Structure-Activity Relationships of Nitro-Substituted Aroylhydrazone Iron Chelators with Antioxidant and Antiproliferative Activities

Article abstract of DOI:10.1021/acs.chemrestox.7b00324

Aroylhydrazone iron chelators such as salicylaldehyde isonicotinoyl hydrazone (SIH) protect various cells against oxidative injury and display antineoplastic activities. Previous studies have shown that a nitro-substituted hydrazone, namely, NHAPI, displayed markedly improved plasma stability, selective antitumor activity, and moderate antioxidant properties. In this study, we prepared four series of novel NHAPI derivatives and explored their iron chelation activities, anti- or pro-oxidant effects, protection against model oxidative injury in the H9c2 cell line derived from rat embryonic cardiac myoblasts, cytotoxicities to the corresponding noncancerous H9c2 cells, and antiproliferative activities against the MCF-7 human breast adenocarcinoma and HL-60 human promyelocytic leukemia cell lines. Nitro substitution had both negative and positive effects on the examined properties, and we identified new structure-activity relationships. Naphthyl and biphenyl derivatives showed selective antiproliferative action, particularly in the breast adenocarcinoma MCF-7 cell line, where they exceeded the selectivity of the parent compound NHAPI. Of particular interest is a compound prepared from 2-hydroxy-5-methyl-3-nitroacetophenone and biphenyl-4-carbohydrazide, which protected cardiomyoblasts against oxidative injury at 1.8 ± 1.2 μM with 24-fold higher selectivity than SIH. These compounds will serve as leads for further structural optimization and mechanistic studies.

Full text of DOI:10.1021/acs.chemrestox.7b00324

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Article
Structure-Activity Relationships of Nitro-Substituted Aroylhydrazone
Iron Chelators with Antioxidant and Antiproliferative Activities
Katerina Hruskova, Eliska Potuckova, Lukas Opalka, Tereza Hergeselova,
Pavlina Haskova, Petra Kovarikova, Tomas Sim#nek, and Katerina Vavrova
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00324 • Publication Date (Web): 16 May 2018
Downloaded from http://pubs.acs.org on May 16, 2018
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StructureꢀActivity Relationships of Nitroꢀ
Substituted Aroylhydrazone Iron Chelators with
Antioxidant and Antiproliferative Activities
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Kateřina Hrušková, Eliška Potůčková, Lukáš Opálka, Tereza Hergeselová, Pavlína Hašková,
Petra Kovaříková, Tomáš Šimůnek*, Kateřina Vávrová*
Charles University, Faculty of Pharmacy in Hradec Králové, Akademika Heyrovského 1203,
500 05 Hradec Králové, Czech Republic
KEYWORDS. iron chelator; hydrazone; oxidative injury; anticancer; structureꢀactivity
relationships
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ABSTRACT. Aroylhydrazone iron chelators such as salicylaldehyde isonicotinoyl hydrazone
(SIH) protect various cells against oxidative injury and/or display antineoplastic activities.
Previous studies have shown that a nitroꢀsubstituted hydrazone, namely, NHAPI, displayed
markedly improved plasma stability, selective antitumor activity, and moderate antioxidant
properties. In this study, we prepared four series of novel NHAPI derivatives and explored
their iron chelation activities, antiꢀ or proꢀoxidant effects, protection against model oxidative
injury in the H9c2 cell line derived from rat embryonic cardiac myoblasts, cytotoxicities to
the corresponding noncancerous H9c2 cells, and antiproliferative activities against the MCFꢀ7
human breast adenocarcinoma and HLꢀ60 human promyelocytic leukemia cell lines. Nitro
substitution had both negative and positive effects on the examined properties, and we
identified new structureꢀactivity relationships. Naphthyl and biphenyl derivatives showed
selective antiproliferative action, particularly in the breast adenocarcinoma MCFꢀ7 cell line,
where they exceeded the selectivity of the parent compound NHAPI. Of particular interest is a
compound prepared from 2ꢀhydroxyꢀ5ꢀmethylꢀ3ꢀnitroacetophenone and biphenylꢀ4ꢀ
carbohydrazide, which protected cardiomyoblasts against oxidative injury at 1.8 ± 1.2 µM
with 24ꢀfold higher selectivity than SIH. These compounds will serve as leads for further
structural optimization and mechanistic studies.
