Synthesis and initial in vitro evaluations of novel antioxidant aroylhydrazone iron chelators with increased stability against plasma hydrolysis

Article abstract of DOI:10.1021/tx100359t

Oxidative stress is known to contribute to a number of cardiovascular pathologies. Free intracellular iron ions participate in the Fenton reaction and therefore substantially contribute to the formation of highly toxic hydroxyl radicals and cellular injur

Full text of DOI:10.1021/tx100359t

ARTICLE
pubs.acs.org/crt
Synthesis and Initial in Vitro Evaluations of Novel Antioxidant
Aroylhydrazone Iron Chelators with Increased Stability against
Plasma Hydrolysis
Katerina Hruskova, Petra Kovarikova, Petra Bendova, Pavlina Haskova, Eliska Mackova, Jan Stariat,
Anna Vavrova, Katerina Vavrova,* and Tomas Simunek*
Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic
ABSTRACT: Oxidative stress is known to contribute to a number
of cardiovascular pathologies. Free intracellular iron ions participate in
the Fenton reaction and therefore substantially contribute to the
formation of highly toxic hydroxyl radicals and cellular injury. Earlier
work on the intracellular iron chelator salicylaldehyde isonicotinoyl
hydrazone (SIH) has demonstrated its considerable promise as an
agent to protect the heart against oxidative injury both in vitro and in
vivo. However, the major limitation of SIH is represented by its labile hydrazone bond that makes it prone to plasma hydrolysis. Hence, in
order to improve the hydrazone bond stability, nine compounds were prepared by a substitution of salicylaldehyde by the respective methyl-
and ethylketone with various electron donors or acceptors in the phenyl ring. All the synthesized aroylhydrazones displayed significant iron-
chelating activities and eight chelators showed significantly higher stability in rabbit plasma than SIH. Furthermore, some of these chelators
were observed to possess higher cytoprotective activities against oxidative injury and/or lower toxicity as compared to SIH. The results of the
present study therefore indicate the possible applicability of several of these novel agents in the prevention and/or treatment of cardiovascular
disorders with a known (or presumed) role of oxidative stress. In particular, the methylketone HAPI and nitro group-containing NHAPI
merit further in vivo investigations.
’ INTRODUCTION
ROS production. The latter can be achieved with Fe-chelating
agents, which have been clinically used for the reduction of
excessive body iron accumulation in disorders such as β-
thalassemia.6,7 Here, effective Fe chelation has been particularly
beneficial due to the reduction of cardiac mortality caused by
excessive myocardial Fe loading and prevention of ROS-
mediated myocardial damage.8 Besides the Fe-overloading con-
ditions, a growing body of evidence suggests that the concept of
Fe chelation may also be useful in preventing Fe-mediated
aggravation of oxidative stress, which can occur locally even in
the presence of normal body Fe stores.9 Indeed, the clinically
approved agent dexrazoxane (ICRF-187) is supposed to reduce
the cardiotoxicity of redox-cycling anthracycline anticancer drugs
by binding free cellular Fe and preventing site-specific oxidative
stress within cardiac tissue.10
Salicylaldehyde isonicotinoyl hydrazone (SIH) is a tridental
chelator selectively forming 2:1 complexes with Fe3þ. Because of
its low molecular weight and optimal lipophilicity, SIH can be
administered orally. It readily enters the cells, firmly chelates the
intracellular LIP, and is therefore able to very efficiently block the
Fe-dependent production of hydroxyl radicals. SIH has been
previously shown as very effective in the protection of guinea pig
or rat isolated cardiomyocytes and H9c2 cells as well as other cell
types against the H2O2-induced cell injury, apparently due to the
An imbalance in cellular redox state, in which the production
of reactive oxygen species (ROS) overwhelms antioxidant capac-
ity, results in the state termed oxidative stress.1 Recent evidence
suggests that oxidative stress is a common denominator in many
aspects of cardiovascular pathogenesis. Its important role is now
established in ischemia-reperfusion myocardial injury, cardiac
arrhythmias, congestive heart failure, myocarditis, anthracycline-
induced cardiomyopathy, atherosclerosis, or hypertension.1-3
Iron (Fe) is an essential element for all living cells. However,
unless appropriately complexed, this transition metal may catalyze
the Fenton reaction which results in the formation of extremely
reactive and toxic hydroxyl radicals that readily attack various
biological molecules. Therefore, organisms are equipped with
specific proteins designed for Fe acquisition, transport, and storage,
as well as with sophisticated mechanisms that maintain the intra-
cellular labile iron pool (LIP) at an appropriate level. Despite these
homeostatic regulations, under certain pathological states, cells face
the threat of Fe toxicity. High cellular levels of superoxide and
peroxide species, as well as low pH (e.g., during ischemia), are able
to release Fe from its storage proteins, increase the redox-active LIP,
and thus further promote the production of ROS.4,5
Despite the well-known pathophysiological role of ROS,
conventional treatment of cardiovascular diseases still lacks the
use of antioxidants due to negative study outcomes. Unfortu-
nately, efforts to reduce oxidative stress have focused primarily
on the use of ROS scavengers, rather than on actual prevention of
Received: October 21, 2010
Published: January 07, 2011
r
2011 American Chemical Society
290
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Chemical Research in Toxicology
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preservation of mitochondrial function and/or lysosomal
integrity.11-14 SIH also efficiently protected H9c2 cells against
oxidative injury induced by tert-butyl hydroperoxide.15 In the
latter study, SIH showed the best ratio of cytoprotective efficiency
and own inherent toxicity of seven Fe chelating agents from several
distinct chemical families including three clinically used drugs: the
bacterial siderophore desferrioxamine (DFO), hydroxypyridinone
deferiprone (L1, CP20) and triazole deferasirox (ICL670A).15 In
addition, SIH has also been shown to efficiently protect against the
cardiotoxicity of anthracycline daunorubicin, both in vitro using
isolated neonatal rat cardiomyocytes16 and in vivo using the chronic
rabbit model of anthracycline-induced heart failure.17 A recent study
demonstrated marked protective efficiency of SIH against cellular
injury by gamma irradiation.18 Low in vivo toxicity and good
tolerability of SIH has been demonstrated following its 10-week
repeated administration to rabbits.19 Furthermore, aroylhydrazone
pro-chelators have been recently developed in which the Fe-binding
groups are chemically masked, and the active chelator SIH is
generated only at sites of ROS production.20-22
Despite these promising pharmacodynamic findings, a pilot
pharmacokinetic study of SIH has revealed the relatively short
biological half-life of this drug candidate following its single
intravenous administration to rabbits,23 apparently due to its
labile hydrazone bond which makes the molecule prone to
hydrolysis.23-25 Other studies showed that this feature is not specific
for SIH; it is rather a class effect of all aroylhydrazones formed by the
coupling of isoniazide with aromatic aldehydes.26,27 Such rapid
plasma clearance results in a need for large bolus doses with transient
very high peak plasma concentrations, which may have been
responsible for the reduction of cardioprotective effectiveness of
SIH upon dose escalation.28 Hence, although SIH has been shown
to be effective and safe, the pharmacokinetic weakness may
represent a serious obstacle for its further development and potential
use in clinical practice. Therefore, the improvement of the plasma
stability of SIH through modification of its chemical structure may
strengthen the therapeutic potential of this class of compounds.
Our ultimate goal is to find an effective chelator with low
toxicity and improved hydrolytic stability compared to those of SIH.
