2,6-Dihydroxybenzaldehyde Analogues of the Iron Chelator Salicylaldehyde Isonicotinoyl Hydrazone: Increased Hydrolytic Stability and Cytoprotective Activity against Oxidative Stress

Article abstract of DOI:10.1021/acs.chemrestox.8b00165

Salicylaldehyde isonicotinoyl hydrazone (SIH) is a small molecule and lipophilic chelating agent that firmly binds ferric ions from the cellular labile iron pool and is able to protect various tissues against oxidative damage. Previously, SIH possessed the best ratio of cytoprotective efficiency to toxicity among various iron chelators, including the desferrioxamine, deferiprone, and deferasirox used in clinical practice. Here, we prepared a series of 2,6-dihydroxybenzaldehyde aroylhydrazones as SIH analogues with an additional hydroxyl group that can be involved in the chelation of metal ions. Compound JK-31 (2,6-dihydroxybenzaldehyde 4-chlorobenzohydrazone) showed the best cytoprotective efficiency among the studied compounds including SIH. This compound significantly protected H9c2 cardiomyoblast cells against oxidative stress induced by various pro-oxidants, such as hydrogen peroxide, tert-butyl hydroperoxide, paraquat, epinephrine, N-acetyl-p-benzoquinone imine (a toxic metabolite of paracetamol), and 6-hydroxydopamine. The exceptional cytoprotective activity of JK-31 was confirmed using epifluorescence microscopy, where JK-31-treated H9c2 cells maintained a higher mitochondrial inner membrane potential in the presence of a lethal dose of hydrogen peroxide than was observed with cells treated with SIH. Hence, this study demonstrates the deleterious role of free iron ions in oxidative injury and the potential of 2,6-dihydroxybenzaldehyde aroylhydrazones in the prevention of various types of cardiac injuries, highlighting the need for further investigations into these compounds.

Full text of DOI:10.1021/acs.chemrestox.8b00165

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Article
2,6-Dihydroxybenzaldehyde analogues of the iron chelator
salicylaldehyde isonicotinoyl hydrazone: Increased hydrolytic
stability and cytoprotective activity against oxidative stress
Hana Jansová, Jan Kubeš, Petra Reimerova, Petra Št#rbová-Kova#íková, Jaroslav Roh, and Tomas Simunek
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00165 • Publication Date (Web): 17 Oct 2018
Downloaded from http://pubs.acs.org on October 17, 2018
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2,6-Dihydroxybenzaldehyde analogues of the iron
chelator salicylaldehyde isonicotinoyl hydrazone:
Increased hydrolytic stability and cytoprotective
activity against oxidative stress
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Hana Jansová, Jan Kubeš, Petra Reimerová, Petra Štěrbová-Kovaříková, Jaroslav Roh*,
Tomáš Šimůnek*
Charles University, Faculty of Pharmacy in Hradec Králové, Akademika Heyrovského
1
203, 500 05 Hradec Králové, Czech Republic
*Authors for correspondence:
Assoc. Prof. Jaroslav Roh
Tel.: +420-495-067-339; Fax: +420-495-067-339
E-mail: Jaroslav.Roh@faf.cuni.cz
Prof. Tomáš Šimůnek
Tel.: +420-495-067-422; Fax: +420-495-067-168
E-mail: Tomas.Simunek@faf.cuni.cz
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Abstract
Salicylaldehyde isonicotinoyl hydrazone (SIH) is a small molecule and lipophilic chelating
agent that firmly binds ferric ions from the cellular labile iron pool, and is able to protect various
tissues against oxidative damage. Previously, SIH possessed the best ratio of cytoprotective
efficiency to toxicity among various iron chelators, including the desferrioxamine, deferiprone and
deferasirox used in clinical practice. Here, we prepared a series of 2,6-dihydroxybenzaldehyde
aroylhydrazones as SIH analogues with an additional hydroxyl group that can be involved in the
chelation of metal ions. Compound JK-31 (2,6-dihydroxybenzaldehyde 4-chlorobenzohydrazone)
showed the best cytoprotective efficiency among the studied compounds including SIH. This
compound significantly protected H9c2 cardiomyoblast cells against oxidative stress induced by
various pro-oxidants, such as hydrogen peroxide, tert-butyl hydroperoxide, paraquat, epinephrine,
N-acetyl-p-benzoquinone imine (a toxic metabolite of paracetamol) and 6-hydroxydopamine. The
exceptional cytoprotective activity of JK-31 was confirmed using epifluorescence microscopy,
where JK-31-treated H9c2 cells maintained a higher mitochondrial inner membrane potential in
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the presence of a lethal dose of hydrogen peroxide than was observed with cells treated with SIH.
Hence, this study demonstrates the deleterious role of free iron ions in oxidative injury and the
potential of 2,6-dihydroxybenzaldehyde aroylhydrazones in the prevention of various types of
cardiac injuries, highlighting the need for further investigations into these compounds.
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Keywords
Salicylaldehyde isonicotinoyl hydrazone; SIH; iron chelation; chelator; oxidative stress; 2,6-
dihydroxybenzaldehyde.