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Introduction
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Iron is an essential element for virtually all organisms. However, this transition metal may
catalyze the Fenton reaction, which yields hydroxyl radicals that are highly reactive and toxic
to various biological macromolecules.1 Therefore, the human body has a finely tuned
mechanism for how to absorb, conserve, recycle and store iron, although its excretion is not
regulated and is restricted to intestinal and skin cell desquamation, minor (e.g., menstrual)
bleeding and urinary excretion.2 Accumulation of iron and subsequent organ damage caused
by ironꢀmediated oxidative stress is connected with numerous illnesses characterized by either
local or generalized iron overload, such as hereditary hemochromatosis, some
neurodegenerative and cardiovascular diseases, and thalassemia and other anemias requiring
repeated blood transfusions.3 Of note, several recent studies have demonstrated the complex
roles of iron in cancer growth. This includes free radical formation with potentially
promutagenic effects and, most importantly, the impaired regulation of iron metabolism,
which can escalate cancer risk and stimulate tumor growth. Tumorigenesis appears to be
associated with regulation of cellular iron intake through an iron efflux pump ferroportin and
its regulator hepcidin . Cancer cells have impaired control of gene expression of proteins that
regulate iron, including differences in miRNA, methylation, and acetylation.4, 5
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Iron chelating agents have been successfully used in diseases with systemic iron overload in
clinical practice.6 In addition to systemic iron overload conditions, a substantial evidence
suggests that iron chelation is also beneficial to prevent ironꢀmediated worsening of oxidative
stress, which can take place locally even with normal body iron levels.7 Furthermore, cancer
cells require increased iron supplementation due to their fast and sustained growth, and the
iron deprivation of cancer cells may represent another promising therapeutic effect of iron
chelators.8
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Salicylaldehyde isonicotinoyl hydrazone (SIH, Figure 1A) is a tridentate chelator that
selectively forms 2:1 complexes with Fe3+. SIH has low molecular weight and optimal
lipophilicity, and can be administered orally.9 It readily penetrates the cell membrane, and
strongly chelates the intracellular labile iron pool; hence, it efficiently prevents the formation
of hydroxyl radicals.9 SIH has previously been shown to effectively protect guinea pig and rat
isolated cardiomyocytes, as well as other cell types, against various types of oxidative
injury.10ꢀ13 In addition, SIH displayed the best ratio of cytoprotection to its inherent toxicity
among seven iron chelating agents of several distinct chemical structures, including three
clinically used drugs: desferrioxamine (DFO), deferiprone (L1, CP20) and deferasirox
(ICL670A).14 Aroylhydrazone iron chelators also showed antiproliferative activity, especially
those of the 2ꢀhydroxyꢀ1ꢀnaphthylaldehyde isonicotinoyl hydrazone (311) or 2ꢀpyridylketone
isonicotinoyl hydrazone (PKIH) class.8
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However, SIH (as well as other aldehydeꢀbased aroylhydrazone chelators) undergoes fast
hydrolysis in plasma and has a short halfꢀlife in vivo 15
.
Recently, the plasma stability of
aroylhydrazones has been substantially increased by alkylation of the hydrazone carbon, i.e.
,
by replacing salicylaldehyde with 2ꢀhydroxyacetophenone (compound HAPI, Figure 1A) or
2ꢀhydroxypropiophenone (HPPI).16 Both HAPI and HPPI retained the cytoprotective activity
of SIH despite having antiproliferative activities that were even higher than that of SIH.16, 17
Further studies on HAPI and HPPI derivatives with modified hydrazide scaffolds showed that
these compounds can be tuned to promote antioxidant (pyridazine derivative) or
antiproliferative properties (naphthyl and biphenyl derivatives).18 In contrast, the reduction of
the C=N bond or the introduction of branching close to this bond decreased both the
antioxidant and antiproliferative properties.19
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Further substitution of the phenol ring in HAPI resulted in the identification of several
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promising substances. In particular, the nitro groupꢀcontaining (E)ꢀN´ꢀ[1ꢀ(2ꢀhydroxyꢀ5ꢀ
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nitrophenyl)ethylidene]isonicotinoylhydrazide (NHAPI; Figure 1A) showed exceptional
plasma stability and selective antitumor activity against the MCFꢀ7 human breast
adenocarcinoma and HLꢀ60 human promyelocytic leukemia cell lines. Although the nitro
group has long been considered to be a source of toxicity and is generally avoided in drug
design, compound NHAPI was one of the least toxic in H9c2 cardiomyoblasts among the
aroylhydrazones studied.16, 17 Of note, several nitro groupꢀcontaining drugs have recently been
introduced into clinical practice.20, 21
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In this work, we further explored the structureꢀactivity relationships in nitroꢀsubstituted
aroylhydrazone iron chelators. We synthesized four series of compounds that were designed
as hybrids of NHAPI16 and compounds described in our most recent work18 by combining the
nitro groupꢀcontaining ketones with hydrazides bearing naphthyl, biphenyl, pyridineꢀ3ꢀyl,
pyridineꢀ4ꢀyl, pyrazineꢀ2ꢀyl and pyridazinꢀ4ꢀyl groups (Figure 1B, compounds a, b, c, d, e
and f, respectively). The first series of compounds, 4a-f, are based on the same ketone as
NHAPI, i.e., 2ꢀhydroxyꢀ5ꢀnitroacetophenone (Figure 1B – series
are homologs of series , with an ethyl group instead of a methyl group at the hydrazone
carbon, while series 10 has the nitro group shifted to position 3 relative to the hydrazone
(Figure 1B). Finally, aroylhydrazones of series are isomers of series 10, with the ethyl group
4). Compounds of series 9
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replaced by two methyl groups, one at the hydrazone carbon and the second in position 5 of
the phenyl ring (Figure 1B).
