds 15 and
16 reduced the oxidative stress but not the cardiotoxicity,
paralleling the intrinsic toxicity of the two H2S-releasing
H2S-DOXOs are less effluxed by Pgp in osteosarcoma-
resistant cells. To shed light on how the presence of H2S-
releasing groups improves the efficacy of doxorubicin in
E
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
The purity determination of compounds. RP-HPLC analyses
were run with a HP 1100 chromatograph system (Agilent
Technologies, Palo Alto, CA, USA) equipped with a quaternary
pump (model G1311A), a membrane degasser (G1379A), and a
diode-array detector (DAD) (model G1315B) integrated into the
HP1100 system. Data were processed using a HP ChemStation system
(Agilent Technologies). The analytical column was a Tracer Excel 120
ODS-B (250 × 4.6 mm, 5 μm; Teknokroma, Barcelona). Compounds
were dissolved in the mobile phase and injected through a 20 μL loop
(Rheodyne, Cotati, CA). The mobile phase consisted of 0.1% aqueous
HCOOH (solvent A) and CH3CN (solvent B) and elution was in
gradient mode: initially 35% of solvent B until 5 min, from 35 to 80%
of solvent B between 5 and 10 min, 80% of solvent B until 20 min, and
from 80 to 35% of solvent B between 20 and 25 min. HPLC retention
times (tR) were obtained at flow rates of 1.0 mL·min−1, and the
column effluent was monitored at 234 and 480 nm referenced against
a 700 nm wavelength. All products displayed purity ≥95% with the
exception of 10 and 12 (93% purity).
General procedure for the synthesis of H2S donor doxorubicins
10−16. To a solution of the appropriate acid (1.35 mmol) in dry
DMF, KF (155 mg, 2.70 mmol) was added in one portion, and the
reaction was vigorously stirred for 15 min. 14-Bromo/chloro
daunorubicin hydrobromide (2) (300 mg, 0.45 mmol) was added
and the reaction was stirred at r.t. until completed (LC control).
Solvent was removed under reduced pressure at 30 °C and the
resulting mixture was separated by flash chromatography (eluent:
gradient from 98/2 to 80/20 CH2Cl2/MeOH) to give a red solid. The
resulting compound was dissolved/suspended in dry THF, and 2 equiv
of HCl solution in dry dioxane were added. The resulting mixture was
stirred for 2 h at r.t., then diluted with Et2O and the precipitate was
filtered, washed with Et2O and dried in desiccators to give a title
compound as a red powder.
Figure 6. Efflux kinetics of DOXO, DOXO + 3, and 10, from drug-
resistant osteosarcoma cells. DOXO-resistant U-2OS/DX580 cells
were incubated for 20 min with increasing concentrations (0−50 μM)
of DOXO, DOXO with the H2S-releasing compound 3, or H2S-
DOXO (10). Cells were washed and tested fluorimetrically for their
intracellular drug content. The procedure was repeated on a second
series of dishes, incubated under the same experimental conditions and
analyzed after 10 min. Data are presented as means SD (n = 3). The
rate of DOXOs efflux (dc/dt) was plotted versus the initial
concentration of the drug. Vmax (nmol/min/mg proteins), and Km
(nmol/mg proteins) values were calculated with the Enzfitter software.
DOXO + 3 vs DOXO: p ≤ 0.001, for concentrations 1−50 μM; 10 vs
DOXO: p ≤ 0.001, for concentrations 0.5−50 μM; 10 vs DOXO + 3:
p ≤ 0.01, for concentrations 0.5−50 μM (not shown). For Km, vs
DOXO: * p 0.01; for Vmax, vs DOXO: * p < 0.001; vs DOXO + 3: ° p
< 0.05.
moieties 7 and 8 present in their structures. Compounds 10
and 11 emerged as the most interesting members of the series.
When tested on H9c2 cells, they did not produce a significantly
higher toxicity than the control. In addition, when tested on
sarcoma cell lines, they displayed significantly more potent
cytotoxic effects than those of the lead. Preliminary studies
carried out on 10, taken as a representative member of the
class, suggest that the increased cytotoxicity is likely due to a
reduced efflux of the product by Pgp. Dedicated investigations
to shed light on the action mechanisms of this new class of
modified doxorubicins, as well as in vivo studies of 10 and 11,
are now in progress.
Doxorubicin 14-[4-(4-phenyl-5-thioxo-5H-[1,2]dithiol-3-yl)]-
benzoate (10). Yield: 52%; pHPLC 93% (tR = 12.2 min); mp 166−
1
170 °C (dec.); H NMR (CD3OD + CDCl3) δ ppm: 1.36 (d, J3
=
HH
6.22 Hz, 3H, ′CH3); 1.91 (m, 1H), 2.06 (m, 1H) (2′CH2); 2.19 (m,
1H), 2.51 (m, 1H) (8CH2); 3.05 (m, 1H), 3.28 (m, 1H) (10CH2); 3.35
(m, 1H, ′CH); 3.56 (m, 1H, ′CHOH); 3.79 (br. s, 1H, ′CHOH);
4.08 (s, 3H, OCH3); 4.28 (m, 1H, ′CH); 5.23 (m, 1H, CH); 5.42
(m, 1H), 5.57 (m, 2H, 1′CH and 14CH2); 7.15 (m, 1H, 2CH); 7.36 (m,
2H), 7.51 (m, 5H), 7.83 (m, 2H), (m, 1H.), 8.01 (m, 2H) (CH Ar.);
MS (ESI+) m/z 856 (M+H)+. HRMS (ESI+) m/z calcd for
C43H37NO12S3 (M+H)+ 856.1551, found 856.1551.