The aim of this first initial study was to synthesize and screen a series
of SIH-derived aroylhydrazones in order to identify the variables
that determine their hydrolytic stability. At the same time, we aimed
at studying the Fe-chelating properties of the evaluated agents in
solution and access to LIP in the cardiac-derived cell line H9c2, and
in vitro assessments of cellular protective actions of the chelators
against model oxidative injury as well as determinations of the
individual toxicities of these substances.
temperature (RT), we added H2O (10 mL), and the mixture was allowed to
crystallize at 4 °C. The product was collected by filtration, washed with H2O,
and dried over P2O5 under reduced pressure to give 0.77 g (77%) of
1
yellowish crystals, mp 240-242 °C (lit.30 235-237 °C). H NMR (300
MHz, Me2SO): δ 13.19 (1H; s; OH); 11.59 (1H; s; NH); 8.79 (2H; d; J =
4.4 Hz; Py); 7.84 (2H; d; J = 5.3 Hz; Py); 7.65 (1H; d; J = 7.6 Hz; Ph); 7.32
(1H; dd; J = 5.9 Hz; J = 7.6 Hz; Ph); 6.92 (1H; d; J = 8.2 Hz; Ph); 6.89 (1H;
d; J = 7.8 Hz; Ph); 2.49 (3H; s; CH3). 13C NMR (75 MHz, Me2SO): δ
163.2; 159.7; 159.0; 150.4; 140.3; 131.8; 129.0; 122.2; 119.4; 118.8; 117.6;
14.5. IR (KBr): νmax 3436; 3116; 1681; 1642; 1610; 1535; 1495; 1304;
1298 cm-1
.
(E)-N0-[1-(2-Hydroxyphenyl)propyliden]isonicotinoylhydra-
zide (HPPI). Isoniazid (0.255 g, 1.9 mmol) and 2-hydroxypropiophenone
(0.28 g, 1.9 mmol) in 96% ethanol (5 mL) were stirred under reflux in a
microwave reactor (400 W) for 30 min. Then, acetic acid (0.25 mL, 4.4
mmol) was added and the reaction continued for a further 30 min. The
reaction mixture was allowed to crystallize at 4 °C. The solid was filtered,
washed with H2O, and recrystallized from ethanol to yield 0.24 g (48%) of
yellowish crystals, mp 244-246 °C (lit.31 248 °C). 1H NMR (300 MHz,
Me2SO): δ 13.28 (1H; s; OH); 11.61 (1H; s; NH); 8.79 (2H; d; J = 5.9 Hz;
Py);7.80(2H;d;J=5.5Hz;Py);7.64(1H;d;J= 7.5 Hz; Ph); 7.31 (1H; dd;
J = 8.4 Hz; J = 7.2 Hz; Ph); 6.92 (1H; d; J = 8.3 Hz; Ph); 6.89 (1H; d; J = 7.2
Hz; Ph); 3.01 (2H; q; J = 7.6 Hz; CH2); 1.14 (3H; t; J = 7.6 Hz; CH3). 13
C
NMR (75 MHz, Me2SO): δ 163.6; 162.7; 159.5; 150.3; 140.5; 131.7; 128.6;
122.4; 118.9; 117.9; 117.9; 19.6; 11.6. IR (KBr): νmax 3436; 3139; 1683;
1603; 1528; 1488; 1279 cm-1
.
(E)-N0-[1-(2,4-Dihydroxyphenyl)ethyliden]isonicotinoylhydra-
zide (2,4DHAPI). Isoniazid (0.51 g, 3.7 mmol), 2,4-dihydroxyacetophe-
none (0.56 g, 3.7 mmol), and acetic acid (1 mL, 17.5 mmol) in 96% ethanol
(10 mL) were stirred under reflux for 9 h. The reaction mixture was cooled to
RT, and after addition of H2O (10 mL), the product was obtained by filtration
and washed with H2O. Recrystallization from ethanol gave 0.35 g (35%) of
yellowish crystals, mp 284-286 °C. 1H NMR (300 MHz, Me2SO): δ 13.38
(2H; s; OH); 11.41 (1H; s; NH); 8.78 (2H; d; J = 5.6 Hz; Py); 7.82 (2H; d;
J = 5.8 Hz; Py); 7.46 (1H; s; Ph); 6.40-6.35 (1H; m; Ph); 6.30-6.25 (1H;
m; Ph); 2.41 (3H; s; CH3). 13C NMR (75 MHz, Me2SO): δ 162.8; 161.1;
160.9; 160.7; 150.4; 140.5; 130.4; 111.6; 107.2; 103.4; 14.4. IR (KBr): νmax
3525; 3087; 1671; 1617; 1507; 1328; 1271 cm-1
.
(E)-N0-[1-(2,6-Dihydroxyphenyl)ethyliden]isonicotinoylhydra-
zide (2,6DHAPI). Isoniazid (0.28 g, 2 mmol), 2,6-dihydroxyacetophe-
none (0.255 g, 1.7 mmol), and acetic acid (0.25 mL, 4.4 mmol) in 96%
ethanol (5 mL) were stirred under reflux in a microwave reactor (400 W)
for 5 h. After cooling the reaction mixture to RT, the resulting product was
filtered off, washed with H2O, and dried over P2O5 affording 0.132 g
1
(26%) of yellowish crystals, mp 245-247 °C. H NMR (300 MHz,
Me2SO): δ 11.41 (2H; s; OH); 11.06 (1H; s; NH); 8.78 (2H; d; J = 4.3
Hz; Py); 7.83 (2H; d; J = 4.3 Hz; Py); 7.05-7.00 (1H; m; Ph); 6.37 (2H;
d; J = 8.1 Hz; Ph); 2.45 (3H; s; CH3). 13C NMR (75 MHz, Me2SO): δ
162.9; 161.3; 158.2; 150.4; 140.5; 131.3; 122.1; 110.5; 107.4; 19.4. IR
’ EXPERIMENTAL PROCEDURES
(KBr): νmax 3369; 3173; 1656; 1603; 1529; 1450; 1286 cm-1
.
(E)-N0-[1-(2-Hydroxy-4-methoxyphenyl)ethyliden]isonicotinoyl-
hydrazide (MHAPI). Isoniazid (0.53 g, 3.9 mmol), 2-hydroxy-4-methoxy-
acetophenone (0.64 g, 3.9 mmol), and acetic acid (0.2 mL, 3.5 mmol) in
96% ethanol (10 mL) were stirred under reflux in a microwave reactor (400
W) for 2 h. The reaction mixture was cooled to 4 °C. The resulting product
was filtered, washed with H2O, and dried over P2O5 giving 0.24 g (24%) of
yellowish crystals, mp 219-222 °C. 1H NMR (300 MHz, Me2SO): δ 13.51
(1H; s; OH); 11.47 (1H; s; NH); 8.74 (2H; d; J = 5.9 Hz; Py); 7.83 (2H; d;
J = 6.0 Hz; Py); 7.56 (1H; d; J = 8.4 Hz; Ph); 6.50 (2H; d; J = 2.6 Hz;
Ph); 6.47 (1H; s; Ph); 3.77 (s; OCH3); 2.45 (3H; s; CH3). 13C NMR
(75 MHz, Me2SO): δ 162.9; 162.2; 161.1; 160.1; 150.4; 140.4; 130.2; 122.2;
112.7; 106.1; 101.7; 55.5; 14.4. IR (KBr): νmax 3346; 3150; 1678; 1618;
Syntheses of Chelators. All chemicals were purchased from
Sigma-Aldrich (Schnelldorf, Germany). Thin layer chromatography was
performed on TLC sheets (silica gel 60 F254) from Merck (Darmstadt,
Germany). Microwave reactions were conducted in a Milestone Micro-
SYNTH Ethos 1600 URM apparatus. Melting points were measured on a
Kofler apparatus and are uncorrected. All products were characterized by IR
(Nicolet Impact 400 spectrophotometer) and NMR (Varian Mercury-Vx
1
BB 300 instrument, H at 300 MHz and 13C at 75 MHz) spectra. The
reference chelator SIH was synthesized as described previously.29
(E)-N0-[1-(2-Hydroxyphenyl)ethyliden]isonicotinoylhydrazide
(HAPI). Isoniazid (0.54 g, 3.9 mmol), 2-hydroxyacetophenone (0.53 g,
3.9 mmol), and acetic acid (1 mL, 17.5 mmol) in 48% ethanol (10 mL) were
stirred under reflux for 9 h. After cooling the reaction mixture to room
1511; 1264; 1138 cm-1
.