The prepared compounds were examined for their ironꢀchelating ability, capability to
mobilize iron and prevent its uptake from transferrin, antiꢀ and proꢀoxidative properties,
ability to protect H9c2 cardiomyoblasts against hydrogen peroxideꢀinduced model oxidative
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injury, inherent toxicity in H9c2 cardiomyoblasts, and antiproliferative activity in the MCFꢀ7
human breast adenocarcinoma and HLꢀ60 human promyelocytic leukemia cell lines. The most
promising compounds were further examined for their stability in plasma in vitro.
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Figure 1. Structures of known aroylhydrazone iron chelators (panel A) and syntheses of the
examined compounds (panel B).
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Experimental procedures
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Chemicals and instrumentation
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Syntheses of the ligands are described in the Supporting Information. Methyl 1ꢀnaphthoate
(2a), 1ꢀnaphthohydrazide (3a), methyl biphenylꢀ4ꢀcarboxylate (2b), biphenylꢀ4ꢀ
carbohydrazide (3b), pyrazineꢀ2ꢀcarbohydrazide (3e), and pyridazineꢀ4ꢀcarbohydrazide (3f)
were prepared as described previously.18 All other chemicals were purchased from Sigmaꢀ
Aldrich (Schnelldorf, Germany), Merck (Darmstadt, Germany) or Penta (Prague, Czech
Republic) and were of the highest available pharmaceutical or analytical grade. Thin layer
chromatography was performed on TLC sheets (silica gel 60 F254) from Merck (Darmstadt,
Germany). Microwave reactions were conducted in a Milestone MicroꢀSYNTH Ethos 1600
URM apparatus (Milestone Inc., Shelton, CT, USA). Melting points were measured on a
Kofler apparatus and are uncorrected. IR spectra were measured on a Nicolet 6700
spectrometer (Thermo Scientific, Waltham, MA, USA). All products were characterized by
NMR (Varian Mercury Vx BB 300 or VNMR S500 NMR spectrometers, Varian, Palo Alto,
CA, USA). Chemical shifts were reported as δ values in parts per million (ppm) and were
indirectly referenced to tetramethylsilane (TMS) via the solvent signal. Elemental analysis
was measured on a CHNSꢀOCE FISONS EA 1110 apparatus (Fisons, Ipswich, UK). MS
experiments (ESI+) were performed with an Agilent 500 Ion Trap LC/MS (Agilent
Technologies, Santa Clara, California, USA). HPLCꢀHRMS experiments (ESI+) were
performed using an HRMS system Acquity UPLC Iꢀclass and a Synapt G2Si QꢀTOF mass
spectrometer (Waters, Milford, MA, USA).
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Cell cultures
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The H9c2 and HLꢀ60 cell lines were purchased from the American Type Culture Collection
(ATCC; Manassas, VA, USA), and the MCFꢀ7 cell line was from the European Collection of
Cell Cultures (ECACC; Salisbury, UK). H9c2 and MCFꢀ7 cells were cultured in Dulbecco’s
modified Eagle’s medium with and without phenol red, respectively, (DMEM; Lonza,
Verviers, Belgium) supplemented with 10% heatꢀinactivated fetal bovine serum (FBS,
Lonza), 1% penicillin/streptomycin solution (Lonza) and 10 mM HEPES buffer (Sigmaꢀ
Aldrich). HLꢀ60 cells were cultured in RPMIꢀ1640 medium (SigmaꢀAldrich) supplemented
with 10% heatꢀinactivated FBS and 1% penicillin/streptomycin solution (Lonza). Each cell
line was cultured in 75 cm2 tissue culture flasks (TPP, Trasadingen, Switzerland) at 37°C in a
humidified atmosphere of 5% CO2. Subconfluent cells (H9c2 and MCFꢀ7) or cell suspensions
(HLꢀ60) were subcultured every 3ꢀ4 days.
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The cells were seeded in 96ꢀwell plates (TPP) at a density of 10,000 cells per well (H9c2
and HLꢀ60) or 5,000 cells per well (MCFꢀ7). H9c2 cells were seeded in the plates 48 h prior
to the addition of a studied compound, and 24 h prior to the experiment, the medium was
exchanged for serumꢀ and pyruvateꢀfree DMEM (SigmaꢀAldrich). MCFꢀ7 cells were seeded
in the plates 24 h prior to the addition of the studied compound.
Proliferation and cytotoxicity studies, protection against oxidative injury
The cytotoxic/antiproliferative activities of the examined hydrazones were studied at
different concentrations after a 72ꢀh incubation. 0.1% DMSO (v/v) was added to increase the
hydrazone solubility in the medium. At this concentration, DMSO had no effect on cellular
proliferation. Model oxidative injury in H9c2 cells was induced by the addition of 200 ꢁM
H2O2 to the culture medium at the same time as the chelator. The protective effect was
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evaluated at various concentrations of the tested substances after 24ꢀh of incubation. Again,
0.1% DMSO (v/v) was used in the culture medium.