6
3
4
4
5
7
EXPERIMENTAL SECTION
■
Chemistry. 1H and 13C NMR spectra were recorded on a
BrukerAvance 300, at 300 and 75 MHz, respectively, using SiMe4 as
internal standard. The following abbreviations indicate peak multi-
plicity: s = singlet, d = doublet, t = triplet, m = multiplet, bs = broad
singlet. ESI MS spectra were recorded on a Micromass Quattro API
micro (Waters Corporation, Milford, MA, USA) mass spectrometer.
Data were processed using a MassLynxSystem (Waters). High
resolution MS spectra were recorded on a Bruker Bio Apex Fourier
transform ion cyclotron resonance (FT-ICR) mass spectrometer
equipped with an Apollo I ESI source, a 4.7 T superconducting
magnet, and a cylindrical infinity cell (Bruker Daltonics, Billerica, MA,
USA). Melting points were determined with a capillary apparatus
Doxorubicin 14-[4-(3-thioxo-3H-1,2-dithiol-4-yl)]benzoate (11).
Yield: 20%; pHPLC 97% (tR = 11.9 min); mp 159−163 °C (dec.); 1H
6
NMR (DMSO-d6) δ ppm: 1.21 (d, J3 = 6.22 Hz, 3H, ′CH3); 1.71
HH
(m, 1H), 1.92 (m, 1H) (2′CH2); 2.12 (m, 1H), 2.38 (m, 1H) (8CH2);
2.89 (d, J2 = 18.3 Hz, 1H), 3.12 (d, J2 = 18.3 Hz, 1H) (10CH2);
HH
HH
4
5
3.62 (m, 1H, ′CHOH); 3.98 (s, 3H, OCH3); 4.29 (m, 1H, ′CH);
4.97 (m, 1H, 7CH); 5.32 (br. s, 1H, 4′CHOH); 5.54 (m, 3H, 1′CH and
14CH2); 5.77 (s, 1H, COH); 7.64 (m, 1H, CH); 7.69 (d, 2H), 7.90
(m, 2H), 8.08 (d, 2H) (CH Ar.); 9.30 (s, 1H, SCH), 13.24 (s, 1H),
14.02 (s, 1H) (2ArOH); MS (ESI+) m/z 780 (M+H)+. HRMS (ESI+)
m/z calcd for C37H33NO12S3 (M+H)+ 780.1238, found 780.1237.
Doxorubicin 14-[2-thioxo-1,3-dithiole-4]carboxylate (12). Yield:
9
2
(Buchi 540) in open capillary. Flash column chromatography was
̈
43%; pHPLC 93% (tR = 13.0 min); mp 190−195 °C (dec.); 1H NMR
performed on silica gel (Merck Kieselgel 60, 230−400 mesh ASTM).
The progress of the reactions was followed by thin-layer
chromatography (TLC) on 5 × 20 cm plates Merck Kieselgel 60
F254, with a layer thickness of 0.20 mm. Anhydrous sodium sulfate
(Na2SO4) was used as drying agent for the organic phases. Organic
solvents were removed under reduced pressure at 30 °C. Synthetic-
purity solvents dichloromethane (DCM), acetonitrile (CH3CN),
methanol (MeOH), diethyl ether (Et2O), diisopropyl ether (i-Pr2O),
dimethylformamide (DMF) and 40−60 petroleum ether (PE) were
used. Dry tetrahydrofuran (THF) was distilled immediately before use
from Na and benzophenone under positive N2 pressure. Dry DMF was
obtained through storage on 4 Å molecular sieves. Commercial
starting materials were purchased from Sigma-Aldrich, Alfa Aesar, and
TCI Europe. For the synthesis of 2−9, see the Supporting
Information.
(DMSO-d6) δ ppm: 1.17 (d, J3 = 6.31 Hz, 3H, ′CH3); 1.70 (m,
6
HH
1H), 1.90 (m, 1H) (2′CH2); 2.09 (m, 1H), 2.30 (m, 1H) (8CH2); 2.88
(d, J2 = 18.0 Hz, 1H), 3.12 (d, J2 = 18.4 Hz, 1H) (10CH2); 3.60
HH
HH
4
5
(m, 1H, ′CHOH); 3.99 (s, 3H, OCH3); 4.24 (m, 1H, ′CH); 4.95
7
4
1
(m, 1H, CH); 5.30 (br. s, 1H, ′CHOH); 5.50 (m, 3H, ′CH and
14CH2); 5.88 (br. s, 1H, COH); 7.67 (m, 1H, CH); 7.93 (m, 2H,
9
2
2CH Ar.); 8.60 (s, 1H, CHS); 13.25 (s, 1H, ArOH); MS (ESI+) m/z
704 (M+H)+; HRMS (ESI+) m/z calcd for C31H29NO12S3 (M+H)+
704.0925, found 704.0929.