Introduction
Iron (Fe) is an indispensable micronutrient for all mammalian cells as well as the most abundant
transition metal in the human body. It catalyzes biochemical reactions of vital importance for
numerous essential cellular processes, such as respiration; syntheses of nucleic acids; proliferation
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and differentiation of cells; as well as regulation of gene expression. An imbalance in Fe
homeostasis may induce numerous disorders. Iron deficiency causes anemia, while Fe overload
results in toxicity because of Fe-mediated oxidative stress. Free Fe ions readily cycle between their
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+
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ferric (Fe ) and ferrous (Fe ) oxidation states and thus catalyze the Haber-Weiss reaction of
superoxide radicals and hydrogen peroxide (H O ) to generate hydroxyl radicals - the most reactive
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and toxic reactive oxygen species (ROS). Additionally, Fe-mediated toxicity may be present even
without systemic Fe overload2-4 and it has been associated with various cardiovascular diseases
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such as ischemia/reoxygenation injury, cardiac dysrhythmias, myocarditis, heart failure, or
cardiotoxicity of diverse redox-cycling agents.5
In addition to conventional therapies for the prevention and/or treatment of cardiovascular
diseases, the use of various antioxidants has been proposed, but negative outcomes have generally
been found.6, 7 Unfortunately, most of the studies assessed various scavengers of already formed
ROS rather than the actual prevention of ROS formation. This may be accomplished with Fe
chelators forming complexes with Fe with no redox activity and thus inhibiting the formation of
hydroxyl radicals.8, 9
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Chelators are now well established in clinical practice to treat systemic Fe overload, where the
key objective is the prevention of cardiac complications caused by long-term Fe deposition.9-11 The
potential of clinically used Fe chelators (desferrioxamine (DFO), deferiprone (L1) and deferasirox
(ICL670A), Fig. 1) has also been examined in various oxidative stress-related pathologies without
systemic Fe overload, such as in neurodegenerative diseases,12-14 skin phototoxicity of UV
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irradiation, cancer,
and AIDS. Another potential application of Fe chelation is the general
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prevention of cardiac ischemia/reperfusion injury and cardiomyocyte oxidative injury, and in
these cases, lipophilic Fe chelators with low molecular weight, including clinically used L1 and
ICL670A as well as experimental compounds, showed promising cardioprotective potentials.20-22
Salicylaldehyde isonicotinoyl hydrazone (SIH) is a tridentate Fe chelator that can readily
penetrate cellular membranes and tightly bind labile Fe ions.23 Previous studies have clearly
demonstrated its potential to protect the heart. SIH protected isolated primary cardiomyocytes and
20, 23, 24
H9c2 cardiomyoblast cells from H O -induced oxidative injury
as well as against the toxicity
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caused by catecholamines and tert-butyl hydroperoxide (t-BHP). It displayed the best ratio of
cytoprotective activity against cellular oxidative injury to its own toxicity among various chelators,
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including the above-mentioned DFO, L1 and ICL-670A. SIH also afforded protection against the
cardiotoxicity of anthracycline daunorubicin (DAU) in vitro as well as in vivo using isolated rat
cardiomyocytes 21 and a model of anthracycline-induced heart failure in rabbits,27, 28 respectively.
However, the main limitation of SIH is its fast degradation in biological environments along with
its relatively short biological half-life.29, 30
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The aim of this study was to design and synthesize novel SIH analogues with increased
cytoprotective properties. The free rotation of the bond between the hydrazone and phenol
fragments can slightly decrease the chelation ability of SIH due to the formation of ineffective
conformers. Thus, we replaced the salicylaldehyde moiety with a symmetrical 2,6-
dihydroxybenzaldehyde group, which minimizes the abovementioned influence of the bond
rotation, and we prepared a series of novel 2,6-dihydroxybenzaldehyde aroylhydrazones (Fig. 2).
Furthermore, the additional hydroxyl group in position 6 adjacent to the hydrazone can also affect
its stability towards hydrolysis. To characterize these novel derivatives, we examined their (1) Fe
chelating activity in solution and in H9c2 rat embryonic cardiomyoblast cells; (2) cytoprotective
potential against H O -induced toxicity to H9c2 cells; (3) inherent toxicity (non-cancerous H9c2
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cells) and anticancer activity using MCF-7 breast adenocarcinoma and HeLa cervical carcinoma
cells; (4) redox properties; and (5) stability against hydrolysis in a cell culture medium.
Experimental procedures
1
General. The prepared compounds were characterized by their melting points, H NMR and
13C NMR spectra. The purities of all reported compounds were determined by elemental analysis.
The chemicals were obtained from following suppliers: Penta (Czech Republic) or Merck
(
Germany). All were of the highest available purity. Merck aluminum plates with silica gel 60 F254
and Merck Kieselgel 60 (0.040-0.063 mm) were used for thin layer chromatography and column
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chromatography, respectively. Melting points were determined with a Büchi B-545 (BUCHI
Labortechnik AG, Flawil, Switzerland) and are uncorrected. H and 13C NMR spectra were
measured on a VNMR S500 NMR spectrometer (Varian, Palo Alto, CA, USA). The elemental
analyses were carried out on an Automatic Microanalyzer EA1110CE (Fisons Instruments S.p.A.,
Milano, Italy). The chelator SIH was prepared as described previously.31
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,6-Dimethoxybenzaldehyde (11): To a solution of 1,3-dimethoxybenzene (10, 5.54 g, 40.1
mmol) in THF (15 mL) was added a solution of n-BuLi in hexane (30 mL, 1.6 mol/L, 48.0 mmol)
at 0 °C over 20 min. The reaction mixture was stirred at rt for 30 min, again cooled to 0 °C and
DMF (3.7 mL, 48 mmol) was added dropwise. The reaction mixture was stirred for 1 h at rt, and
then the mixture was concentrated to approximately half its volume under reduced pressure. Then,
10% aqueous HCl (50 mL) was added at 0 °C, and the reaction mixture was stirred for 12 h. The
resulting mixture was extracted with EtOAc (3 × 50 mL), and the combined organic phases were
washed with brine, dried over Na SO and evaporated. The product (11) was purified using column
2
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chromatography (mobile phase: hexane/ethyl acetate, 4:1 – 2:1) and recrystallized from aq. EtOH.