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The cell viabilities of H9c2 and MCFꢀ7 cells were evaluated by a neutral red assay, which
measures the incorporation of the dye into the viable lysosomes.22 The optical density of the
neutral red dye was measured using a Tecan Infinite 200M plate reader (Tecan Group,
Männedorf, Switzerland) at λ = 540 nm. Viabilities of HLꢀ60 cells were evaluated by the
ability of active mitochondria to reduce yellow 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT; SigmaꢀAldrich) to purple formazan according to the
manufacturers' instruction. The optical density of MTT was measured at λ = 570 nm with the
subtraction of the λ = 690 nm background using a Tecan Infinite 200M plate reader. The
viabilities of hydrazoneꢀtreated cells were expressed as a percentage of untreated controls
(100%).
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Determinations of iron chelating efficiencies in solution
To assess iron chelation potential of the new hydrazones in solution, calcein fluorescence23
was measured. Calcein is a fluorescent probe that forms complexes with iron ions, causing
fluorescence quenching. The addition of the ironꢀchelating agent to the Feꢀcalcein removes
iron from its calcein complex with a new complex forming. This is manifested by an
increased fluorescence. A complex of calcein (free acid, 20 nM) with ferrous ammonium
sulfate (FAS; 200 nM) was prepared in HEPESꢀbuffered saline (150 mM NaCl, 40 mM
HEPES; pH 7.2). Calcein and FAS were stirred continuously in the dark for 45 min, after
which > 90% of fluorescence was quenched. Next, 995 ꢁl of the complex was transferred into
a stirred cuvette, and a baseline was measured. After 100 s, 5 ꢁl of studied compound solution
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was added yielding a final concentration of 5 ꢁM. Fluorescence intensity change was
measured as a function of time at 25°C using a Perkin Elmer LS50B fluorimeter (Perkin
Elmer, Waltham, MA, USA) at λex = 486 nm and λem = 517 nm for 350 s. The iron chelation
efficiency in solution was expressed as the percentage of efficiency of the reference chelator
SIH (100%).
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Ascorbate oxidation assay for analysis of redox activity of iron complexes
The ascorbate oxidation assay was employed to evaluate the redox activities of the iron
complexes of chelators in buffered solution using an established protocol.24, 25 In brief, 100
ꢁM ascorbic acid was prepared immediately prior to the experiment and incubated either
alone or in the presence of 10 ꢁM Fe3+ (added as ferric chloride in a 50ꢀfold molar excess in
500 ꢁM citrate) and chelators. Chelators were evaluated at iron binding equivalents (IBE) of
0.1 (excess of Fe), 1 (fully charged Feꢀchelator complexes) and 3 (excess of free chelator).
The chelators EDTA and DFO were used as positive and negative controls, respectively, as
their redox activity has been well characterized.26 The decrease in absorbance at 265 nm,
which is proportional to ascorbate oxidation, was measured after 10 and 40 min incubation at
room temperature using the Tecan Infinite 200M plate reader (Tecan, Austria). The decrease
in absorbance between the two time points was calculated and expressed as a percentage of
the control group without chelator (100%).
The effects of hydrazones on mobilizing cellular 59Fe
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Human transferrin (Tf) (SigmaꢀAldrich) was labeled with 56Fe or 59Fe (PerkinElmer,
Massachusetts, USA) to produce 56Fe2ꢀTf or 59Fe2ꢀTf, respectively, as previously described.27,
28 Unbound 59Fe was removed by exhaustive vacuum dialysis against a large excess of 0.15 M
NaCl buffered at pH 7.4 with 1.4% NaHCO3 by standard methods.27, 28
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To study the potencies of the prepared hydrazones to mobilize 59Fe from MCFꢀ7 cells, Fe
efflux experiments were carried out using established techniques.25, 29 In brief, after preꢀ
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labeling the cells with FeꢀTf (0.75 ꢁM) for 3 h at 37°C, the cell cultures were washed four
times with iceꢀcold phosphate buffered saline (PBS) and were then incubated with each
chelator (25 ꢁM) for 3 h at 37°C. The overlying medium containing released 59Fe was then
separated from the cells. Radioactivity was measured in both the cell pellet and supernatant
using a γꢀscintillation counter (Wallac Wizard 3, Turku, Finland).
Effect of the hydrazones on preventing cellular 59Fe uptake from 59FeꢀTf
The ability of the compounds to prevent cellular 59Fe uptake from 59FeꢀTf was examined
using standard techniques.25, 29 In brief, MCFꢀ7 cells were incubated with 59FeꢀTf (0.75 ꢁM)
for 3 h at 37°C in the presence of the chelators (25 ꢁM). The cells were then washed four
times with iceꢀcold PBS, and internalized 59Fe was determined via established methods by
incubating the cell monolayer for 30 min at 4°C with the general protease Pronase (1 mg/ml;
SigmaꢀAldrich). The cells were then harvested from the monolayer with a plastic spatula and
centrifuged for 1 min/12,000 g. The supernatant represents membraneꢀbound, Pronaseꢀ
sensitive 59Fe that was released by the protease, while the Pronaseꢀinsensitive fraction
28, 29
represents internalized 59Fe.25,
The amount of internalized Fe was expressed as a
percentage of Fe internalized by the control (untreated) cells (100%).