Doxorubicin 14-[4-(5-Thioxo-5H-[1,2]dithiol-3-yl)phenoxy]-
acetate (13). Yield: 45%; pHPLC 95% (tR = 14.2 min); mp 159−
162 °C (dec.); 1H NMR (DMSO-d6) δ ppm: 1.16 (d, J3HH = 6.59 Hz,
3H, 6′CH3); 1.71 (m, 1H), 1.89 (m, 1H) (2′CH2); 2.08 (m, 1H), 2.29
(m, 1H) (8CH2); 2.87 (d, J2 = 17.9 Hz, 1H), 3.09 (d, J2 = 18.0
HH
HH
F
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Hz, 1H) (10CH2); 3.60 (m, 1H, 4′CHOH); 3.98 (s, 3H, OCH3); 4.23
(m, 1H, 5′CH); 4.95 (m, 1H, 7CH); 5.09 (s, 2H, CH2COO); 5.29 (br.
s, 1H, 4′CHOH); 5.44 (m, 3H, 1′CH and 14CH2); 5.76 (s, 1H,
9COH); 7.13 (d, 2H, CH Ar.); 7.65 (m, 1H, 2CH); 7.78 (s, 1H, SCH);
7.90 (m, 4H, CH Ar.); 13.23 (s, 1H), 14.02 (s, 1H) (2ArOH); MS
(ESI+) m/z 810 (M+H)+. HRMS (ESI+) m/z calcd for C38H35NO13S3
(M+H)+ 810.1343, found 810.1342.
the serum. The sample was sonicated, vortexed, and centrifuged for 10
min at 2150 g. The clear supernatant was filtered through 0.45 μm
PTFE filters and analyzed by RP-HPLC. All experiments were
performed at least in triplicate.
In both the assays the HPLC analyses were performed with a HP
1100 chromatograph system (Agilent Technologies, Palo Alto, CA,
USA) in the experimental conditions previously described. The
reverse-phase HPLC procedure allowed separation and quantitation of
H2S-DOXOs and of degradation products (DOXO, H2S donor
substructure); quantitation of H2S-DOXOs and of DOXO was done
using calibration curve obtained with standards solutions chromato-
graphed in the same conditions in a concentration range of 1−200 μM
(r2 > 0.99).
Doxorubicin 14-[2(S)-2-acetylamino-3-allylsulfanyl]propionate
(14). Yield: 44%; pHPLC 97% (tR = 10.3 min); mp 143−146 °C
1
(dec.); H NMR (DMSO-d6) δ ppm: 1.16 (d, J3 = 6.59 Hz, 3H,
HH
6′CH3); 1.71 (m, 1H), 1.89 (m, 4H) (2′CH2 and CH3CONH); 2.08
(m, 1H), 2.27 (m, 1H) (8CH2); 2.71 (dd, 1H), 2.83 (d, J2 = 18.0
Hz, 1H), 2.94 (dd, 1H), 3.07 (d, J2 = 18.3 Hz, 1H) (1H0CHH2 and
HH
The half-lives (t1/2) of all the H2S-DOXOs were determined by
fitting the data with one phase exponential decay equation using Prism
software vers. Five (Graph Pad, San Diego, CA, USA).
SCH2CH(NHAc)COO); 3.19 (d, 2H, SCH2CHCH2); 3.60 (m, 1H,
5
4′CHOH); 3.98 (s, 3H, OCH3); 4.24 (m, 1H, ′CH); 4.57 (m, 1H,
CHCOO); 4.94 (m, 1H, 7CH); 5.13 (m, 2H, CHCH2); 5.29 (br. s,
4
1
3H, ′CHOH and 14CH2); 5.47 (m, 1H, ′CH); 5.74 (m, 2H, CH
CH2 and 9COH); 7.65 (m, 1H, 2CH); 7.91 (m, 2H, CH Ar.); 8.48 (d,
1H, NH); 13.22 (s, 1H, ArOH); MS (ESI+) m/z 729 (M+H)+. HRMS
(ESI+) m/z calcd for C35H40N2O13S (M+H)+ 729.2324, found
729.2326.
H2S release in culture medium and human serum. To 1900
μL of culture medium (DMEM or IMDM) or human serum preheated
at 37 °C, 60 μL of dansylazide solution (10 mM in Ethanol) and 40 μL
of H2S-DOXO stock solution (10 mM in DMSO) were added to have
an initial H2S-DOXO concentration of 200 μM. Compounds 10 and
11 were incubated at half concentration due to their low solubility.
The solution was incubated at 37 0.5 °C, and at fixed time (1, 4, and
24 h) 200 μL of reaction mixture was withdrawn and diluted with 200
μL of CH3CN to have a final concentration of compound 100 μM.