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Chemical Research in Toxicology
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(E)-N0-[1-(5-Chloro-2-hydroxyphenyl)ethyliden]isonicotinoyl-
hydrazide (CHAPI). Isoniazid (0.24 g, 1.8 mmol) and 5-chloro-2-hydroxy-
acetophenone (0.29 g, 1.7 mmol) in 96% ethanol (5 mL) were stirred under
reflux in a microwave reactor (200 W) for 1 h. Acetic acid (0.25 mL, 4.4
mmol) was added and the reaction continued for a further 1 h. The resulting
solid was filtered and washed with H2O. The filtrate was collected and cooled
overnight at 4 °C to obtain another portion of product. The combined solids
were recrystallized from ethanol and dried over P2O5 affording 0.23 g (46%)
of yellowish crystals, mp 236-238 °C (lit. 242 °C31). 1H NMR (300 MHz,
Me2SO): δ 13.26 (1H; s; OH); 11.66 (1H; s; NH); 8.79 (2H; d; J = 5.6 Hz;
Py);7.84(2H;d;J= 6.0 Hz; Py); 7.66 (1H; d; J= 2.6 Hz; Ph); 7.34 (1H; dd;
J = 2.6 Hz; Ph); 6.95 (1H; d; J = 8.8 Hz; Ph); 2.49 (3H; s; CH3). 13C NMR
(75 MHz, Me2SO): δ 163.4; 158.2; 157.6; 150.4; 140.1; 131.3; 128.2; 122.4;
122.3; 120.9; 119.4; 14.6. IR (KBr): νmax 3443; 3177; 1682; 1602; 1527;
Table 1. Chromatographic Conditions Used in the Determina-
tion of the Stability of the New Chelators and SIH in Plasmaa
mobile phase composition
pH of
ratio
UV
chelator
SIH
aqueous partb
organic part
(v/v) (nm)
I.S.
6
6
6
6
6
6
6
3
6
6
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH/ACNc 50:50
MeOH/ACNc 40:60
53:47
55:45
60:40
54:46
55:45
288
288
288
326
300
328
285
288
288
325
o-108
o-108
o-108
SIH
HAPI
HPPI
2,4HAPI
2,6HAPI
MHAPI
CHAPI
NHAPI
A2,4DHAPI
AHCI
o-108
SIH
1289; 675 cm-1
.
SIH
(E)-N0-[1-(2-Hydroxy-5-nitrophenyl)ethyliden]isonicotinoyl-
hydrazide (NHAPI). Isoniazid (0.23 g, 1.7 mmol) and 2-hydroxy-5-nitro-
acetophenone (0.31 g, 1.7 mmol) in 96% ethanol (5 mL) were stirred at
reflux in a microwave reactor (400 W) for 1 h. After the mixture was cooled to
4 °C, the resulting product was obtained by filtration and washed with H2O.
The recrystallization from ethanol gave 0.317 g (63%) of orange crystals, mp
274-276 °C. 1H NMR (300 MHz, Me2SO): δ 14.39 (1H; s; OH); 11.86
(1H; s; NH); 8.80 (2H; d; J = 5.7 Hz; Py); 8.46 (2H; d; J = 2.8 Hz; Py);
8.21-8.17 (1H; m; Ph); 7.90-7.85 (1H; m; Ph); 7.11 (1H; d; J = 9.1 Hz;
Ph); 2.56 (3H; s; CH3). 13C NMR (75 MHz, Me2SO): δ 164.6; 163.6;
157.5; 150.4; 139.9; 139.3; 127.0; 125.0; 122.3; 119.5; 118.6; 14.6. IR (KBr):
MeOH
MeOH/ACNc 50:50
MeOH 42:58
58:42
SIH
SIH
SIH
a I.S. = internal standard; ACN = acetonitrile; MeOH = methanol. b 10
mM Na2HPO4 and 2 mM EDTA. c A mixture of MeOH/ACN in a 1:1
ratio (v/v).
were processed using LC solution software, version 1.21 SP1 (Shimadzu,
Duisburg, Germany).
The analyses of the Fe chelators were performed on an analytical
column (LiChrospher 250 mm ꢀ 4.6 mm, RP-18, 5 μm) protected with
a guard column, both purchased from Merck (Darmstadt, Germany).
The mobile phase was composed of a mixture of 10 mM phosphate
buffer (with the addition of 2 mM EDTA) and either methanol or
methanol/acetonitrile mixture in differing ratios. A flow rate of
1.0 mL/min, a column temperature of 25 °C, and an injection volume of
20 μL were used for the analysis. The quantification was made at the
maximum absorption wavelength of the particular chelator. Either SIH
or pyridoxal 2-chlorobenzoyl hydrazone (o-108) was used as the internal
standard (I.S.). Detailed chromatographic conditions for each chelator
are given in Table 1.
The linearity, precision, and accuracy of the methods were tested by
the analysis of the sets of plasma samples spiked with different amounts
of the chelators. Selectivity was confirmed by an analysis of blank plasma
samples. All methods fulfilled the general validation criteria for bioana-
lytical methods.32 SIH was analyzed using a previously developed and
validated HPLC method.27
Assessments of Chelator Stabilities against Hydrolysis in
Rabbit Plasma. The drug-free plasma was spiked with a standard
solution of each chelator (1 mg/mL) to obtain a concentration of
100 μM. It was incubated in a water bath set at 37 ( 0.5 °C and mixed
(120 rpm) during the entire experiment. At defined time intervals,
0.3 mL of plasma was transferred into Eppendorf tubes, and I.S. was
added. The samples were treated by precipitation with acetonitrile
(0.4 mL); the clear supernatant was injected onto the column. The
stabilities of the chelators were determined as the percent decrease in the
initial concentration with time.
νmax 3458; 3146; 1661; 1614; 1516; 1289 cm-1
.
(E)-N0-[1-(5-Acetyl-2,4-dixydroxyphenyl)ethyliden]isonicotinoyl-
hydrazide (A2,4DHAPI). Isoniazid (0.22 g, 1.6 mmol), 2,4-diacetylresor-
cinol (0.31 g, 1.6 mmol), and acetic acid (0.5 mL, 8.8 mmol) in 96% ethanol
(5 mL) were stirred at reflux in a microwave reactor for 2.5 h. After cooling the
reaction mixture to 4 °C, the resulting solid was filtered, washed with H2O, and
recrystallized from ethanol to give 0.437 g (87%) of orange crystals, mp
294-300 °C. 1H NMR (300 MHz, Me2SO): δ 14.33 (1H; s; OH); 12.61
(1H; s; OH); 11.62 (1H; s; NH); 8.78 (2H; d; J = 4.4 Hz; Py); 8.10 (1H; s;
Ph); 7.83 (2H; d; J = 6.0 Hz; Py); 6.36 (1H; s; Ph); 2.64 (3H; s; CH3); 2.54
(3H; s; CH3). 13C NMR (75 MHz, Me2SO): δ 203.0; 166.0; 164.6; 163.2;
159.2; 150.4; 140.1; 133.9; 122.2; 113.5; 112.8; 104.0; 27.2; 14.4. IR (KBr):
νmax 3447; 3131; 1682; 1645; 1585; 1263 cm-1
.