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Stability in plasma
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Heparinized drugꢀfree rabbit plasma was spiked with a standard solution of each chelator in
DMSO (1 mg/ml) to obtain a concentration of 100 ꢁM. The plasma was incubated at 37°C in
a Thermomixer comfort (Eppendorf, Germany) for 10 h and gently shaken (500 rpm) during
the experiment. Fifty microliters of plasma was taken at 0, 1, 2, 4, 6, 8 and 10 h, spiked with
an internal standard and precipitated with methanol (200 ꢁl). The clear supernatant was then
analyzed.
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HPLC assays were performed on an LC 20A Prominence (Shimadzu, Duisburg, Germany)
chromatographic system with a PDA detector. Separation was achieved on an Ascentis® C18
column (100 × 3 mm, 3 µm) protected with a guard column (2 × 4 mm, 3 µm, both from
SigmaꢀAldrich, Schnelldorf, Germany). The mobile phase was composed of 10 mM NaH2PO4
with 2 mM EDTA and methanol in either a 20:80 (4a and 4b) or 25:75 (5b) ratio.
Quantification was performed at 279 nm. A flow rate of 0.3 ml/min, a column temperature of
25°C and an injection volume of 20 µL were used. Either (E)ꢀN´ꢀ[1ꢀ(2ꢀ
hydroxyphenyl)propylidene]biphenylꢀ4ꢀcarbohydrazide
or
(E)ꢀN´ꢀ[1ꢀ(2ꢀ
hydroxyphenyl)propylidene]ꢀ1ꢀnaphthoylhydrazide were used as internal standards for the
analysis of 4a-b and 5b, respectively. All methods fulfilled the general validation criteria for
bioanalytical methods.
Data analysis
The concentrations of chelators inducing a 50% proliferation decrease (IC50) or 50%
cellular protection (EC50) were calculated using CalcuSyn 2.0 software (Biosoft, Cambridge,
UK). SigmaStat for Windows 3.5 (Systat Software, San Jose, CA, USA) was used for
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statistical analysis. Data are presented as a mean ± SD of a given number of experiments.
Statistical significance was determined using ANOVA with a Bonferroni post hoc test
(comparisons of multiple groups against a corresponding control). The results were
considered to be statistically significant at p < 0.05.
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Synthesis
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9
The target aroylhydrazones were synthesized via acetic acidꢀcatalyzed condensation of a
ketone with the respective hydrazide in a microwave reactor at 100 W or in an autoclave at
110°C (Figure 1). Ketones 7 and 8 were prepared by nitration of 2ꢀhydroxypropiophenone in
glacial acetic acid; the two positional isomers (7 and 8) were separated by column
chromatography. Hydrazides 3a, 3b, 3e and 3f were synthesized from the corresponding
methyl esters as described previously.18 The calculated values of molecular weight (MW),
lipophilicity (logP) and acidity of the phenol group (pKa) are given in Table 1.
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Table 1. The chemical properties of the studied compounds, their protective effects against model oxidative injury of H9c2 cardiomyoblasts,
their inherent toxicities to H9c2 cells, and their antiproliferative potential in MCFꢀ7 and HLꢀ60 cell lines compared to the lead compounds SIH
and NHAPI.
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Comp.
Ratio
Ratio
EC50 H9c2
(ꢁM)
TC50 H9c2 IC50 MCFꢀ7 IC50 HLꢀ60
MW logP pKa
TC50 H9c2/ TC50 H9c2/
IC50 MCFꢀ7 IC50 HLꢀ60
(ꢁM)
(ꢁM)
(ꢁM)
SIH a
241.3 1.64
9.2
7.8
7.8
7.8
7.8
7.8
7.8
5.4
5.4
5.4
5.4
5.3
18.2
68.5
28.9 ± 6.7
241.5 ± 52.9
153.9 ± 3.6
45.0 ± 1.6
37.1 ± 9.7
> 200*
12.4 ± 0.6
19.4 ± 6.0
4.7 ± 0.4
3.0 ± 0.7
18.1 ± 2.5
28.3 ± 7.6
> 200*
5.7 ± 0.5
2.3 ± 0.5
5.5 ± 1.3
1.6 ± 0.2
5.1 ± 2.0
24.6 ± 4.0
> 200*
2.3
12.6
33.1
15.2
2.1
5.1
104
28.2
27.6
7.2
NHAPI (4c) 300.3 0.97
349.4 3.20
375.4 3.52
300.3 0.97
301.3 0.09
301.3 3.41
363.4 3.62
389.4 3.93
314.3 1.39
314.3 1.39
315.3 0.51
NA
4a
4b
4d
4e
4f
22.8 ± 2.2
NA
NA
> 7
> 8
NA
> 200*
NA
2.3
NA
5.9
12.4 ± 12.1
1.8 ± 1.2
NA
16.2 ± 4.5
69.7 ± 3.9
69.7 ± 0.8
44.9 ± 7.4
> 200*
7.2 ± 1.5
6.8 ± 1.8
53.0 ± 7.2
22.3 ± 1.8
28.4 ± 6.5
2.8 ± 0.3
6.0 ± 1.9
10.8 ± 1.9
9.1 ± 3.1
21.0 ± 5.6
5a
5b
5c
5d
5e
10.5
1.3
11.7
6.5
61.2 ± 3.4
NA
2.0
4.9
> 7
> 10
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315.3 3.82
363.4 3.66
389.4 3.97
314.3 1.43
314.3 1.43
363.4 3.66
389.4 3.97
314.3 1.43
314.3 1.43
5.4
7.7
7.8
7.8
7.8
5.3
5.4
5.4
5.4
NA
10.3 ± 1.7
1.4 ± 1.0
NA
> 200*
35.9 ± 2.7