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Yield: 88% (5.9 g) as a yellowish solid. Mp 96-97 °C (lit. 95.5-97 °C). H NMR (500 MHz,
1
3
CDCl ) δ 10.50 (s, 1H), 7.44 (t, J = 8.5 Hz, 1H), 6.57 (d, J = 8.5 Hz, 2H), 3.88 (s, 6H). C NMR
3
(
126 MHz, CDCl ) δ 189.32, 162.10, 135.84, 114.25, 103.79, 55.97.
3
3
4
2
,6-Dihydroxybenzaldehyde (DHBA): AlCl (1.30 g, 9.76 mmol) was mixed with CH Cl (15
3 2 2
mL) at 0 °C. To this suspension, a solution of 2,6-dimethoxybenzaldehyde (11, 0.5 g, 3.0 mmol)
in CH Cl (10 mL) was added dropwise at rt, and the mixture was stirred at rt for 24 h. Then, 10%
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aqueous HCl (25 mL) was carefully added to the mixture under vigorous stirring. After 10 min, the
aqueous phase was separated and extracted with EtOAc (3 × 25 mL). The combined organic phases
were dried over Na SO and evaporated. The product (DHBA) was purified using column
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chromatography (mobile phase: hexane/ethyl acetate, 7:1). Yield: 63% (0.26 g) as a yellowish
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solid. Mp 154-156 °C (lit. 156-157 °C). H NMR (500 MHz, DMSO) δ 11.25 (s, 2H), 10.25 (s,
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1H), 7.35 (t, J = 8.3 Hz, 1H), 6.36 (d, J = 8.3 Hz, 2H). C NMR (126 MHz, DMSO) δ 194.50,
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62.23, 139.11, 110.18, 106.83.
General procedure for the preparation of 2,6-dihydroxybenzaldehyde hydrazones 1-9. 2,6-
Dihydroxybenzaldehyde (DHBA, 0.07 g, 0.5 mmol) and the appropriate hydrazide (0.75 mmol)
were dissolved in ethanol (96%, 5 mL) and five drops of conc. acetic acid were added. The reaction
mixture was refluxed for 2-24 h and then cooled to 5 °C and kept at this temperature as long as
crystallization was observed (1-5 days). In some cases (products 3 and 9), the solution was diluted
with water to initiate crystallization. Finally, the product was isolated by filtration, washed with
cold ethanol and then dried.
2,6-Dihydroxybenzaldehyde isonicotinoyl hydrazone (1): Commercially available isoniazide
was used. The reaction mixture was refluxed for 24 h. Yield: 97 % as a white solid. Mp 283-285
1
°
C. H NMR (500 MHz, DMSO) δ 12.33 (s, 1H), 11.08 (s, 2H), 8.97 (s, 1H), 8.80 (d, J = 5.3 Hz,
1
3
2
H), 7.87 – 7.83 (m, 2H), 7.12 (t, J = 8.2 Hz, 1H), 6.38 (d, J = 8.2 Hz, 2H). C NMR (126 MHz,
DMSO) δ 161.12, 158.70, 150.58, 147.77, 139.87, 132.90, 121.62, 106.74, 106.22. Anal. Calcd.
for C H N O : C, 60.70; H, 4.31; N, 16.33; Found: C, 60.39; H, 4.33; N, 16.28.
1
3
11
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,6-Dihydroxybenzaldehyde nicotinoyl hydrazone (2): Commercially available nicotinoyl
hydrazide was used. The reaction mixture was refluxed for 6 h. Yield: 81% as a white solid. Mp
1
271-272 °C. H NMR (500 MHz, DMSO-d ) δ 12.28 (s, 1H), 11.09 (s, 2H), 9.11 (d, J = 2.3 Hz,
6
1
4
H), 8.95 (s, 1H), 8.78 (dd, J = 4.9, 1.7 Hz, 1H), 8.30 (dt, J = 8.1, 2.0 Hz, 1H), 7.60 (dd, J = 7.9,
1
3
.9 Hz, 1H), 7.11 (t, J = 8.2 Hz, 1H), 6.38 (d, J = 8.2 Hz, 2H). C NMR (126 MHz, DMSO) δ
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61.24, 158.64, 152.65, 148.79, 147.18, 135.57, 132.73, 128.62, 123.83, 106.73, 106.27. Anal.
Calcd. for C H N O : C, 60.70; H, 4.31; N, 16.33; Found: C, 60.31; H, 4.52; N, 15.92.
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,6-Dihydroxybenzaldehyde benzohydrazone monohydrate (3): The benzohydrazide used in
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this reaction was prepared according to a published procedure. The reaction mixture was refluxed
for 24 h. The final solution was diluted with water to initiate the crystallization. Yield: 57% as a
1
white solid. Mp 249-251 °C. H NMR (500 MHz, DMSO) δ 12.10 (s, 1H), 11.12 (s, 2H), 8.95 (s,
1
H), 7.99 – 7.88 (m, 2H), 7.63 – 7.58 (m, 1H), 7.57 – 7.51 (m, 2H), 7.10 (t, J = 8.2 Hz, 1H), 6.38
1
3
(d, J = 8.2 Hz, 2H). C NMR (126 MHz, DMSO) δ 162.65, 158.57, 146.58, 132.79, 132.48,
1
1
32.16, 128.73, 127.77, 106.69, 106.40. Anal. Calcd. for C H N O .H O: C, 61.31; H, 5.15; N,
14 12 2 3 2
0.21; Found: C, 61.35; H, 5.18; N, 10.13.
,6-Dihydroxybenzaldehyde 4-methylbenzohydrazone monohydrate (4): The 4-
2
methylbenzohydrazide used in this reaction was prepared according to a published procedure.35
1
The reaction mixture was refluxed for 2 h. Yield: 92% as a white solid. Mp 255-257 °C. H NMR
(500 MHz, DMSO) δ 12.04 (s, 1H), 11.12 (s, 2H), 8.94 (s, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.34 (d,
1
3
J = 8.0 Hz, 2H), 7.09 (t, J = 8.2 Hz, 1H), 6.37 (d, J = 8.2 Hz, 2H), 2.38 (s, 3H). C NMR (126
MHz, DMSO) δ 162.50, 158.55, 146.29, 142.28, 132.41, 129.92, 129.28, 127.80, 106.69, 106.45,
2
9
1.23. Anal. Calcd. for C H N O .H O: C, 62.49; H, 5.59; N, 9.72; Found: C, 62.37; H, 5.65; N,
15 14 2 3 2
.68.
2
,6-Dihydroxybenzaldehyde 4-chlorobenzohydrazone monohydrate (5, JK-31): The 4-
3
5
chlorobenzohydrazide used in this reaction was prepared according to a published procedure. The
reaction mixture was refluxed and stirred for 24 h. Yield: 53% as a white solid. Mp 276-278 °C.