(E)-N0-(1-(7-Hydroxy-2-oxo-2H-chromen-8-yl)ethylidene)iso-
nicotinohydrazide (AHCI). Isoniazid (0.21 g, 1.5 mmol) and 8-ace-
tyl-7-hydroxycoumarine (0.32 g, 1.6 mmol) in 96% ethanol (5 mL) were
stirred at reflux in microwave reactor (400 W) for 20 min. The product
was filtered off, washed with H2O, and dried over P2O5 to give 0.303 g
1
(61%) of yellowish crystals, mp 327-330 °C. H NMR (300 MHz,
Me2SO): δ 11.90 (1H; s; OH); 11.40 (1H; s; NH); 8.78 (2H; d; J = 5.2
Hz; Py); 7.98 (2H; d; J = 9.5 Hz; Py); 7.83 (1H; d; J = 5.4 Hz; Naph);
7.59 (1H; d; J = 8.6 Hz; Naph); 6.93 (1H; d; J = 8.5 Hz; Naph); 6.25
(1H; d; J = 9.5 Hz; Naph); 2.44 (2H; s; CH2); 2.26 (3H; s; CH3). 13
C
NMR (75 MHz, Me2SO): δ 163.2; 160.1; 159.8; 155.0; 154.9; 153.3;
150.4; 145.1; 140.8; 130.2; 122.2; 113.7; 112.9; 111.6; 111.6; 19.3. IR
(KBr): νmax 3350; 3063; 1718; 1693; 1607; 1557; 1276 cm-1
.
Cell Culture. The H9c2 cell line, derived from embryonic rat heart
tissue using selective serial passages,33 was purchased from the American
Type Culture Collection (Manassas, VA, U.S.A.; catalog # CRL-1446).
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM,
Lonza, Belgium) supplemented with 10% heat inactivated fetal bovine
serum (FBS, Lonza, Belgium), 1% penicillin/streptomycin solution
(Lonza, Belgium), and 10 mM HEPES buffer (Sigma, Germany) in
75 cm2 tissue culture flasks (TPP, Switzerland) at 37 °C in a humidified
atmosphere of 5% CO2. Subconfluent cells were subcultured every 3-4
days. For cytotoxicity experiments with neutral red (NR) as well as for
experiments with acetoxymethyl ester of calcein green, cells were seeded
in 96-well plates (TPP) at a density of 10,000 cells per well. For
Calculation of Molecular Descriptors. Molecular models of
the prepared aroylhydrazones were energy-minimized using a modified
MM2 force field computation provided by ChemBio3D Ultra 11.0
(Cambridgesoft, Cambridge, MA, U.S.A.). From these models, various
molecular descriptors were calculated. The logarithm of acid dissocia-
tion constant values (pKa) was calculated using ACD/Laboratories
Software V11.02 (Toronto, Canada).
HPLC Instruments and Methods. HPLC analyses were per-
formed on a chromatographic system LC 20A Prominence (Shimadzu,
Duisburg, Germany) consisting of a DGU-20A3 degasser, two LC-20
AD pumps, a SIL-20 AC autosampler, a CTO-20AC column oven, a
PDA-detector, and a CBM-20AC communication module. The data
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Chemical Research in Toxicology
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morphological assessments, cells were seeded at a density of 75,000 cells
per well in 12-well plates (TPP). Twenty-four hours prior to the
experiments, the medium was changed to serum- and pyruvate-free
DMEM (Sigma, Germany).
order to dissolve lipophilic chelators, Me2SO (0.1% v/v) was present in
the culture medium of all groups. At this concentration Me2SO had no
effect on cellular viability. Individual cytotoxicities of chelators were
evaluated following 24 h and 72 h of incubations.
Changes in cellular morphology were evaluated using an epifluores-
cence inverted microscope Eclipse TS100 (Nikon, Japan) equipped with
a digital cooled camera (1300Q, VDS Vossk€uhler, Germany) and
software NIS-Elements AR 2.20 (Laboratory Imaging, Czech Republic).
H9c2 cells were seeded on 12-well plates (75,000 cells per well) and
incubated under conditions identical to those described above for NR
uptake assays. Furthermore, cellular death was visualized using nuclei
double staining with Hoechst 33342 and propidium iodide (PI; Molec-
ular Probes) that are well established and sensitive procedures to
determine apoptosis and necrosis.14,16 Hoechst 33342 is a blue-fluor-
escent probe (λex= 360 nm; λem= 460 nm) staining all nuclei. In
apoptotic cells, chromatin condensation occurs, and apoptotic cells
can thus be identified as those with condensed and more intensely
stained chromatin. The red-fluorescent (λex = 560 nm; λem= 630 nm)
DNA-binding dye, PI, is unable to cross the plasma membrane of living
cells but readily enters necrotic (or late-stage apoptotic) cells and stains
their nuclei red. Cells were loaded with 10 μg/mL of Hoechst 33342 and
1 μg/mL of PI for 20 min at room temperature and sample images taken
using the microscope setup described above.
Determinations of Iron Chelating Efficiencies in Solution
and Access to Cellular Labile Iron Pools. To assess the Fe
chelation efficiencies of the newly synthesized agents in solution,
measurements of calcein fluorescence intensity34 were used. Calcein is
a fluorescent probe readily forming complexes with Fe ions, which
quench its fluorescence. Addition of Fe-chelating agent to the Fe-calcein
leads to the removal of Fe from this complex with new complex
formation. This is accompanied by an increase in fluorescence intensity.
The complex of calcein (free acid, 20 nM) with ferrous-diammonium
sulfate (FAS; 200 nM) was prepared in HEPES buffered saline (150 mM
NaCl and 40 mM HEPES; pH 7.2). Calcein and FAS were continuously
stirred for 45 min in the dark after which >90% of fluorescence was
quenched. Then 995 μL of the complex solution was pipetted into a
stirred cuvette, and the baseline measurement was started. After 100 s, 5
μL of assessed potential Fe chelator solution was added yielding a final
concentration of 5 μM. It should be noted, that as the ferrous ions (from
FAS) get oxidized to ferric before the actual measurement, the assay is in
effect looking at ferric iron binding.
The change in fluorescence intensity was measured as a function of
time at 25 °C using a Perkin-Elmer LS50B fluorimeter (Perkin-Elmer, U.
S.A.) at λex = 486 nm and λem = 517 nm for 350 s. The Fe chelation
efficiency in solution was expressed as a percentage of efficiency of the
reference chelator SIH (100%).
Intracellular ROS Generation Measurements Using 20,70-
Dichlorodihydrofluorescein-diacetate (H2DCF-DA). The in-
tracellular generation of ROS was assessed using the probe, 20,70-
dichlorodihydrofluorescein-diacetate (H2DCF-DA; Molecular Probes/
KRD, Prague, Czech Republic). Cells were washed with buffer contain-
ing 116 mM NaCl, 5.3 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 1.13 mM
NaH2PO4, 5 mM glucose, and 20 mM HEPES (pH 7.4) and loaded with
10 μM H2DCF-DA solution for 30 min at 37 °C. Cellular esterases
cleave the acetyl groups to produce the cell membrane-impermeable
compound H2DCF, which becomes highly fluorescent when oxidized by
ROS (particularly hydroxyl radicals and other highly oxidizing species)
to DCF.37 Following the addition of the tested substance(s), changes in
intracellular fluorescence intensity (λex = 488 nm; λem = 530 nm) were
followed every 15 min for 1 h at 37 °C using the plate reader referenced
above.
Data Analysis. The statistical software, SigmaStat for Windows 3.5
(SPSS, U.S.A.), was used in this study. Data are presented as the mean (
SE of a given number of experiments. Statistical significance was
determined using Student’s t test or ANOVA with a Bonferroni posthoc
test (comparisons of multiple groups against a corresponding control).
Data not displaying a normal distribution were evaluated using the
nonparametric Mann-Whitney Rank Sum Test or Kruskal-Wallis
ANOVA on Ranks with Dunn’s test. Changes of chelator concentrations
with time were evaluated with the Paired t test. Results were considered
to be statistically significant when p < 0.05. The chelator concentration
inducing 50% viability loss (TC50) and the chelator concentration
leading to 50% protection from t-BHP (EC50) were calculated using
CalcuSyn 2.0 software (Biosoft, Cambridge, U.K.). Pearson pro-
duct-moment correlation analysis was used to determine dependences
between various pairs of variables.