1.0 ± 0.4
28.2 ± 4.2
44.7 ± 7.8
23.1 ± 4.5
7.0 ± 4.0
> 100*
> 200*
5.1 ± 1.3
3.5 ± 0.4
21.3 ± 3.8
14.2 ± 2.3
6.4 ± 1.1
2.8 ± 0.2
56.5 ± 2.6
27.8 ± 4.1
> 200*
5.1 ± 0.9
2.3 ± 1.5
4.7 ± 2.1
4.6 ± 2.0
4.1 ± 0.7
1.6 ± 0.4
23.9 ± 1.8
15.6 ± 1.6
NA
7.0
NA
7.0
0.4
6.0
9.8
5.7
4.4
> 3
6.4
5f
9a
0.3
9b
9
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1.3
9c
NA
3.2
9d
8.5 ± 2.2
2.7 ± 0.1
NA
3.6
10a
10b
10c
10d
2.5
> 1.8
3.6
93.4 ± 6.2
> 100*
a Data from Refs.16, 17; * limited solubility beyond this concentration; NA = not active
MW, molecular weight; logP, the logarithm of the octanol/water partition coefficient; and pKa, the negative logarithm of the acid dissociation
constant calculated using ChemBioOffice Ultra 13.0. The EC50 values (concentration that reduced the toxicity induced by 200 µM H2O2 to 50%
of the untreated control) were calculated after a 24ꢀh incubation with H9c2 cells. The TC50 values (concentration that reduced cellular viability to
50% of the untreated control) were calculated after 72ꢀh of incubation with H9c2 cells. The IC50 values (concentration that reduced cellular
proliferation to 50% of the untreated control) were calculated after 72ꢀh of incubation with MCFꢀ7 or HLꢀ60 cells. The ratios of the
concentrations (TC50 in H9c2 cells/IC50 in cancer cells) indicate the selectivity of the antiproliferative action. Mean ± SD; n ≥ 4 experiments.
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The ability to chelate iron ions in solution
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The iron binding dynamics of the synthesized aroylhydrazones compared to those of the model
and wellꢀestablished iron chelator SIH were assayed using a weak chelator calcein, because iron
complexation quenches its fluorescence (Figure 2A). The studied compounds compete with
calcein for the iron ions; thus, the fluorescence of free (dequenched) calcein is proportional to the
chelating efficiency of the studied substances. The chelation activities of the new compounds
were expressed as a percentage of the efficiency of the SIH (100%). Compounds 4a, 4e, 4f, 9a,
9b and 9d showed an ability to chelate iron comparable to that of SIH (92ꢀ97%). In contrast,
biphenylꢀcontaining compounds 4b and 5b were apparently much weaker iron chelators with
only 41% and 29%, respectively, of the SIH activity. In general, the compounds of series 5 and
10, i.e., those with a nitro group in position 3, showed 10ꢀ19% lower iron binding than the
corresponding compounds of series 4 and 9 with 5ꢀnitro substitution.
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The ability to mobilize cellular 59Fe and prevent cellular 59Fe uptake from 59Feꢀtransferrin
The ability of the aroylhydrazones to mobilize the cellular 59Fe was expressed as the
percentage of total 59Fe loaded into MCFꢀ7 cells (Figure 2B). In agreement with the calcein
assay, biphenyl compound 4b had a weak ability to mobilize cellular iron. In contrast, compound
5b, which had a low iron binding ability in solution, released 40% of cellular iron, whereas
compounds 4f, 5f and 9b, which showed good iron chelation in solution, did not significantly
mobilize cellular iron. All naphthyl derivatives (4a, 5a, 9a, and 10a), as well as compounds 4d-e,
5e and 9c-d, mobilized more than 50% of the cellular iron.
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For the ability of the novel compounds to influence iron metabolism in the cell, their capability
to prevent the cellular uptake of 59Fe from 59Feꢀlabeled transferrin27, 30 is also important. The
uptake was expressed as a percentage of 59Fe internalized by MCFꢀ7 cells from a control
medium without a chelator (100%; Figure 2C). The uptake correlated with the ability of the
ligand to mobilize iron, and the best results were found using compounds 4a, 4d-e, 5a, 5c-e, 9a,
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9c-d and 10a. The low ability of compounds 4b and 5b to prevent the uptake of iron from Feꢀ
Tf can be explained by their low chelation capacity. Nevertheless, compounds 9b, 10c, 4f and 5f
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59
were not able to mobilize the Fe from MCFꢀ7 cells or to prevent the uptake of Fe from 59Feꢀ
Tf despite displaying chelation activities of 69ꢀ94% compared to SIH in the calcein assay. A
plausible explanation of this effect may be their poor ability to cross cellular membranes.