The mixture was vortexed, centrifuged for 10 min at 2150 × g. The
clear supernatant was filtered through 0.45 μm PTFE filters and
analyzed by RP-HPLC. All experiments were performed at least in
triplicate. HPLC analyses were performed with a HP 1200
chromatograph system (Agilent Technologies, Palo Alto, CA, USA)
equipped with a quaternary pump (model G1311A), a membrane
degasser (G1322A), a UV detector, MWD (model G1365D) and a
fluorescence detector (model G1321A) integrated in the HP1200
system. Data analysis was done using a HP ChemStation system
(Agilent Technologies). The sample was eluted on Tracer Excel 120
ODSB C18 (250 × 4.6 mm, 5 μm; Teknokroma); injection volume
was 20 μL. The mobile phase consisted of 0.1% aqueous HCOOH and
CH3CN 20/80 v/v; elution was in isocratic mode at a flow rate of 0.8
mL/min. The signals were obtained on fluorescence using an
excitation and emission wavelength of 340 and 535 nm, respectively
(gain factor = 10). Data manipulation was performed by Agilent
ChemStation. The values obtained from integration of the peak of
dansyl amide were interpolated in a calibration line, prepared using
NaHS as a standard, so the concentration of dansyl amide in each
sample is an index of H2S amount.
Cell lines. Rat H9c2 cardiomyocytes and human DOXO-sensitive
U-2OS cells were purchased from ATCC (Manassas, VA) and cultured
in DMEM medium. The corresponding variants with increasing
resistance to DOXO (U-2OS/DX30, U-2OS/DX100, U-2OS/
DX580), selected by culturing parental cells in a medium with 30,
100, 580 ng/mL DOXO, were generated as described elsewhere.50
Culture media were supplemented with 10% FBS, 1% penicillin-
streptomycin, and 1% L-glutamine. Cell cultures were maintained in a
humidified atmosphere at 37 °C and 5% CO2.
Intracellular DOXOs accumulation. The amount of DOXOs in
cell lysates was measured spectrofluorimetrically, as described
elsewhere,13 using a Synergy HT Multi-Detection Microplate Reader
(Bio-Tek Instruments, Winooski, VT). Excitation and emission
wavelengths were 475 and 553 nm, respectively. A cell-free blank
was prepared for each set of experiments, and its fluorescence was
subtracted from that measured in the presence of cells. Fluorescence
was converted to nmol DOXOs/mg cell proteins using a previously
prepared calibration curve.
Doxorubicin 14-(3-allyldisulfanyl)propionate (15). Yield: 42%;
pHPLC 98% (tR = 12.4 min); mp 135−138 °C (dec.); 1H NMR
6
(DMSO-d6) δ ppm: 1.15 (d, J3 = 6.31 Hz, 3H, ′CH3); 1.56 (m,
HH
1H), 1.74 (m, 1H) (2′CH2); 2.05 (m, 1H), 2.29 (m, 1H) (8CH2); 2.71
(d, J2 = 12.4 Hz, 1H) (10CHH), 2.88 (m, 4H, CH2CH2), 3.06 (m,
HH
1H) (10CHH); 3.40 (d, 2H, SCH2CH); 3.57 (m, 1H, 4′CHOH); 3.98
5
7
(s, 3H, OCH3); 4.16 (m, 1H, ′CH); 4.94 (br. s, 1H, CH); 5.21 (m,
4H, 14CH2, CHCH2); 5.60 (br. s, 1H, ′CH); 5.81 (m, 1H, CH
1
CH2) 7.64 (m, 1H, 2CH); 7.92 (m, 2H, CH Ar.); MS (ESI+) m/z 704
(M+H)+. HRMS (ESI+) m/z calcd for C33H37NO12S2 (M+H)+
704.1830, found 704.1829.
Doxorubicin 14-(4-thiocarbamoyl)benzoate (16). Yield: 51%;
pHPLC 99% (tR = 10.5 min); mp 179−183 °C (dec.); 1H NMR
6
(DMSO-d6) δ ppm: 1.20 (d, J3 = 6.31 Hz, 3H, ′CH3); 1.72 (m,
HH
1H), 1.91 (m, 1H) (2′CH2); 2.12 (m, 1H), 2.37 (m, 1H) (8CH2); 2.89
(d, J2 = 18.9 Hz, 1H), 3.13 (d, J2 = 18.9 Hz, 1H) (10CH2); 3.62
HH
HH
4
5
(m, 1H, ′CHOH); 3.98 (s, 3H, OCH3); 4.28 (m, 1H, ′CH); 4.96
7
4
1
(m, 1H, CH); 5.31 (br. s, 1H, ′CHOH); 5.50 (m, 3H, ′CH and
14CH2); 5.82 (s, 1H, COH); 7.65 (m, 1H, CH); 7.96 (m, 6H, CH
Ar.); 9.75 (s, 1H), 10.12 (s, 1H) (NH2); MS (ESI+) m/z 707 (M+H)+.
HRMS (ESI+) m/z calcd for C35H34N2O12S (M+H)+ 707.1905, found
707.1906.
9
2
Chemicals. Fetal bovine serum (FBS) and culture medium were
supplied by Invitrogen Life Technologies (Carlsbad, CA); human
serum (sterile filtered from human male AB plasma) was supplied by
Sigma-Aldrich; plasticware for cell cultures was from Falcon (Becton
Dickinson, Franklin Lakes, NJ). Electrophoresis reagents were from
Bio-Rad Laboratories (Hercules, CA); the protein content of cell
monolayers and lysates was assessed with the BCA kit from Sigma
Chemical Co (St. Louis, MO). Doxorubicin and reagents not specified
were from Sigma Chemical Co.