1H NMR (500 MHz, DMSO) δ 12.18 (s, 1H), 11.11 (s, 2H), 8.94 (s, 1H), 7.97 (d, J = 8.6 Hz, 2H),
1
3
7
.62 (d, J = 8.6 Hz, 2H), 7.10 (t, J = 8.2 Hz, 1H), 6.37 (d, J = 8.2 Hz, 2H). C NMR (126 MHz,
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1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
DMSO) δ 161.62, 158.63, 146.91, 137.06, 132.65, 131.53, 129.73, 128.89, 106.73, 106.36. Anal.
Calcd. for C H ClN O .H O: C, 54.47; H, 4.24; N, 9.07; Found: C, 54.20; H, 4.30; N, 8.93.
1
4
11
2
3
2
2
,6-Dihydroxybenzaldehyde 3-chlorobenzohydrazone monohydrate (6): The 3-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
5
chlorobenzohydrazide used in this reaction was prepared according to a published procedure. The
1
reaction mixture was refluxed for 4 h. Yield: 76% as a white solid. Mp 226-228 °C. H NMR (500
MHz, DMSO) δ 12.18 (s, 1H), 11.09 (s, 2H), 8.95 (s, 1H), 8.00 (t, J = 1.9 Hz, 1H), 7.93 – 7.89 (m,
1
H), 7.69 – 7.66 (m, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.10 (t, J = 8.2 Hz, 1H), 6.38 (d, J = 8.2 Hz,
1
3
2H). C NMR (126 MHz, DMSO) δ 161.23, 158.64, 147.15, 134.79, 133.56, 132.71, 131.99,
1
30.79, 127.49, 126.64, 106.73, 106.32. Anal. Calcd. for C H ClN O .H O: C, 54.47; H, 4.24;
14 11 2 3 2
N, 9.07; Found: C, 54.11; H, 4.27; N, 9.11.
2
,6-Dihydroxybenzaldehyde 4-bromobenzohydrazone monohydrate (7): The 4-
3
6
bromobenzohydrazide used in this reaction was prepared according to a published procedure. The
1
mixture was refluxed for 2 h. Yield: 85% as a white solid. Mp 276-278 °C. H NMR (500 MHz,
DMSO) δ 12.17 (s, 1H), 11.10 (s, 2H), 8.94 (s, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 8.6 Hz,
1
3
2
H), 7.10 (t, J = 8.2 Hz, 1H), 6.37 (d, J = 8.2 Hz, 2H). C NMR (126 MHz, DMSO) δ 161.71,
158.60, 146.91, 132.62, 131.87, 131.80, 129.86, 126.00, 106.70, 106.34. Anal. Calcd. for
C H BrN O .H O: C, 47.61; H, 3.71; N, 7.93; Found: C, 47.75; H, 3.73; N, 7.84.
1
4
11
2
3
2
2
,6-Dihydroxybenzaldehyde pyrazine-2-carbohydrazone (8): The pyrazine-2-carbohydrazide
3
7
used in this reaction was prepared according to a published procedure. The mixture was refluxed
1
and stirred for 4 h. Yield: 72% as a white solid. Mp 320 °C (with decomposition). H NMR (500
MHz, DMSO) δ 12.70 (s, 1H), 11.12 (s, 2H), 9.26 (d, J = 1.5 Hz, 1H), 9.14 (s, 1H), 8.92 (d, J =
1
3
2
.5 Hz, 1H), 8.79 (dd, J = 2.5, 1.5 Hz, 1H), 7.11 (t, J = 8.2 Hz, 1H), 6.37 (d, J = 8.2 Hz, 2H). C
NMR (126 MHz, DMSO) δ 159.52, 158.80, 148.65, 148.10, 144.43, 144.35, 143.59, 132.82,
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06.72, 106.45. Anal. Calcd. for C H N O : C, 55.81; H, 3.90; N, 21.70; Found: C, 55.46; H,
1
2
10
4
3
3
.85; N, 21.34.
,6-Dihydroxybenzaldehyde cyclohexanecarbohydrazone (9): The cyclohexanecarbohydrazide
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
5
used in this reaction was prepared according to a published procedure. The reaction mixture was
refluxed for 7 h. The final solution was diluted with water to initiate the crystallization. Yield: 77
1
%
as a white solid. Mp 226-230 °C. H NMR (500 MHz, DMSO) δ 11.53 (s, 1H), 11.00 (s, 2H),
8
.64 (s, 1H), 7.05 (t, J = 8.2 Hz, 1H), 6.33 (d, J = 8.2 Hz, 2H), 2.24 – 2.14 (m, 1H), 1.80 – 1.70 (m,
1
3
4H), 1.67 – 1.59 (m, 1H), 1.46 – 1.34 (m, 2H), 1.31 – 1.12 (m, 3H). C NMR (126 MHz, DMSO)
δ 171.25, 158.36, 144.73, 132.19, 106.63, 106.29, 42.80, 29.11, 25.53, 25.34. Anal. Calcd. for
C H N O : C, 64.11; H, 6.92; N, 10.68; Found: C, 63.75; H, 6.93; N, 10.49.
1
4
18
2
3
3
8
Cell culture. The H9c2 cardiomyoblast cell line was obtained from the American Type
Culture Collection (ATCC, USA). Cells were cultured as described previously.39, 40 Twenty-four
hours before the experiments, the medium was replaced with serum- and pyruvate-free DMEM
(Sigma, Germany). Serum deprivation was used to stop cellular proliferation to mimic the situation
in post-mitotic cardiomyocytes. However, to maintain the same protein content, the medium was
supplemented with the corresponding amount of BSA (4.7 mg/mL). Pyruvate-free medium was
used because pyruvate is an antioxidant and may interfere with ROS-induced toxicity.
The MCF-7 human breast adenocarcinoma cell line (ECACC; Salisbury, UK) and the human
cervical carcinoma (HeLa) cell line (ATCC; Manassas, VA, USA) were cultured as described
previously.41
DMSO was used to dissolve the lipophilic compounds. The final concentration of DMSO was
0
.2 % in all experimental groups, which had no effect on cellular viability.