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Figure 2. Iron chelation and mobilization by the studied compounds. (A) The chelation
dynamics of the compounds in solution using the calcein assay for 360 s expressed as the
percentage of the chelation activity of the reference chelator SIH (100%). Mean ± SD; n ≥ 3
experiments. # significant difference compared to SIH at p < 0.05 (ANOVA). (B) The abilities of
the hydrazones to promote 59Fe mobilization from 59Fe prelabeled MCFꢀ7 cells and (C) to inhibit
the uptake of 59Fe from 59FeꢀTf by MCFꢀ7 cells after a 3ꢀh incubation. Mean ± SD; n ≥ 3
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#
experiments. Statistical significance (ANOVA, p < 0.05): compared to the reference chelator
5
6
SIH. * compared to the control (untreated) group.
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Proꢀoxidant/antioxidant activities of ironꢀchelator complexes
Proꢀoxidant properties of iron chelators have been previously identified as one of the plausible
mechanisms of their antiproliferative effect.25 The ability to form redoxꢀactive complexes was
therefore assessed by an ascorbate oxidation method.24, 25 The rate of ascorbate oxidation was
calculated as the percentage of the control group (ascorbate with free Fe3+, Figure 3). Three wellꢀ
established chelators were used as antioxidant (DFO, SIH) or proꢀoxidant (EDTA) controls.26
The iron complexes of DFO and SIH demonstrated a typical antioxidant profile: the ascorbate
oxidation was lower at an ironꢀbinding equivalent (IBE) of 3 (i.e., excess of chelator) than at an
IBE of 0.1 (i.e., excess iron). In contrast, the iron complex of EDTA showed a proꢀoxidant
profile, as it led to higher ascorbate oxidation at an IBE of 3 (429% of control) compared to that
at an IBE of 0.1 (155% of control).
Our model compound SIH showed antioxidant properties comparable to DFO (11% of control
at an IBE of 3). Similar activities were found for compounds 4d-f, 5c-5f and 10c-d with the level
of oxidized ascorbate at an IBE of 3 reaching 22ꢀ57% of control. In contrast, biphenyl
derivatives 4b, 5b and 10b were apparently proꢀoxidant with the levels of oxidized ascorbate at
an IBE of 3 reaching 133ꢀ159% of control. All other compounds were neither antioxidant nor
proꢀoxidant, as the levels of oxidized ascorbate were similar to those of the control at all IBEs.
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Figure 3. Proꢀoxidant or antioxidant properties of the studied hydrazones assessed by their
effects on Feꢀinduced oxidation of ascorbic acid at pH 7.4. Compounds were examined at iron
binding equivalents (IBEs) of 0.1 (excess of Fe), 1 (ironꢀchelator complexes with fully filled
coordination spheres) and 3 (excess of free chelator). DFO and SIH were used as negative
(antioxidant) controls, and EDTA was used as a positive (proꢀoxidative) control. The results are
expressed as the percentage of the control group in the absence of chelator (100%). Mean ± SD;
n ≥ 3 experiments. * significant differences compared to the control group (Fe3+ with ascorbate)
at p < 0.05 (ANOVA).
Prevention of H2O2ꢀinduced oxidative injury to cardiacꢀderived H9c2 cells
The ability of the studied substances to protect H9c2 cells from hydrogen peroxideꢀinduced
model oxidative stress was evaluated after 24ꢀh coꢀincubation of the cells with 200 ꢁM H2O2 and
various concentrations of the tested compounds (Figure 4, Supplementary Figure S1 and Table
1). The level of protection was expressed as halfꢀmaximal effective concentration (EC50), which
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is the concentration that reduced the cytotoxicity induced by H2O2 to 50% compared to the
control.
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9
Heterocyclic aroylhydrazones did not show any protective activities (except for nicotinic acid
derivatives 5d and 10d, which increased cell viability to 50% at concentrations over 60 µM).
Naphthyl derivatives showed protection at concentrations similar to SIH, except for 4a, which
was inactive. The most promising results were found for the biphenyl compounds, particularly
5b, 9b and 10b, which showed protective activities in the µM range (EC50 values of 1.4ꢀ2.7 µM),
that is, at approximately 10ꢀfold lower concentrations than SIH.16 At higher concentrations of the
ligands, the cell viabilities decreased, which was probably caused by the toxicity of the ligands
(Figure 4). Compound 5b increased cell viability by over 75% at concentrations of 3ꢀ30 µM,
which was the best activity/toxicity ratio among the studied compounds (Figure 4C).
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Figure 4. Protection of H9c2 cells against model oxidative damage induced by H2O2 (200 µM)
exposure for 24 h. Only the compounds that significantly protected H9c2 cells (4b, 5a, 5b, 5d,
9a, 9b, 10a, 10b and 10d) are shown. The results for the other compounds (4a, 4d-f, 5c-f and
10c) are included in the Supplementary content (Supplementary Figure S1). Mean ± SD; n ≥ 4
experiments. * significant differences compared to the H2O2 group without hydrazone coꢀ
treatment at p < 0.05 (ANOVA).