The experiments analyzing iron chelating efficiencies in cultured cells
were performed according to Glickstein et al.35 with slight modifications.
H9c2 cells were seeded on 96-well plates (10,000 cells per well).
Twenty-four hours before the experiments, cells were loaded with 100
μM ferric-ammonium citrate (FAC) in serum-free medium. The cells
were then washed and, to prevent potential interferences (especially
with regard to various trace elements), the medium was replaced with a
buffer prepared from Millipore-filtered (demineralized) water, contain-
ing 116 mM NaCl, 5.3 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 1.13 mM
NaH2PO4, 5 mM glucose, and 20 mM HEPES (pH 7.4). Cells were then
loaded for 30 min at 37 °C with 1 μM cell-permeable acetoxymethyl
ester of calcein green (Molecular Probes/KRD, Prague, Czech Repub-
lic) and washed. Cellular esterases cleave the acetoxymethyl groups to
render the cell membrane-impermeable calcein green, whose fluores-
cence was quenched by FAC. Intracellular fluorescence (λex = 488 nm;
λem = 530 nm) was then followed in time (1 min before and 10 min after
the addition of chelator) at 37 °C using an Infinite 200 M plate reader
(Tecan, Austria).
Cytotoxicity Studies. Cellular viability was determined using a
cytotoxicity assay based on the ability of viable cells to incorporate
neutral red (NR). NR (Sigma, Germany) is a weak cationic dye that
readily penetrates cell membranes by nonionic diffusion, accumulating
intracellularly in lysosomes.36 For vital stainings, after the experimental
incubations, half the volume of medium from each well was removed,
and the same volume of medium with NR was added (final concentra-
tion 40 μg/mL). After 3 h of incubation at 37 °C, the supernatant was
discarded, and the cells were fixed in 0.5% formaldehyde with 1% CaCl2
for 15 min, then were washed twice with PBS, and coagulated with 1%
acetic acid in 50% ethanol, which released accumulated NR into the fluid
where its optical density was measured at λ = 540 nm using a Tecan
Infinite 200 M plate reader (Tecan, Austria). The viability of experi-
mental groups was expressed as the percentage of untreated controls
(100%).
’ RESULTS
Stabilities of Chelators against Plasma Hydrolysis. Stabil-
ities of the studied chelators in rabbit plasma in vitro were
assessed by HPLC. The concentrations of the chelators were
followed for 10 h, during which more than 50% degradation was
observed for most of the compounds tested (Figure 2A-J).
While the concentration of SIH decreased to 10% of its initial
value already after 180 min of incubation, the novel analogues
(except for 2,6DHAPI) were significantly more stable, with more
Model oxidative injury was induced in H9c2 cells by their 24-h
exposure to 200 μM tert-butyl hydroperoxide (t-BHP). The protective
action of various iron chelators was assayed at different concentrations;
chelators were added to cells 10 min before their exposure to t-BHP. In
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Figure 1. Chemical structures of SIH and the newly synthesized
aroylhydrazone iron chelators.
than 50% of the initial concentrations still present at 180 min
(Figure 2K). In the case of 2,6DHAPI, its concentration
decreased to below the quantification limit of the HPLC method
already at 360 min into the experiment. NHAPI was the most
stable novel analogue, as less than 6% and 22% of its initial
concentration underwent hydrolysis after 180 and 600 min,
respectively. The sample chromatograms demonstrating the
improved NHAPI stability in rabbit plasma in comparison with
SIH are shown in Figure 3.
Iron-Chelating Efficiencies in Solution and Cell Culture.
As seen in Figure 4A, all the nine newly synthesized aroylhy-
drazones showed high Fe-chelating activities in solution, which
did not differ significantly from the reference chelator SIH. It
should be noted though that at the 5 μM concentration the tested
chelators were provided in large excess over calcein, and there-
fore this assay may have not discerned differences in their iron
affinity.
In fact, diverse activities were detected with respect to the
access to intracellular LIP in the H9c2 cardiomyoblast cell line.
All the novel compounds displayed lower rates of intracellular
calcein dequenching, and the unsubstituted ketone-derived
analogues HAPI and HPPI were the only two agents which did
not differ significantly from the parent chelator SIH (Figure 4B).
Protection from tert-Butyl Hydroperoxide-Induced Cellu-
lar Toxicity by Chelators. Incubation of H9c2 cells for 24 h
with 200 μM t-BHP resulted in pronounced toxicity. Peripheral
membrane blebbing was followed by the loss of spindle-like cell
shape and rounding up of cells (Figure 5). Furthermore, severe
nuclear condensation was conspicuous, and the nuclei were
brightly stained with PI indicating cellular necrosis (or late-stage
apoptosis). Complete loss of cellular viability was also clear from
the NR uptake test (Figure 6). Co-treatment with either SIH or
all of the newly synthesized Fe-chelating agents was able to
preserve cellular morphology (Figure 5) as well as afford
statistically significant increases in cellular viability (Figure 6),
although at different concentrations. As compared to SIH, higher
Figure 2. Stabilities of SIH and the new aroylhydrazone iron chelators
in rabbit plasma. SIH (A), HAPI (B), HPPI (C), 2,4DHAPI (D),
2,6DHAPI (E), MHAPI (F), CHAPI (G), NHAPI (H), A2,4DHAPI
(I), or AHCI (J) were incubated in rabbit plasma at 37 °C, and their
concentration was followed in time using HPLC. Panel K shows the
comparison of the remaining relative concentrations of the chelators
following 180 min plasma incubations. Statistical significance: *, against
initial concentration at t = 0 (paired t test; p e 0.05); $, comparison
against SIH (ANOVA; p e 0.05).
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Figure 3. Sample chromatograms showing concentrations of chelators SIH (A) and NHAPI (B) in rabbit plasma at time 0 and following the 180 min
incubations at 37 °C. I.S. = internal standard.
cells with chelators for 24 h, i.e., the same time-period as that used
for the cytoprotection experiments. All chelators were compared
at 100 μM concentration, which is the solubility limit for most of
them. As can be seen in Figure 7B, only SIH and CHAPI led to
significant reduction of viability to approximately 55% of the
control values. One hundred micromolar HAPI showed no
sign of toxicity. Also with MHAPI, NHAPI, and A2,4DHAPI,
the viability reduction (e15%) was insignificant as compared to
the control and significantly less pronounced than that observed
with SIH.
Given the marked differences in toxicities and limited solubil-
ity of the lipophilic agents, longer treatment periods were
necessary to obtain TC50 values (i.e., chelator concentrations
inducing 50% viability loss). Thus, the next set of experiments
was performed with 72 h incubations. All chelators decreased
cellular viability in a concentration-dependent manner; the
results are shown in Figure 8, and the TC50 values summarized
in Table 2. In addition, Figure 7C depicts a comparison of toxicities
of all the chelators at 100 μM. NHAPI and AHCI were the least
toxic agents; even incubations of cells with their 100 μM concentra-
tion did not induce a significant decrease in cell viability. In fact, their
toxicities were very similar following the 24 and 72 h incubations. In
all of the other agents, significant viability reduction below 50%
occurred. The highest toxicities were observed with 2,4DHAPI,
MHAPI, and CHAPI, with significantly lower viability observed
from 3 μM concentrations.
Intracellular ROS Production. In the first set of experiments
performed, intracellular ROS formation was induced by 200 μM
t-BHP, and this resulted in a marked and statistically significant
increase in intracellular H2DCF oxidation (Figure 9A). With the
exception of 2,4DHAPI, coincubations of t-BHP with all aroyl-
hydrazone chelators resulted in a reduction in fluorescence
intensity. SIH and MHAPI were the least effective agents,
decreasing the rate of ROS formation only insignificantly, while
2,6DHAPI was the most potent, yielding a significant reduction
of the fluorescence intensity comparable to that of the untreated
control cells. Interestingly, 2,4DHAPI significantly augmented
the t-BHP-induced ROS formation.