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Calcein assay for measuring chelating activity of studied compounds in HBS buffer.42
Determinations of the calcein fluorescence intensity were used to evaluate the Fe chelation
3
9
efficiencies of the studied compounds as described previously by our group. Solution of Fe-
chelator under investigation was added to give a final concentration of 100 µM. The change in
fluorescence intensity was measured at 25 °C for 10 min using a Tecan plate reader. The Fe-
chelation activity of a given compounds is expressed as a percentage of the efficiency of SIH
(100 %).
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Calcein-AM assay for determining the cell membrane permeability of studied compounds
and their access to the intracellular labile Fe pool. The experiments were performed according
4
3
39, 40
to Glickstein et al. with some modifications as described previously by our group.
The
intracellular fluorescence was monitored as a function of time (1 min before and 10 min after the
addition of the studied compounds at a concentration of 100 µM; λ = 488 nm; λ = 530 nm)
ex
em
using a Tecan Infinite 200 M micro-plate spectrophotometer (Tecan, Austria).
Neutral red uptake assay for the assessment of cellular viability. The cellular viabilities of
H9c2, MCF-7 and HeLa cells were determined using a neutral red assay as described previously
by our group.39, 40 H9c2 cells (10,000 cells per well) were incubated with the studied compounds
and H O or other pro-oxidants for 24 h to measure their cytoprotective effects. The toxicities
2
2
(
H9c2 cells) and antiproliferative effects (MCF-7 and HeLa cells) of the tested compounds were
measured after 72 h of incubation. The viability of each group was expressed as a percentage
relative to the untreated control (100 %).
Ascorbate oxidation assay for the analysis of the redox activity of the Fe-chelator
complexes. The ability of the Fe complexes of the novel SIH analogues to mediate the oxidation
of ascorbate was examined using an established protocol44, 45 with step-by-step procedure described
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in our previous study. The solutions were assayed at Fe-binding equivalents (IBE) of 0.1 (excess
Fe), 1 (complexes of Fe with the studied compounds) and 3 (excess of the studied compounds).
4
6
The redox activities of Fe chelators EDTA and DFO have been well characterized, and therefore
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
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3
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4
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5
5
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5
6
0
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2
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8
9
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9
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7
8
9
0
they were used as the positive and negative controls, respectively. The decrease in absorbance
between the two time points (T10 – T ) was calculated and is expressed as the decrease in
40
absorbance.
Stability study. A Nexera X2 UHPLC instrument with a photodiode array detector (Shimadzu,
Japan) was emplyed to assay concentrations of the SIH analogues. The data were processed using
LabSolutions software (v. 5.55, 2013, Shimadzu, Japan). An Ascentis C18 column (100 x 3 mm,
3
µm) with the corresponding guard column (both Supelco, PA, USA) were used as a stationary
phase. The column oven was set at 25 °C. The mobile phase consisted of a component A (an
aqueous solution of 5 mM NaH PO and 1 mM EDTA; without pH correction) and a component
2
4
B (methanol). Their ratios (v/v) as well as detection wavelengths used for the assay were optimized
for each compound individually (Table S2). A flow rate of mobile phase was 0.3 mL/min. Ten µL
of the samples were injected onto the column. The selectivity was tested by comparing the
chromatograms obtained from analysis of the blanks with that monitored for the spiked samples.
The linearity, precision and accuracy of the assay were evaluated for both media (with and without
4
7
BSA) according to the FDA guidelines on Bioanalytical Method Validation over the range of
1
0 – 100 µM and fit the criteria well.
An appropriate volume of the standard solution of each compound (100 mM in DMSO) was
added to preheated aliquots (n = 3) of the cell culture medium with or without BSA. The final
concentration of the compound in the media was 100 µM. These samples were incubated at 37 °C
for 24 h. At selected times (0, 0.5, 1, 3, 6 and 24 h), 100 µL aliquots of the medium without BSA
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5
6
were diluted with methanol (1:1) and injected onto the column, whereas 100 µL aliquots of the
medium with BSA were transferred to Eppendorf tubes, and BSA proteins were precipitated with
300 µL of ice-cold methanol. Centrifugation (10,000 rpm, 10 min, 4 °C) was used to divide
proteins from the supernatant which was analyzed.
0
1
2
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9
0
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0
Determination of the mitochondrial inner membrane potential. Photomicrographs were
3
9
obtained as described previously by our group. The red and green fluorescence was quantitated
using a Tecan Infinite 200 M plate reader (Tecan, Austria).
Measurements of the inner tryptophan fluorescence. The ability of SIH and JK-31 to bind
to BSA was determined by measuring of quenching of the inner tryptophan fluorescence.48, 49 BSA
was dissolved in buffer (120 mM NaCl, 10 mM KCl, and 30 mM TRIS at pH = 7.4) to a final
concentration of 0.6 µM. The fluorescence (λ = 280 nm; λ = 350 nm) was measured 1 min
ex
em
before and 10 min after the addition of warfarin (used as a positive control) and SIH and JK-31 at
concentrations of 10, 30, 100 and 300 µM using a spectrophotometer Tecan Infinite 200 M (Tecan,
Austria).
Data analysis. SigmaStat for Windows 3.5 (SPSS, USA) was used for statistical analysis. The
data are presented as the mean ± standard deviation (SD). Statistical significance was assessed
using ANOVA with a Bonferroni post hoc test. Half-maximal toxic concentration (TC ), half-
5
0
maximal inhibitory concentration (IC ) and half-maximal effective concentration (EC ) were
5
0
50
calculated using CalcuSyn 2.0 software (Biosoft, UK). The graphs were prepared using GraphPad
Prism 7 for Windows (GraphPad Software, USA).
Results and Discussion
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Chemistry. 2,6-Dihydroxybenzaldehyde (DHBA) was prepared according to previously
published procedures with some modifications. In the first step, 1,3-dimethoxybenzene (10) was
lithiated with BuLi and subsequently formylated using N,N-dimethylformamide (DMF).32
Cleavage of the resulting 2,6-dimethoxybenzaldehyde (11) under mild reaction conditions using
excess aluminum chloride at room temperature gave DHBA in moderate yield (Scheme 1).34
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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0
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7
8
9
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9
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2
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5
6
7
8
9
0
The 2,6-dihydroxybenzaldehyde aroylhydrazones 1-8 and alkanoylhydrazone 9 were prepared
via the condensation of DHBA with the corresponding hydrazides in boiling ethanol (Scheme 2).