Stability in culture medium and in human serum. Before
addition of the compound, culture medium (DMEM or IMDM) was
preheated at 37 °C. Then solution of each H2S-DOXO in DMSO was
added to culture medium to have a final concentration of compound
200 μM. The obtained solution was incubated at 37 0.5 °C, and at
appropriate time intervals, an aliquot of 200 μL of sample were
collected and diluted with the same amount of acetonitrile containing
0.1% HCOOH to final concentration of 100 μM. The obtained
mixture was vortexed, filtrated (PTFE filters, 0.45 μM) and
immediately analyzed by RP-HPLC. All experiments were performed
at least in triplicate.
Cytotoxicity assays. To verify the cytotoxic effect of DOXOs, the
extracellular medium was centrifuged at 12,000 × g for 5 min to pellet
cellular debris, while cells were washed with fresh medium, detached
with trypsin/EDTA, resuspended in 0.2 mL of 82.3 mM triethanol-
amine phosphate-HCl (pH 7.6) and sonicated on ice with two 10 s
bursts. Lactic dehydrogenase (LDH) activity was measured in
extracellular medium and cell lysate, as reported elsewhere.16 The
The stability of the H2S-DOXOs to the esterase was evaluated by
incubating the compounds in human serum; the solution of compound
(10 mM in DMSO) was added to human serum preheated at 37 °C;
the final concentration of the compound was 100 μM. The solution
was incubated at 37 0.5 °C, and at appropriate time intervals, an
aliquot of 300 μL of reaction mixture was withdrawn and added to 300
μL of acetonitrile containing 0.1% HCOOH, in order to deproteinize
G
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
reaction was monitored for 6 min, measuring absorbance at 340 nm
with a Synergy HT Multi-Detection Microplate Reader, and was linear
throughout the measurement time. Both intracellular and extracellular
enzyme activities were expressed in μmol NADH oxidized/min/dish.
Extracellular LDH activity was calculated as the percentage of the total
LDH activity occurring in the dish.
To determine the IC50 of DOXO, the cell viability was measured by
the neutral red staining method, as reported elsewhere.42 The
absorbance of untreated cells was considered as 100% viability; the
results are expressed as percentages of viable cells versus untreated
cells. IC50 was considered as the concentration of each drug that
reduces to 50% the cell viability versus untreated cells.
ROS measurement. The amount of intracellular ROS was
measured fluorimetrically as described elsewhere.43 1 × 106 cells
were resuspended in a final volume of 0.5 mL PBS, incubated for 30
min at 37 °C with 5 μM of the fluorescent probe 5-(and-6)-
chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate-acetoxymeth-
yl ester (DCFDA-AM), centrifuged at 13,000 × g at 37 °C and
resuspended in 0.5 mL PBS. The fluorescence of each sample, an
indicator of ROS levels, was read at 492 nm (λ excitation) and 517 nm
(λ emission). The results were expressed as nmol ROS/mg cell
proteins.
ABBREVIATIONS USED
■
DOXO, doxorubicin; Pgp, P-glycoprotein; ROS, reactive
oxygen species; PLP, pyridoxal-5′-phosphate; CBS, cystathio-
nine β-synthase; CSE, cystathionine γ-lyase; RNS, reactive
nitrogen species; ER, endoplasmatic reticulum; MAPK,
mitogen-activated protein kinase; CRT, calreticulin; DMEM,
Dulbecco’s Modified Eagle Medium; IMDM, Iscove’s Modified
Dulbecco’s Medium; DNS-Az, dansyl azide; LDH, lactic
dehydrogenase; OHCbl, hydroxocobalamin; ABCB1, ATP
binding cassette B1; FBS, fetal bovine serum; BCA,
bicinchoninic acid; DCFDA-AM, 5-(and-6)-chloromethyl-
2′,7′-dichlorodihydro-fluorescein diacetate-acetoxymethyl
ester; BSA, bovine serum albumin
REFERENCES
■
(1) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L.
Anthracyclines: molecular advances and pharmacologic developments
in antitumor activity and cardiotoxicity. Pharmacol. Rev. 2004, 56,
185−229.
Doxorubicin efflux. DOXOs efflux kinetics was measured as
described elsewhere.51 Cells were incubated for 20 min with increasing
(0−50 μM) concentrations of parent or synthetic DOXOs, then
washed and analyzed for intracellular DOXO concentration. A second
series of dishes, incubated in the same experimental conditions, were
left for a further 10 min at 37 °C, then washed and tested for
intracellular drug content. The difference of DOXO concentration
between the two series, expressed as nmol DOXOs extruded/min/mg
cell proteins, was plotted versus initial drug concentration. Values were
fitted to the Michaelis−Menten equation to calculate Vmax and Km,
using the Enzfitter software (Biosoft Corporation, Cambridge, United
Kingdom).
(2) Chabner, A.; Amrein, P. C.; Druker, B. J.; Michaelson, M. D.;
Mitsiades, C. S.; Goss, P. E.; Ryan, D. P.; Ramachandra, S.;
Richardson, P. G.; Supko, J. G.; Wilson, W. H. Antineoplastic Agents.
In Goodman & Gilman’s, The Pharmacological Basis of Therapeutic;
Brunton, L., Lazo, J. S., Parker, K. L., Eds.; McGraw-Hill: New York,
Chicago, 2006; pp 1315−1403.