In the next series of studies, the cells were incubated for 60 min
with the Fe donor, FAC (5 μM), either alone or in combination with
the studied chelators (all at 10 μM concentrations, corresponding to
the 1:2 Fe/chelator binding equivalent ratio). As demonstrated in
Figure 9B, in cells incubated with FAC, the ROS production
significantly increased as compared to that of the control. Co-
incubations with all chelators (except for 2,4DHAPI) resulted in
reduction of the fluorescence intensity as compared to that of
uncomplexed Fe, which reached statistical significance in the case
of HAPI and 2,6DHAPI. Interestingly, incubation of cells with the Fe
Figure 4. Iron chelation efficiencies of SIH, the newly synthesized
agents and their access to cellular labile iron pool. (A) Displacement of iron
from its complexes with calcein in buffered solution (pH 7.4) measured
as an increase in fluorescence 10 min after the addition of chelator.
(B) Displacement of intracellular labile iron from its complex with calcein
in H9c2 cells 10 min after the addition of Fe chelators to extracellular
medium. Iron chelating efficiencies are displayed as a percentage of SIH
(100%). Results are presented as the mean ( SE (n = 4 experiments).
Statistical significance (ANOVA; p e 0.05): $, compared to SIH.
protective efficiencies (resulting in lower EC50 values) were
observed with HAPI, 2,4DHAPI, and CHAPI. In contrast,
2,6DHAPI, NHAPI, A2,4DHAPI, and AHCI had higher EC50
values as compared to those of SIH (Table 2). When compared
at a single concentration (100 μM), which is the highest concen-
tration at which most of the lipophilic agents (including SIH)
could be dissolved, comparable protective actions of all of the
chelators were observed, which did not differ significantly from
those of SIH (Figure 7A).
Toxicities of Individual Chelators. In order to assess the
cytotoxic effects of the tested chelators, we first incubated H9c2
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Figure 5. Cellular morphology and apoptosis/necrosis nuclear staining with Hoechst 33342 (blue) and propidium iodide (red) after a 24 h incubation of H9c2
cardiomyoblasts with 200 μM tert-butyl hydroperoxide and its combinations with iron chelators SIH and HAPI (both 30 μM). Scale bars 100 μm.
complexes of the two latter agents resulted in even lower levels of
fluorescence than in control (Fe-free) cells (Figure 9B). Combina-
tion of FAC and 2,4DHAPI induced significant increase in cellular
ROS levels.
been used as a prototypic inducer of oxidative stress in numerous
previous cellular experiments. Once inside cells, t-BHP generates
tert-butoxy radicals, which induce various toxic effects including
peroxidation of lipids, depletion of intracellular reduced glu-
tathione, modification of protein thiols, or disturbance of calcium
homeostasis.1,42 t-BHP induces oxidative stress gradually over
time and therefore induces sustained oxidative challenge to cells.
Of note, t-BHP has been previously shown to release nonheme,
nonferritin iron from rat liver microsomes.43 Additional studies
are nevertheless needed to examine the ROS formation and iron-
mediated toxicity in cells exposed to stressors such as drugs that
release iron from ferritin or heme sources.
The purpose of intracellular Fe chelation is to prevent redox-active
Fe from engaging in radical-mediated cellular damage.9,44,45 Pre-
viously, SIH has shown considerable potential in preventing various
ROS-mediated pathologies both in vitro and in vivo. However, rapid
plasma hydrolysis of SIH as well as other aldehyde-derived aroylhy-
drazones represents a serious obstacle to the effective in vivo
application of these promising drug candidates.
Hence, the main purpose of our current study was to increase
the chelator stability in plasma as compared to that of labile SIH
while maintaining its favorable pharmacodynamic properties.
The mechanism of the hydrolytic cleavage of the hydrazone
bond involves protonation of the imine CdN nitrogen followed
by a nucleophilic attack of water and cleavage of the C-N bond.
Therefore, our first hypothesis was that the replacement of the
aldimine hydrogen in SIH by a bulkier and electron-donating
alkyl group would decrease the probability of attack of water on
the CdN carbon and thus improve the compound stability. This
was indeed confirmed; both methylketone-derived HAPI and
ethylketone-derived HPPI were shown as significantly more
stable and resistant against hydrolysis in plasma than SIH.
The second approach was to modify the substituents on the
phenyl ring to investigate their effects on the hydrazone bond
stability. The literature reports, particularly on this issue, were
somewhat contradictory. For example, replacement of pyridine
In Figure 9C, the effects of chelators on spontaneous cellular
ROS production are shown. 2,4DHAPI displayed a significant
increase in DCF fluorescence as compared to both control and
SIH-treated cells. All the other nine examined chelators did not
significantly affect the spontaneous cellular ROS production.
Molecular Descriptors and Correlations. To obtain a basic
insight into what molecular descriptors are important in inter-
preting the activity of the prepared compounds, correlation
analyses were performed. Because of the small data set, no
attempts at a detailed QSAR analysis were carried out; we only
aimed at identifying any possible trends. All of the prepared
aroylhydrazones comply with Lipinski’s Rule of Five;38 they have
molecular weights from 241 to 323 g/mol, log P values of
1.4-3.1, up to 7 hydrogen bond acceptors, and up to 3 hydrogen
bond donors. Both the ability of the compounds to protect the
cells against the action of t-BHP and their toxicity significantly
correlated with their polar surface area and pKa of the phenolic
hydroxyl (Figure 10). A significant relationship was also found
between the protective efficacy of the compounds and their
toxicity (r = 0.88, r2 = 0.78,and p < 0.001). No significant corre-
lations with other parameters were observed.
’ DISCUSSION
Aroylhydrazone Fe chelators represent an interesting group of
biologically active molecules.39 However, to date, these agents
have not been thoroughly screened, and their structure-activity
relationships were evaluated only for their ability to mobilize Fe
from Fe overloaded cells39,40 and their potential anticancer
effects.41 This study aimed at the assessment of their antioxidant
properties against model oxidative injury of cardiomyoblast-
derived cells induced by t-BHP. This organic peroxide is one of
the most common membrane-permeable oxidant agents and has
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Figure 6. Effects of various concentrations of iron chelators SIH (A), HAPI (B), HPPI (C), 2,4DHAPI (D), 2,6DHAPI (E), MHAPI (F), CHAPI (G),
NHAPI (H), A2,4DHAPI (I), or AHCI (J) on cellular toxicity induced in H9c2 cells by a 24 h incubation with 200 μM tert-butyl hydroperoxide (t-BHP).
Studies were performed using the neutral red uptake test, and the results are presented as mean ( SE (n g 4 experiments). Statistical significance
(ANOVA; p e 0.05): *, compared to the control (untreated) group; #, compared to the 200 μM t-BHP group.