Upon cooling the reaction mixtures, pure products 1, 2 and 4-8 crystallized from the reaction
mixtures in sufficient purity. In the case of hydrazones 3 and 9, a small amount of water was added
to the cold reaction mixture to initiate the crystallization.
Determination of Fe-chelating efficiency in solution. Chelation efficiencies of the studied
compounds were firstly examined in a cell-free system. The increase in fluorescence (induced by
the displacement of Fe ions from the calcein complex ) was monitored for 10 min after the addition
of the studied compounds (all 100 µM). As apparent from Fig. 3, all of the studied compounds
(1-9) displayed strong chelating activities, with the exception of starting substance DHBA.
Compounds 1, 3 and 8 showed rapid chelation, while the effects of compounds 4, 5, 6 and 7, with
substituted benzoyl groups, had slower onsets (Fig. 3A); however, all of these compounds
displayed better chelating activity than parent chelator SIH at t = 10 min (Fig. 3B). Isonicotinoyl
derivative 1 and 4-chlorobenzoyl derivative 5 (JK-31) exhibited the highest efficacies. The only
alkanoyl derivative, 2,6-dihydroxybenzaldehyde cyclohexanecarbohydrazone (9), showed a
significantly lower chelating efficiency than SIH.
Determination of cell membrane permeability and access to the intracellular labile Fe.
The abilities of the studied compounds to access the labile Fe pool inside the H9c2 cells were
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1
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2
2
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5
5
6
compared using the calcein-AM assay. Increases in the intracellular fluorescence were measured
during the 10-min incubation of the cells with the studied compounds (all 100 µM concentration).
The results are shown as the percentages of the chelating effectiveness relative to reference chelator
SIH at t = 10 min (Fig. 4). In previous studies, SIH was more effective and faster at dequenching
intracellular calcein than clinically used Fe chelators L1 and ICL670A.26, 39 All of the newly
prepared compounds showed chelating activities comparable to SIH except for compound 4, which
displayed a chelating efficiency that was slightly but significant lower (18%) than that of SIH.
DHBA showed a low, but significant chelating activity compared to control (11 % of the chelating
activity of SIH).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
Inherent toxicity of the studied compounds. The own toxicities of each compound toward
H9c2 cells were examined using the neutral red uptake assay. To compare the individual agents,
their TC values (the concentrations causing 50 % viability loss compared to untreated control
5
0
cells) were determined. After 24 h, the toxicities of the studied a were very low (TC > 300 µM;
5
0
data not shown), therefore we incubated the cells with the studied compounds for 72 h. (Table 1).
All of the studied agents induced dose-dependent decreases in cellular viability (Fig. 5). Compound
6
was the only compound that exhibited significantly higher toxicity than chelator SIH; TC (6) =
50
6
.5 µM and TC (SIH) = 15.3 µM. Hydrazones 8 and 9 and starting substance DHBA caused lower
50
reductions in cell viability. Nonetheless, the majority of the studied hydrazones (1-5, 6, 7) displayed
inherent toxicities similar to that of parent chelator SIH.
Cytoprotective potential of the studied compounds against the oxidative stress. The ability
of the studied compounds SIH, DHBA and hydrazones 1-9 to protect H9c2 cells against oxidative
stress induced by 200 µM H O was evaluated by the neutral red uptake assay after 24 h (Fig. 6).
2
2
The EC values, i.e., concentrations reducing the H O toxicity to half of the viability of untreated
5
0
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2
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control cells, and their cytoprotective effectiveness at concentrations of 30 µM and 100 µM are
summarized in Table 1.
SIH and hydrazones 6-8 protected cells against the toxicity caused by H O in similar dose-
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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4
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4
4
4
5
5
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5
5
5
5
5
5
5
6
0
1
2
3
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5
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7
8
9
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8
9
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7
8
9
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5
6
7
8
9
0
dependent manner. They protected H9c2 cells significantly at a concentration of 30 µM. However,
even in the highest achievable concentration (600 µM), they were not able to increase cellular
viability above half of the untreated control cells, and therefore we could not calculate the EC50
values. Compounds 1-5 exhibited better protective properties than SIH. Similarly, they
significantly protected cells at a concentration of 30 µM, but they were more efficient. Compound
5
(JK-31) significantly protected cells at a concentration of 10 µM and showed the lowest EC50
value. Therefore, the protective properties of this compound were studied in more detail, and its
effectiveness was compared with that of SIH (see below). Alkanoyl derivative 9 and DHBA
displayed lower protective effects than SIH.
In this study, we corroborated the previously reported ability of SIH to protect H9c2 cells from
model oxidative stress.39, 40 However, here we used DMEM supplemented with bovine serum
albumin (BSA) to maintain the same protein content as in the medium with fetal bovine serum
(FBS). In previous studies, DMEM was employed without serum or albumin, which could cause
some discrepancies between the previous results and those presented herein. Therefore, we
determined the stability and cytoprotective effects in DMEM without BSA as well and found that
SIH and halobenzoyl hydrazones 5, 6 and 7 displayed better cytoprotective effect in DMEM
without BSA supplementation (Fig. S1; Table S1). These results are in agreement with the
increased stability of compounds 5, 6 and 7 in DMEM without BSA (Fig. S2). As SIH exhibited
similar stabilities in both media, we assume that its protein binding caused the decrease of its
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1
1
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2
2
2
2
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6
cytoprotective effect in BSA-containing medium. This hypothesis is supported by the ability of
SIH to quench tryptophan fluorescence in BSA (Fig. S3).