(3) Buzdar, A. U.; Marcus, C.; Smith, T. L.; Blumenschein, G. R.
Early and delayed clinical cardiotoxicity of doxorubicin. Cancer 1985,
55, 2761−2765.
(4) Doroshow, J. H.; Davies, K. J. A. Redox cycling of anthracyclines
by cardiac mitochondria. II. Formation of superoxide anion, hydrogen-
peroxide, and hydroxyl radical. J. Biol. Chem. 1986, 261, 3068−3074.
(5) Simunek, T.; Sterba, M.; Popelova, O.; Adamcova, M.; Hrdina,
R.; Gersl, V. Anthracycline-induced cardiotoxicity: overview of studies
examining the roles of oxidative stress and free cellular iron. Pharmacol.
Rep. 2009, 61, 154−171.
(6) Doroshow, J. H.; Locker, G. Y.; Myers, C. E. Enzymatic defenses
of the mouse heart against reactive oxygen metabolites: alterations
produced by doxorubicin. J. Clin. Invest. 1980, 65, 128−135.
(7) Granados-Principal, S.; Quiles, J. L.; Ramirez-Tortosa, C. L.;
Sanchez-Rovira, P.; Ramirez-Tortosa, M. C. New advances in
molecular mechanisms and the prevention of adriamycin toxicity by
antioxidant nutrients. Food Chem. Toxicol. 2010, 48, 1425−1438.
(8) Berthiaume, J. M.; Wallace, K. B. Adriamycin-induced oxidative
mitochondrial cardiotoxicity. Cell Biol. Toxicol. 2007, 23, 15−25.
(9) Kashfi, K.; Israel, M.; Sweatman, T. W.; Seshadri, R.; Cook, G. A.
Inhibition of mitochondrial carnitine palmitoyltransferases by
adriamycin and adriamycin analogues. Biochem. Pharmacol. 1990, 40,
1441−1448.
Statistical analysis. All data in text and figures are given as means
SD. The results were analyzed by one-way analysis of variance
(ANOVA) and Tukey’s test. p < 0.05 was considered significant.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00184.
Synthesis of intermediate compounds, supplementary
biological tests, HPLC chromatograms of final com-
pounds, 1H and 13C NMR spectra, and hydrolytic profile
of H2S-DOXOs (PDF)
Molecular formula strings for compounds 3−23 (CSV)
(10) Chegaev, K.; Riganti, C.; Rolando, B.; Lazzarato, L.; Gazzano,
E.; Guglielmo, S.; Ghigo, D.; Fruttero, R.; Gasco, A. Doxorubicin-
antioxidant co-drugs. Bioorg. Med. Chem. Lett. 2013, 23, 5307−5310.
(11) Jean, J. R.; Tulumello, D. V.; Riganti, C.; Liyanage, S. U.;
Schimmer, A. D.; Kelley, S. O. Mitochondrial targeting of doxorubicin
eliminates nuclear effects associated with cardiotoxicity. ACS Chem.
Biol. 2015, 10, 2007−2015.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: +39-011-6705857. E-mail: chiara.riganti@unito.it.
*Tel: +39-011-6707850. E-mail: roberta.fruttero@unito.it.
Notes
(12) Gottesman, M. M. Mechanisms of cancer drug resistance. Annu.
Rev. Med. 2002, 53, 615−627.
The authors declare no competing financial interest.
(13) Riganti, C.; Miraglia, E.; Viarisio, D.; Costamagna, C.;
Pescarmona, G.; Ghigo, D.; Bosia, A. Nitric oxide reverts the
resistance to doxorubicin in human colon cancer cells by inhibiting
the drug efflux. Cancer Res. 2005, 65, 516−525.
(14) Fruttero, R.; Crosetti, M.; Chegaev, K.; Guglielmo, S.; Gasco,
A.; Berardi, F.; Niso, M.; Perrone, R.; Panaro, M. A.; Colabufo, N. A.
Phenylsulfonylfuroxans as modulators of multidrug-resistance asso-
ciated protein-1 and P-glycoprotein. J. Med. Chem. 2010, 53, 5467−
5475.
ACKNOWLEDGMENTS
■
This work was supported by grants from Italian Ministry of
University and Research; Future in Research Program (FIRB
2011; grant: RBFR12SOQ1), Italian Association for Cancer
Research (AIRC IG grants), and University of Turin (ricerca
locale 2014), 5‰ contributions to Orthopaedic Rizzoli
Institute.
H
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(15) Chegaev, K.; Riganti, C.; Lazzarato, L.; Rolando, B.; Guglielmo,
S.; Campia, I.; Fruttero, R.; Bosia, A.; Gasco, A. Nitric oxide donor
doxorubicins accumulate into doxorubicin-resistant human colon
cancer cells inducing cytotoxicity. ACS Med. Chem. Lett. 2011, 2,
494−497.
(16) Riganti, C.; Rolando, B.; Kopecka, J.; Campia, I.; Chegaev, K.;
Lazzarato, L.; Federico, A.; Fruttero, R.; Ghigo, D. Mitochondrial-
targeting nitrooxy-doxorubicin: a new approach to overcome drug
resistance. Mol. Pharmaceutics 2013, 10, 161−174.