in SIH (and also in PIH) by a phenyl ring markedly increased
their hydrolytic stability.46 This was explained by an increased
electron density at the hydrazone CdN carbon and, conse-
quently, its lower susceptibility to the attack of water after the
removal of the strong electron-withdrawing nitrogen. This seems
to be in contrast with our results; however, it should be noted
that the aforementioned structural alterations46 were on the
other side of the hydrazone bond and might have influenced the
distribution of the electron density on the hydrazone CdN bond
differently. Nevertheless, compound NHAPI, containing the
strongest electron acceptor of all the novel chelators, was the
most stable compound in our study. This effect was consistently
found in all of the studied hydrazones containing electron-
withdrawing groups, i.e., acetyl in A2,4DHAPI increased its
stability compared to that of 2,4DHAPI, and CHAPI bearing
chlorine in position 5 was more stable than HAPI, while those
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Table 2. Selected Characteristics of the Evaluated Iron Che-
latorsa
MW
log
PSA
(Å2)
EC50
TC50
chelator
SIH
(g/mol)
P
pKa
(μM)
(μM)
241
255
269
271
271
285
290
300
313
323
2.1
2.1
2.6
1.4
1.4
2.2
3.1
2.5
2.0
2.2
75
8.5
8.5
8.5
8.2
7.6
7.8
8.0
5.6
7.2
6.3
18.2
11.4
17.8
9.2
29.7
12.2
29.2
2.7
HAPI
75
HPPI
75
2,4DHAPI
2,6DHAPI
MHAPI
CHAPI
NHAPI
A2,4DHAPI
AHCI
95
95
48.3
21.2
9.5
31.2
9.5
84
75
4.8
121
112
100
68.5
39.9
56.6
>100b
73.3
>100b
a MW, molecular weight; log P, logarithm of octanol/water partition
coefficient; PSA, polar surface area calculated using ChemBioOffice
Ultra 11.0 (Cambridgesoft, Cambridge, MA); pKa, logarithm of acid
dissociation constant of the phenolic hydroxyl calculated using ACD/
Labs Software V11.02 (Toronto, Canada); EC50, concentration of
chelator reducing the toxicity induced by 24-h incubation with tert-butyl
hydroperoxide to 50% of the control (untreated) cells; TC50, chelator
concentration inducing 50% viability reduction compared to control
(untreated) cells following 72 h incubation. Neutral red uptake assay;
mean values of n g 4 experiments. b Exceeds solubility limit.
with electron donors displayed stability similar to that of
unsubstituted HAPI. A possible explanation of these results is
that electron acceptors decrease the electron density on CdN
nitrogen and decrease its protonation, which is consistent with a
previous study on hydrazone basicity.47 Thus, protonation of the
CdN nitrogen seems to be the key factor in the hydrolysis of the
methylketone-based hydrazones. It should be noted that the
effects of certain substituents on hydrazone stability also depend
on pH; for example, the nitro group increased the hydrazone
hydrolysis rate at lower pH but decreased it in a neutral or basic
environment.48 Interestingly, it was found that 2,6DHAPI was
much less stable compared to its position isomer 2,4DHAPI.
This observation could be explained by the steric effect of
hydroxyls in both o-positions. In general, aromatic hydrazones
are more stable to acidic hydrolysis than the aliphatic ones due to
a conjugation of the π electrons of the CdN bond with the
aromatic ring.49 However, such conjugation requires coplanarity
of the aromatic ring with the CdN bond, which may be hindered
by repulsion between the methyl group and one of the o-
hydroxyls.
Figure 7. (A) Protective effects of various aroylhydrazone iron chela-
tors (100 μM) against cellular injury induced to H9c2 cells by a 24 h
incubation with tert-butyl hydroperoxide (t-BHP; 200 μM). (B) Effects
of a 24 h incubation with these chelators at 100 μM concentration on
cellular viability. (C) Effects of a 72 h incubation with these chelators at
100 μM concentration on cellular viability. Studies were performed
using the neutral red uptake test, and the results are presented as mean (
SE (n g 4 experiments). Statistical significance (ANOVA; p e 0.05): *,
compared to the control (untreated) group; #, compared to the 200 μM
t-BHP group; $, compared to the SIH group.
The marked improvement in plasma stability of the ketone-
derived SIH analogues is an important, but by no means
sufficient, outcome of this study. The newly synthesized analo-
gues had to retain their Fe-chelating, antioxidant, and cytopro-
tective activities as well as possess acceptable specific toxicity. As
all of the three structural moieties of SIH responsible for Fe
binding remained unchanged, all these agents displayed expec-
tably high Fe-chelating activity in solution, comparable to that of
SIH. However, in terms of access to intracellular LIP, the new
chelators displayed lower efficiencies as compared to that of SIH.
The decrease in the chelation of intracellular free Fe was
significant in all the agents except for the unsubstituted HAPI
and HPPI. The lowest access to cellular Fe was observed in
2,4DHAPI and 2,6DHAPI. In contrast, most of the new agents
were shown to block intracellular ROS production more effi-
ciently than SIH. Interestingly, the highest antioxidant action was
observed with 2,6DHAPI, whereas its position isomer 2,4DHAPI
displayed a totally opposite effect: it rather augmented the
cellular ROS burst. It should be noted that the higher increase
in ROS production was observed when the cells were incubated
with 2,4DHAPI alone (Figure 8C) than with the 2,4DHAPI-Fe
combination (Figure 8B), suggesting that this agent may pro-
mote ROS formation per se, rather than by forming a redox-
active Fe complex. Interestingly, this pro-oxidant property of
2,4DHAPI was not shared by either its position isomer 2,6DHAPI
or A2,4DHAPI, which contains an additional acetyl in position 5
compared to 2,4DHAPI. This may be because the hydroxyl in
position 4 together with free positions 3 and 5 of the phenyl ring
of 2,4DHAPI may resemble the phenol-type substitution pattern
in the B ring of flavonoids such as apigenin, naringenin, or
naringin, which act as pro-oxidants without involving a transition
metal-catalyzed autoxidation.50
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Figure 8. Effects of a 72 h incubation with iron chelators SIH (A), HAPI
(B), HPPI (C), 2,4DHAPI (D), 2,6DHAPI (E), MHAPI (F), CHAPI
(G), NHAPI (H), A2,4DHAPI (I), or AHCI (J) on the cellular viability
of H9c2 cells. Studies were performed using the neutral red uptake, and
the results are presented as the mean ( SE (n g 4 experiments).
Statistical significance (ANOVA; p e 0.05): *, compared to the control
(untreated) group.
Despite all of the differences discussed above, all of these
agents were able to protect the cells against the model oxidative
injury induced by t-BHP, although with variable efficiencies.
Notably, the protective efficiencies of the studied hydrazones
(expressed as the EC50 values) correlated with their molecular
polar surface area. This value is defined as the sum of the surface
areas of polar atoms and serves as a useful parameter for the
prediction of passive transport through cellular membranes.51,52
All of the studied compounds have polar surface area values from
75 to 121 Å2, which is within the limit of 140 Å2 for penetrating
cell membranes.53 Thus, we suppose that the observed changes
in protective efficiencies and toxicities of the studied compounds
compared to SIH are related to their different abilities to
penetrate into the cells. Of note, it is commonly assumed that
such correlations are sigmoidal. However, in this study, linear
relationships were observed. This may be explained by two
factors: first, the compounds are structurally related, and second,
Figure 9. Intracellular reactive oxygen species production in H9c2 cells
loaded with H2DCF-DA (diacetate ester of reduced 20,70-dichloro-
fluorescein) and exposed for 60 min to tert-butyl hydroperoxide (t-
BHP), ferric ammonium citrate (FAC), and/or various aroylhydra-
zone Fe chelators. Following intracellular acetate group cleavage, the
increase in fluorescence intensity (λex = 488; λem = 520 nm) is
proportional to probe oxidation by reactive oxygen species to green
fluorescent DCF. (A) Effects of chelators on an oxidative burst
induced by incubation with 200 μM t-BHP; (B) effects of iron
chelators on probe oxidation induced by incubation with 5 μM
FAC; (C) effects of Fe chelators alone (all 10 μM). Results are
presented as the mean ( SE (n = 4 experiments). Statistical sig-
nificance (ANOVA; p e 0.05): *, compared to the control (sponta-
neous H2DCF oxidation); #, compared to the t-BHP group; §, compared
to the FAC group; $, compared to the SIH group.
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In this study, the individual toxicities of the tested compounds
toward H9c2 cells were determined following 24- and 72-h incuba-
tions. Interestingly, with the exception of CHAPI, the increase in
chelator stability resulted in the decrease of their toxicity following
the 24-h cellular treatments as compared to SIH. This may be
related to the lower release of toxic isoniazid by the new chelators, as
compared to labile SIH. In contrast, following the long (72 h)
incubations, enhancement in the toxicities occurred in most of the
agents, except for AHCI and NHAPI. The highest inherent long-
term toxicity (the lowest TC50) as well as the worst ratio of
protective efficiency and own toxicity (EC50/TC50) was observed
with 2,4DHAPI, which may be caused by its pro-oxidant properties.