Evaluation of the redox activity of Fe complexes of the studied compounds to catalyze the
oxidation of ascorbate. The ascorbate oxidation assay was used to assess the Fe complexes of the
chelators with respect to affect the oxidation of a physiological substrate, ascorbate. The chelator
DFO was used as a negative (antioxidant) control, whereas EDTA was used as a positive (pro-
oxidant) control. As previously described, the Fe-DFO complex exhibited a typical antioxidant
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
effect, whereas the Fe-EDTA complex promoted higher ascorbate oxidation (Fig. 7). The Fe-SIH
complex as well as the Fe complexes with compounds 1, 2, 3, 5, 6, 7 and 8 displayed antioxidant
activities similar to that of the Fe complex with DFO. Compounds 4 and 9 significantly reduced
ascorbate oxidation only at an IBE of 3, and DHBA did not significantly influence ascorbate
oxidation. None of the studied compounds, except EDTA, exhibited redox cycling at any IBE
expressed as an increase in ascorbate oxidation (Fig. 7).
Stability of the studied compounds in cell culture medium. The stabilities of novel SIH
analogues 1-9 were assessed using HPLC analysis following 24 h of incubation of the compounds
in DMEM supplemented with BSA without cells (Fig. 8). The initial concentration of the studied
compounds was 100 µM, which reflected the maximal plasmatic concentration (108 µM)
3
0
determined in the pilot in vivo pharmacokinetics study with SIH in rabbits. For clarity, we divided
the studied compounds into three groups according to their structure. The first group included SIH
and its closest derivatives, 1 and 2, which each bear an (iso)nicotinoyl group (Fig. 8A). All these
compounds showed similar rapid degradation profiles. After only 3 h, approximately 50 % of these
compounds had decomposed, and by the end of the experiment, near complete decomposition of
these compounds was observed. Thus, unexpectedly, the additional hydroxyl group in the 6-
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position of the 2,6-dihydroxybenzaldehyde moiety did not influence the stability of the adjacent
hydrazone bond. The second group consists of unsubstituted and monosubstituted
benzohydrazones (compounds 3-7; Fig. 8B). These derivatives displayed higher stabilities than
SIH. The concentrations of these compounds had been reduced to approximately half of the initial
concentration after 12-16 h of incubation, and by the end of the experiment, approximately 25 %
of the compounds remained. Similar stability profiles were also exhibited by compound 8 and 9
(Fig. 8C), and their concentrations decreased by approximately 50% after 12-16 h of incubation.
The greater stability of derivatives 3-7 and 9 can be attributed to their higher lipophilicity when
compared to those of SIH or derivatives 1 and 2 (Table 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparison of the cytoprotective properties of SIH and compound 5 (JK-31). The JC-1
probe was employed to determine the mitochondrial inner membrane potentials (ΔΨ values), and
m
it was observed by epifluorescence microscopy (Fig. 9) and quantified spectrofluorometrically
(Fig. 10). H9c2 cells were incubated in DMEM with BSA for 24 h with 100 µM SIH or the most
active analogue (JK-31) alone or in combination with H O (200 µM). SIH and JK-31 induced
2
2
slight but significant reductions in the red fluorescence intensity and slight increases in green
fluorescence, indicating slow and transient losses of ΔΨ . SIH showed a decrease of approximately
m
2
0 % relative to the control group, while JK-31 showed a decrease of only 10 %. The H O -treated
2 2
cells displayed a conspicuous transition to green fluorescence, indicating complete ΔΨm
dissipation, whereas SIH and JK-31 significantly protected the H9c2 cells from the H O -induced
2
2
ΔΨ loss with JK-31 providing significantly greater protection than SIH.
m
The cytoprotective efficiencies of SIH and JK-31 were also evaluated on H9c2 cells against the
toxicity of other pro-oxidants that are used as the basic models of oxidative damage in various
pathological conditions (Fig. 11). We observed that SIH and particularly JK-31 significantly
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9
1
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5
5
5
5
5
5
5
6
protected H9c2 cells against the viability reduction induced by all the tested pro-oxidant
xenobiotics in a dose-dependent manner. The excellent cytoprotective efficacy of JK-31 was
observed in the experiments in which the H9c2 cells were exposed to tert-butyl hydroperoxide
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
t-BHP), a model oxidative stressor that is more stable than H O (Fig. 11B), and N-acetyl-p-
2 2
benzoquinone imine (NAPQI), a toxic metabolite of the analgesic paracetamol (Fig. 11C). NAPQI
is an ROS that causes oxidative injury by binding cellular macromolecules, which leads to
5
1
subsequent cell death. JK-31 significantly protected H9c2 cells at concentrations as low as
0 µM, while SIH showed protective effects starting at 10 µM (NAPQI) or 100 µM (t-BHP). In
1
these experiments, JK-31 always displayed significantly higher protective efficiencies than SIH at
10, 30 and 100 µM concentrations.
Two catecholamines, epinephrine (EPI; 100 µM; Fig. 11D) and 6-hydroxydopamine (6OHDA;
1
00 µM; Fig. 11E), were also used in this study because there is an increasing evidence that the
5
2
catecholamine-induced cardiac injury involves the Fe-mediated ROS formation. Previous studies
have shown the promising cytoprotective properties of SIH against oxidative damage induced by
2
5
the catecholamines EPI and isoprenaline to H9c2 cells. Compared with another study conducted
under the same conditions but with Fe chelators used in clinical practice (DFO, L1 and ICL670A),
2
2
SIH achieved better protective efficacy than the clinically used Fe chelators. In this study, JK-31
also significantly reduced the cytotoxicity caused by both of the studied catecholamines and
displayed a higher effectiveness than SIH at all concentrations with significant cytoprotective
effects. As catecholamines undergo spontaneous oxidation resulting in the formation of reactive
redox cycling intermediates, 53 we assessed the protective effect of SIH and JK-31 against
oxidative damage caused by EPI and 6OHDA that had been preoxidized for 24 h (oxEPI; Fig. 11F,
and ox6OHDA; Fig. 11G, respectively). JK-31 significantly protected H9c2 cells starting at
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concentrations of 10 µM and always displayed significantly higher protective potentials than SIH
except at 300 µM in the experiment with ox6OHDA.