(17) Gazzano, E.; Chegaev, K.; Rolando, B.; Blangetti, M.;
Annaratone, L.; Ghigo, D.; Fruttero, R.; Riganti, C. Overcoming
multidrug resistance by targeting mitochondria with NO-donating
doxorubicins. Bioorg. Med. Chem. 2016, 24, 967−975.
(18) Kabil, O.; Banerjee, R. Redox Biochemistry of hydrogen sulfide.
J. Biol. Chem. 201028529, 21903−21907.10.1074/jbc.R110.128363
(19) Mathai, J. C.; Missner; Kuegler, A. P.; Saparov, S. M.; Zeidel, M.
L.; Lee, J. K.; Pohl, P. No facilitator required for membrane transport
of hydrogen sulphide. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 16633−
16638.
insight into physiological effects of H2S: reaction with peroxynitrite
and formation of a new nitric oxide donor, sulfinyl nitrite. Biochem. J.
2012, 441, 609−621.
(35) Wang, X.-Y.; Yang, C.-T.; Zheng, D.-D.; Mo, L.-Q.; Lan, A.-P.;
Yang, Z.-L.; Hu, F.; Chen, P.-X.; Liao, X.-X.; Feng, J.-Q. Hydrogen
sulphide protects H9c2 cells against doxorubicin-induced cardiotox-
icity through inhibition of endoplasmatic reticulum stress. Mol. Cell.
Biochem. 2012, 363, 419−426.
(36) Guo, R.; Lin, J.; Xu, W.; Shen, N.; Mo, L.; Zhang, C.; Feng, J.
Hydrogen sulphide attenuates doxorubicin-induced cardiotoxicity by
inhibition of the p38 MAPK pathway in H9c2 cells. Int. J. Mol. Med.
2013, 31, 644−650.
(37) Liu, M.-H.; Zhang, Y.; Lin, X.-L.; He, J.; Tan, T.-P.; Wu, S.-J.;
Yu, S.; Chen, L.; Chen, Y.-D.; Fu, H.-Y.; Yuan, C.; Li, J. E. Hydrogen
sulphide attenuates doxorubicin-induced cardiotoxicity by inhibiting
calreticulin expression in H9c2 cells. Mol. Med. Rep. 2015, 12, 5197−
5202.
(38) Jacob, C. Perspective on recent developments on sulphur-
containing agents and hydrogen sulphide signalling. Planta Med. 2008,
74, 1580−1592.
(20) Szabo, C. Hydrogen sulphide and its therapeutic potential. Nat.
Rev. Drug Discovery 2007, 6, 917−935.
(39) Zhao, Y.; Biggs, T. D.; Xian, M. Hydrofen sulphide (H2S)
releasing agents: chemistry and biological applications. Chem.
Commun. 2014, 50, 11788−11805.
(21) Wang, R. Two’s company, three’s a crowd: can H2S be the third
endogenous gaseous transmitter. FASEB J. 2002, 16, 1792−1798.
(22) Caliendo, G.; Cirino, G.; Santagata, V.; Wallace, J. L. Synthesis
and biological effects of hydrogen sulfide (H2S): development of H2S
releasing drugs as pharmaceuticals. J. Med. Chem. 2010, 53, 6275−
6286.
(40) Kashfi, K.; Olson, K. R. Biology and therapeutic potential of
hydrogen sulphide and hydrogen sulphide-releasing chimeras. Biochem.
Pharmacol. 2013, 85, 689−703.
(41) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer,
D. J.; Wang, B. A fluorescent probe for fast and quantitative detection
of hydrogen sulphide in blood. Angew. Chem., Int. Ed. 2011, 50, 9672−
9675.
(42) Riganti, C.; Kopecka, J.; Panada, E.; Barak, S.; Rubinstein, M.
The role of C/EBP-β LIP in multidrug resistance. J. Natl. Cancer Inst.
2015, 107, 46−59.
(43) Riganti, C.; Gazzano, E.; Gulino, G. R.; Volante, M.; Ghigo, D.;
Kopecka, J. Two repeated low doses of doxorubicin are more effective
than a single high dose against tumors overexpressing P-glycoprotein.
Cancer Lett. 2015, 360, 219−226.
(23) Tan, B. H.; Wong, P. T. H.; Bian, J.-S. Hydrogen sulfide: a novel
signaling molecule in the central nervous system. Neurochem. Int. 2010,
56, 3−10.
(24) Liu, Y.-H.; Yan, C.-D.; Bian, J.-S. Hydrogen sulphide: a novel
signalling molecule in the vascular system. J. Cardiovasc. Pharmacol.
2011, 58, 560−569.
(25) Wang, R. Hydrogen sulfide: a new EDRF. Kidney Int. 2009, 76,
700−704.
(26) Ali, M. Y.; Ping, C. Y.; Mok, Y. Y. P.; Ling, L.; Whiteman, M.;
Bhatia, M.; Moore, P. K. Regulation of vascular nitric oxide in vitro and
in vivo; a new role for endogenous hydrogen sulphide. Br. J.
Pharmacol. 2006, 149, 625−634.
(27) Whiteman, M.; Li, L.; Kostetski, I.; Chu, S. H.; Siau, J. L.; Bhatia,
M.; Moore, P. K. Evidence for the formation of a novel nitrosothiol
from the gaseous mediators nitric oxide and hydrogen sulphide.