Indeed, some of the aroylhydrazone chelators may, apart from Fe
deprivation, induce toxicity due to oxidative stress induction.56
Nevertheless, as shown by the DCF assay, other studied agents
seem unlikely to act as pro-oxidants.
In some agents, a certain discrepancy has been observed
between the results of cytoprotection experiments against t-BPH
toxicity and the experiments where the intracellular ROS formation
was evaluated using the H2DCF-DA assay. For instance, SIH was
very effective in protecting the cells according to the first experiment
(Figure 6A), but the results from the second one seem to suggest that
it does not have a significant effectonROSgeneration(Figure9A). It
is possible that apart from forming the alkoxyl radicals t-BHP may
activate various peroxidases to compound I or II1 that eventually
oxidize H2DCF to DCF, and the various chelating agents may affect
DCF levels by interfering with the catalytic cycle of peroxidases.
In conclusion, all of the newly synthesized SIH analogues were
shown as significantly more resistant against the hydrazone bond
hydrolysis, with the exception of 2,6DHAPI. At the same time,
these agents retained their ability to protect the cardiac-derived
cells against Fe-mediated oxidative injury. In particular, the novel
chelator HAPI shows promise due to the absence of short-term
toxicity and improved antioxidant and cytoprotective action with
preserved access to LIP as compared to the reference chelator
SIH. HAPI might be especially useful in acute situations, e.g., for
short periods of treatment during which temporary Fe depriva-
tion could be tolerable, such as in ischemia/reperfusion injury.
On the other hand, due to its negligible long-term toxicity and
high plasmastability, NHAPI may be beneficial in animal experiments
requiring long-term chelator exposure, such as in studies on athero-
sclerosis or heart failure. Further detailed studies are necessary to
confirm the cardioprotective efficacies and toxicities of these novel
drug candidates using more complex in vivo models, as well as to
determine their potential clinical implications.
Figure 10. Scatterplots and correlations between protective efficiencies
(EDC50 values, panels A and C) and long-term toxicities (TC50 values,
panels B and D), and the molecular parameters of the studied aroylhy-
drazones including polar surface area (PSA, panels A and B) and
acidity of the phenolic hydroxyl (pKa, panels C and D). r, Pearson
product-moment correlation coefficients; p, statistical significance
of the correlation.
they possess medium polar surface area values, which would be
located at the central linear part of the sigmoidal curve.
The assumption that the ability of the studied aroylhydrazones
to protect cells against the action of t-BHP is related to their
penetration through the cell membrane is further supported by
the strong correlation observed between this ability and the pKa
of the phenolic hydroxyl. Since pKa values range from 5.6 to 8.5,
the ratio of negatively charged/neutral molecules will be starkly
different at physiological pH 7.4. For example, introduction of a
nitro group in NHAPI resulted in a markedly increased acidity of
the phenolic hydroxyl compared to that of HAPI due to the
strongly electron-withdrawing properties of the nitro group.
Thus, this compound is ∼99% ionized at pH 7.4 and cannot
be expected to penetrate the lipophilic cell membrane readily,
which is in agreement with its low protective effect. These
pronounced differences in ionization at around pH 7.4 may also
explain the lack of correlation of the protective activities and/or
toxicities of the chelators with their log P.
’ AUTHOR INFORMATION
In order to fully assess the therapeutic potential of these new
aroylhydrazones, it was also necessary to determine their own
individual toxicities. It is known that the use of Fe chelators in
states without Fe overload is associated with the obvious risk of
toxicity due to the removing or withholding of Fe from critical
Fe-containing proteins and/or the interaction with normal Fe
metabolism. On the one hand, the low stability of SIH and other
aldehyde-derived aroylhydrazones may have led to the under-
estimation of the pharmacological potential of these chelators in
previous studies. However, on the other hand, the desired
increase in stability may also potentiate the risk of the toxicity
of these new chelators, especially in long-term treatment.9 In fact,
improved hydrolytic stability is an important feature of novel
cytotoxic thiosemicarbazone Fe chelators, which are currently
studied for the treatment of cancer.54,55
Corresponding Author
*(K.V.) Department of Inorganic and Organic Chemistry,
Charles University in Prague, Faculty of Pharmacy, Heyrovskeho
1203, 500 05 Hradec Kralove, Czech Republic. Tel: þ420-495-067-
497. Fax: þ420-495-067-166. E-mail: Katerina.Vavrova@faf.cuni.cz.
(T.S.) Department of Biochemical Sciences, Charles University in
Prague, Faculty of Pharmacy, Heyrovskeho 1203, 500 05 Hradec
Kralove, Czech Republic. Tel: þ420-495-067-422. Fax: þ420-495-
067-168. E-mail: Tomas.Simunek@faf.cuni.cz.
Funding Sources
This study was supported by the Charles University in Prague (grants
SVV 2010/261/001 and 2010/261/003) and Ministry of Education
of the Czech Republic (Research Project MSM0021620822).
300
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Chemical Research in Toxicology
ARTICLE
’ ACKNOWLEDGMENT
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We thank Mrs. Alena Pakostova for her technical assistance in
the cell-culture laboratory.
’ ABBREVIATIONS
A2,4DHAPI, (E)-N0-[1-(5-acetyl-2,4-dixydroxyphenyl)ethyliden]-
isonicotinoylhydrazide; AHCI, (E)-N0-(1-(7-hydroxy-2-oxo-2H-
chromen-8-yl)ethylidene)isonicotinohydrazide; CHAPI, (E)-N0-
[1-(5-chloro-2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide;
DCF, dichlorofluorescein; DFO, desfferioxamine; 2,4DHAPI, (E)-
N0-[1-(2,4-dihydroxyphenyl)ethyliden]isonicotinoylhydrazide; 2,6-
DHAPI, (E)-N0-[1-(2,6-dihydroxyphenyl)ethyliden]isonicotinoyl-
hydrazide; DMEM, Dulbecco’smodified Eagle’smedium;EC50, con-
centration of chelator reducing the toxicity induced by 24-h incuba-
tion with tert-butyl hydroperoxide to 50% of the control (untreated)
cells; FAC, ferric-ammonium citrate; FAS, ferrous-diammonium sul-
fate;FBS, fetal bovine serum;Fe, iron;HAPI, (E)-N0-[1-(2-hydro-
xyphenyl)ethyliden]isonicotinoylhydrazide;H2DCF-DA, 20,70-di-
chlorodihydrofluorescein-diacetate; HPPI, (E)-N0-[1-(2-hydroxy-
phenyl)propyliden]isonicotinoylhydrazide; ICL670A, deferasirox;
ICRF-187, dexrazoxane; I.S., internal standard; L1 or CP20, defer-
iprone; LIP, labile iron pool; log P, logarithm of octanol/water parti-
tion coefficient; MHAPI, (E)-N0-[1-(2-hydroxy-4-methoxyphenyl)-
ethyliden]isonicotinoylhydrazide; MW, molecular weight; NHAPI,
(E)-N0-[1-(2-hydroxy-5-nitrophenyl)ethyliden]isonicotinoylhy-
drazide; NR, neutral red; PI, propidium iodide; PSA, polar surface
area; QSAR, quantitative structure-activity relationship; ROS, reac-
tive oxygen species; RT, room temperature; SIH, salicylaldehyde
isonicotinoyl hydrazone; t-BHP, tert-butyl hydroperoxide; TC50, con-
centration of the chelator inducing 50% viability reduction compared
to the control (untreated) cells following 72 h of incubation.
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peroxide. J. Inorg. Biochem. 102, 2130–2135.
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