We also investigated the protective effects of JK-31 against the cytotoxicity induced by the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
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4
4
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4
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4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
8
9
0
1
2
3
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0
1
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0
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1
2
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0
5
4
redox cycling herbicide paraquat (Fig. 11H) and anthracycline anticancer agent daunorubicin
(DAU; Fig. 11I). Anthracyclines induce ROS formation through several pathways, including one-
electron reduction leading to the formation of a semiquinone free radical. They may also increase
the redox-active Fe pool by generating superoxide radicals, which can induce the Fe release from
5
5
ferritin. SIH significantly reduced their toxicities starting at a concentration of 100 µM, while
JK-31 was effective from 10 µM (paraquat) or 30 µM (DAU). JK-31 provided greater protection
than SIH at all tested concentrations, but a statistically significant difference was only observed at
30 and 100 µM concentrations of paraquat.
9
Antiproliferative effects. Fe chelators have shown potential in anticancer therapies. Cancer
56
cells have a high Fe demand due to their quick replication rate. Limiting the Fe available to cancer
cells may therefore block their proliferation. The antiproliferative effects of SIH and its analogues
1
-9 were studied on MCF-7 breast adenocarcinoma cells (Fig. 12) after 72 h of incubation.
Previous studies have shown that chelator SIH demonstrated moderate cytotoxic activity compared
to some other anticancer drugs.57, 58 In this study, SIH was used as a control for the newly prepared
analogues. Only compounds 5 (JK-31) and 7 showed higher antiproliferative activities than SIH.
The remaining compounds (1-4, 6, 8, and 9) had similar or lower antiproliferative activities than
SIH. As expected, DHBA displayed the lowest activity (Table 2).
Similar results were also obtained with HeLa cervical carcinoma cells (Fig. 12). DHBA and
compound 8 exhibited poor antiproliferative activities. On the other hand, compounds 5, 7 and 4
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5
6
showed better antiproliferative activities than SIH. Other compounds displayed effects similar to
that of SIH (Table 2).
0
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Conclusions
In clinical practice, chelators of Fe represent a well-established therapy of diseases with chronic
5
9
Fe overload, especially the β-thalassemia. Chelation therapy is now being specifically evaluated
1
1
1
1
1
1
1
1
1
1
2
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0
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0
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0
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9
0
1
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3
4
5
6
7
8
9
0
6
0
and optimized for myocardial Fe removal and/or prevention of myocardial Fe accumulation. Fe
chelators have also been studied in cardiovascular diseases without systemic Fe overload to reduce
Fe-mediated intramyocardial oxidative stress. Earlier studies suggested that the experimental Fe
chelator SIH had the most promising properties in this field. However, the free rotation of the bond
between the hydrazone and phenol fragments in SIH can hinder its chelation ability due to the
formation of ineffective conformers. Therefore, we designed and prepared a series of 2,6-
dihydroxybenzaldehyde-type analogues of SIH and assessed their structure-activity relationships.
We found that all of the prepared compounds (1-9) showed excellent chelating properties
comparable to that of the parent SIH. Regarding their cytoprotective activities in H9c2
cardiomyoblast cells, only alkanoylhydrazone 9 showed a significantly reduced ability to protect
H9c2 cardiomyoblasts from model H O -induced toxicity. All other aroylhydrazones (1-8) showed
2
2
comparable or better cytoprotective activities than the parent SIH. Thus, an aromatic R substituent
greatly enhances the cytoprotective properties. It should be noted that 2,6-dihydroxybenzaldehyde,
the common metabolite of compounds 1-9, was not responsible for any of the biological activities
of compounds 1-9 evaluated in this study. The R substituent also plays a major role in the hydrolytic
stability of the studied hydrazones in aqueous media: (iso)nicotinoyl hydrazones 1 and 2 are
significantly less stable than the more lipophilic aroylhydrazones 3-7.
Among the studied compounds, analogue 5 (JK-31, 2,6-dihydroxybenzaldehyde 4-
chlorobenzohydrazone) showed the most promising properties. JK-31 displayed a cytotoxicity and
chelating activity similar to those of SIH, but it exhibited increased stability in cellular media and
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9
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6
better cytoprotective properties against oxidative damage of H9c2 cells induced by H O . JK-31
2
2
protected half of the H9c2 cells against the toxic effects of H O at a concentration of 18.2 µM,
2
2
whereas SIH was uncapable to protect 50 % of cells even at 600 µM. Moreover, JK-31 showed a
significantly higher cytoprotective efficiency than SIH against various pro-oxidant xenobiotics,
including tert-butyl hydroperoxide, the redox cycling herbicide paraquat, parent and preoxidized
epinephrine and 6-hydroxydopamine, and the toxic metabolite of paracetamol N-acetyl-p-
benzoquinone imine. Thus, JK-31 can serve as a new lead hydrazone-type antioxidant Fe chelating
agent for further structural refinements and in vitro and in vivo studies.
0
1
2
3
4
5
6
7
8
9
0
1
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Acknowledgements
This
study
was
supported
by
the
project
EFSA-CDN
(No.
CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF, Czech Science Foundation (project 13-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
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4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5008S) and the Charles University (project SVV 260 416).
Abbreviations
6
OHDA, 6-hydroxydopamine; BSA, bovine serum albumin; DAU, daunorubicin; DFO,
desferrioxamine; DHBA, 2,6-dihydroxybenzaldehyde; DMEM, Dulbecco’s modified Eagle’s
medium; EDTA, ethylenediaminetetraacetic acid; EPI, epinephrine; FAC, ferric ammonium
citrate; Fe, iron; IBE, Fe-binding equivalents; ICL670A, deferasirox; L1, deferiprone; NAPQI, N-
acetyl-p-benzoquinone imine; ox6OHDA, 24 h-preoxidized 6-hydroxydopamine; oxEPI, 24 h-
preoxidized epinephrine; PDA, photodiode array; ROS, reactive oxygen species; SIH,
salicylaldehyde isonicotinoyl hydrazone; t-BHP, tert-butyl hydroperoxide.
Supporting information
Cytoprotective effects and stabilities of compounds 1-9 in DMEM medium without BSA
1
13
supplementation, chromatographic conditions, H and C NMR spectra of final compounds 1-9.
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6
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