Biochem. Biophys. Res. Commun. 2006, 343, 303−310.
(28) Bertova, A.; Cacanyiova, S.; Kristek, F.; Krizanova, O.; Ondrias,
K.; Tomaskova, Z. The hypothesis of the main role of H2S in coupled
sulphide-nitroso signalling pathway. Gen. Physiol. Biophys. 2010, 29,
402−410.
(29) Filipovic, M. R.; Miljkovic, J. L.; Nauser, T.; Royzen, M.; Klos,
K.; Shubina, T.; Koppenol, W. H.; Lippard, S. J.; Ivanovic-Burmazovic,
I. Chemical characterization of the smallest S-nitrosothiol, HSNO;
cellular cross-talk of H2S and S-nitrosothiols. J. Am. Chem. Soc. 2012,
134, 12016−12027.
(30) Elrod, J. W.; Calvert, J. W.; Morrison, J.; Doeller, J. E.; Kraus, D.
W.; Tao, L.; Jiao, X.; Scalia, R.; Kiss, L.; Szabo, C.; Kimura, H.; Chow,
C.-W.; Lefer, D. J. Hydrogen sulfide attenuates myocardial ischemia-
reperfusion injury by preservation of mitochondrial function. Proc.
Natl. Acad. Sci. U. S. A. 2007, 104, 15560−15565.
(31) Calvert, J. W.; Jha, S.; Gundewar, S.; Elrod, J. W.;
Ramachandran, A.; Pattillo, C. B.; Kevil, C. G.; Lefer, D. J. Hydrogen
sulfide mediates cardioprotection through Nrf2 signaling. Circ. Res.
2009, 105, 365−374.
(32) Guo, W.; Kan, J.-T.; Cheng, Z.-y.; Chen, J.-F.; Shen, Y.-Q.; Xu,
J.; Wu, D.; Zhu, Y.-Z. Hydrogen sulfide as an endogenous modulator
in mitochondria and mitochondria dysfunction. Oxid. Med. Cell.
Longevity 2012, 2012, 1−9.
(44) Truong, D.; Hindmarsh, W.; O’Brien, P. J. The molecular
mechanisms of diallyl disulphide and diallyl sulphide induced
hepatocyte cytotoxicity. Chem.-Biol. Interact. 2009, 180, 79−88.
(45) Rharass, T.; Gbankoto, A.; Canal, C.; Kursunluoglu, G.; Bijoux,
A.; Panakova, D.; Ribou, A.-C. Oxidative stress does not play a primary
role in the toxicity induced with clinical doses of doxorubicin in
myocardial H9c2 cells. Mol. Cell. Biochem. 2016, 413, 199−215.
(46) Hattinger, C. M.; Fanelli, M.; Tavanti, E.; Vella, S.; Ferrari, S.;
Picci, P.; Serra, M. Advance in emerging drugs for osteosarcoma.
Expert Opin. Emerging Drugs 2015, 20, 495−514.
̀
(47) Baldini, N.; Scotlandi, K.; Barbanti-Brodano, G.; Manara, M. C.;
Maurici, D.; Bacci, G.; Bertoni, F.; Picci, P.; Sottili, S.; Campanacci, M.;
Serra, M. Expression of P-glycoprotein in high-grade osteosarcomas in
relation to clinical outcome. N. Engl. J. Med. 1995, 333, 1380−1385.
(48) Fanelli, M.; Hattinger, C. M.; Vella, S.; Tavanti, E.; Michelacci,
F.; Gudeman, B.; Barnett, D.; Picci, P.; Serra, M. Targeting ABCB1
and ABCC1 with their specific inhibitor CBT-1® can overcome drug
resistance in osteosarcoma. Curr. Cancer Drug Targets 2016, 16, 261−
274.
(49) Apetoh, L.; Mignot, G.; Panaretakis, T.; Kroemer, G.; Zitvogel,
L. Immunogenicity of anthracyclines: moving towards more
personalized medicine. Trends Mol. Med. 2008, 14, 141−151.
(50) Serra, M.; Scotlandi, K.; Manara, M. C.; Maurici, D.; Lollini, P.
L.; De Giovanni, C.; Toffoli, G.; Baldini, N. Establishment and
characterization of multidrug-resistant human osteosarcoma cell lines.
Anticancer Res. 1993, 13, 323−329.
(51) Kopecka, J.; Salzano, G.; Campia, I.; Lusa, S.; Ghigo, D.; De
Rosa, G.; Riganti, C. Insights in the chemical components of liposomes
responsible for P-glycoprotein inhibition. Nanomedicine 2014, 10, 77−
87.
(33) Lowicka, E.; Beltowski, J. Hydrogen sulfide (H2S): the third gas
of interest for pharmacologists. Pharmacol. Rep. 2007, 59, 4−24.
(34) Filipovic, M. R.; Miljkovic, J.; Allgaeuer, A.; Chaurio, R.;
Shubina, T.; Herrmann, M.; Ivanovic-Burmazovic, I. Biochemical
I
DOI: 10.1021/acs.jmedchem.6b00184
J. Med. Chem. XXXX, XXX, XXX−XXX