Inherent toxicities in cardiacꢀderived H9c2 cells
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The inherent toxicities of the studied hydrazones were determined after their 72ꢀh incubation
with the (nonꢀcancerous) H9c2 cells and are expressed as halfꢀmaximal toxic concentration
(TC50) values (Figures 5ꢀ6 and Table 1). Toxicity after a 24ꢀh incubation was not determined
because the shortꢀterm toxicities of aroylhydrazones are usually low and the TC50 values could
not be calculated due to their limited solubility.16 All pyrazine and pyridazineꢀbased hydrazones
(4e-f and 5e-f) and compounds 10c-d revealed very low toxicities even after 72 h; they did not
reach TC50 values within their solubility limits. Additionally, compounds 4a, 5b and 5c
displayed toxicities above 60 ꢁM. Other compounds showed comparable toxicities to SIH,
except for the biphenylꢀcontaining propiophenones 9b and 10b, which were toxic to cardiacꢀ
derived H9c2 cells at concentrations of 1ꢀ7 µM.
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Although the protection and toxicity values are not directly comparable because they were
determined at different time points (and hence overestimate toxicity), the TC50/EC50 ratio can
give an estimate of the selectivity of the protective action. Compound 5b showed the most
advantageous combination of protection (1.8 ± 1.2 µM) and toxicity (69.7 ± 3.9 µM) to H9c2
cells, which gives a TC50/EC50 ratio of 39. This ratio is much higher than 1.6 for SIH or 3.5 for
NHAPI.16
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Figure 5. Cytotoxicities of series 4 and 5 of the hydrazones towards nonꢀcancerous and
cancerous cell lines. The viabilities of H9c2 cardiomyoblasts were assessed after a 72ꢀh
incubation with the tested compounds. The antiproliferative activity was determined after
incubation of MCFꢀ7 breast adenocarcinoma cells and HLꢀ60 promyelocytic leukemia cells with
the compounds for 72 h. Mean ± SD; n ≥ 4 experiments. * significant differences compared to
the control (untreated) group at p < 0.05 (ANOVA).
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Figure 6. Cytotoxicities of series 9 and 10 of the hydrazones towards nonꢀcancerous and
cancerous cell lines. The viabilities of H9c2 cardiomyoblasts were assessed after a 72ꢀh
incubation with the tested compounds. The antiproliferative activity was determined after
incubation of MCFꢀ7 breast adenocarcinoma cells and HLꢀ60 promyelocytic leukemia cells with
the compounds for 72 h. Mean ± SD; n ≥ 4 experiments. * significant differences compared to
the control (untreated) group at p < 0.05 (ANOVA).
Antiproliferative activity towards MCFꢀ7 and HLꢀ60 cancer cell lines
The antiproliferative activities of the examined hydrazones against two cancerous cell lines,
human breast adenocarcinoma MCFꢀ7 and human promyelocytic leukemia HLꢀ60, were assessed
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Chemical Research in Toxicology
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after a 72ꢀh incubation with the tested compounds (Figures 5 and 6 and Table 1). The MCFꢀ7
and HLꢀ60 cancer cell lines used in the present study have been widely used in various
experimental panels of malignant cells and represent model solid and hematological cancer cell
types, respectably. They have also been used in our previous studies17, 18 and therefore allow
direct comparisons of the present results with our previous data. Compounds with naphthyl (4a,
5a, 9a and 10a) and biphenyl moieties (4b, 5b, 9b and 10b) showed antiproliferative potential at
µM concentrations (1.6ꢀ7.2 µM) in both MCFꢀ7 and HLꢀ60 cells. Similar IC50 values were also
found for pyridine derivatives 4d, 5c, 5d, 9c and 9d in the HLꢀ60 cell line. Pyrazines 4e and 5e
had IC50 values >20 µM, whereas pyridazines 4f and 5f did not show any toxicity towards the
studied tumor cell lines.
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Considering the inherent toxicities of the studied hydrazones in H9c2 cardiomyoblasts, many
of them showed higher selectivities for the malignant cell lines than SIH. No compound showed
better selectivity than NHAPI towards HLꢀ60 cells. However, naphthyl and biphenyl derivatives
of NHAPI, i.e., 4a and 4b, with TC50/IC50 ratios of 33 and 15, respectively, exceeded the
selectivity of NHAPI for MCFꢀ7 cells.
For most of the novel compounds, their antiproliferative activity was higher against the HLꢀ60
cell line than against the MCFꢀ7 cell line. This effect could also be observed for the model
compound NHAPI, whose IC50 values were 19.1 ± 6.0 ꢁM for MCFꢀ7 cells and 2.3 ± 0.5 ꢁM for
HLꢀ60 cells (i.e., 8 times lower than that for the MCFꢀ7 cells). Among the newly synthesized
aroylhydrazones, a striking contrast between the toxicities of the two cell lines was found for
compounds 4d, 5c, 9c and 9d. In our previous work investigating compounds containing the
same hydrazides with ketones without a nitro group, no marked differences in the toxicities to
both the MCFꢀ7 and HLꢀ60 cell lines were observed.16
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