In vitro antioxidant activity of thiazolidinone derivatives of 1,3-thiazole and 1,3,4-thiadiazole

Article abstract of DOI:10.1016/j.cbi.2018.03.013

The initial steps in preclinical drug developing research concern the synthesis of new compounds for specific therapeutic use which needs to be confirmed by in vitro and then in vivo testing. Nine thiazolidinone derivatives (numerically labeled 1–9) classified as follows: 1,3-thiazole-based compounds (1 and 2); 1,3,4-thiadiazole based compounds (3 and 4); substituted 5-benzylideno-2-adamantylthiazol[3,2-b][1,2,4]triazol-6(5H)ones (5–8); and an ethylaminothiazole-based chalcone (9), were tested for antioxidant activity (AOA) by using three in vitro assays: DPPH (1,1-diphenyl-2-picrylhydrazyl scavenging capacity test); FRAP (ferric reducing antioxidant power test); and TBARS (thiobarbituric acid reactive substances test). Compounds 1–4 and 9 in particular are newly synthesized compounds. Also, traditional antioxidants Vitamins E and C and α-lipoic acid (α-LA) were tested. The results of DPPH testing: Vitamin C 94.35%, Vitamin E 2.99% and α-LA 1.57%; compounds: 4 33.98%; 2 18.73%; 1 15.62%; 5 6.59%; 3 4.99%; 6–9 demonstrated almost no AOA. The results of TBARS testing (% of LPO inhibition): Vitamin C 62.32%; Vitamin E 36.29%; α-LA 51.36%; compounds: 1 62.11%; 5 66.71%; 9 60.93%; 4, 6 and 7 demonstrated ～50%; 3 and 8 displayed ～38%; 2 23.51%. By FRAP method, Vitamins E and C showed equal AOA, ～100%, unlike α-LA (no AOA), and AOA of the tested compounds (expressed as a fraction of the AOA of Vitamin C) were: 2 and 4–75%; 8, 3 and 1–45%; 5–7 and 9–27%. Different red-ox reaction principles between these assays dictate different AOA outcomes for a single compound. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Phenyl-functionalized benzylidene, amino-carbonyl functional domains and chelating ligand properties of the thiazolidinone derivatives correlated with AOA.

Full text of DOI:10.1016/j.cbi.2018.03.013

Chemico-Biological Interactions 286 (2018) 119–131
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
In vitro antioxidant activity of thiazolidinone derivatives of 1,3-thiazole and
1
,3,4-thiadiazole
Mirjana Djukica,∗, Mara Fesatidou , Iakovos Xenikakis , Athina Geronikaki ,
b
b
b
c
d
a
a
e
Violina T. Angelova , Vladimir Savic , Marta Pasic , Branislav Krilovic , Dusan Djukic ,
Borko Gobeljic , Marina Pavlica , Ana Djuric , Ivan Stanojevic , Danilo Vojvodic , Luciano Saso
Department for Toxicology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia
Laboratory of Pharm. Chemistry, School of Pharmacy, Aristotle, University of Thessaloniki, 54124 Thessaloniki, Greece
Department of Chemistry, Faculty of Pharmacy, Medical University of Sofia, Dunav 2, 1000 Sofia, Bulgaria
Department of Organic Chemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11221 Belgrade, Serbia
School of Medicine, University of Belgrade, Dr.Subotica 8, 11221 Belgrade, Serbia
Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia
a
e
a
f
f
g
a
b
c
d
e
f
g
Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Rome, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
The initial steps in preclinical drug developing research concern the synthesis of new compounds for specific
therapeutic use which needs to be confirmed by in vitro and then in vivo testing.
Nine thiazolidinone derivatives (numerically labeled 1–9) classified as follows: 1,3-thiazole-based compounds
1
,3-thiazole,1,3,4-thiadiazole
Thiazolidinone
DPPH
FRAP
(
1 and 2); 1,3,4-thiadiazole based compounds (3 and 4); substituted 5-benzylideno-2-adamantylthiazol[3,2-b]
1,2,4]triazol-6(5H)ones (5–8); and an ethylaminothiazole-based chalcone (9), were tested for antioxidant ac-
tivity (AOA) by using three in vitro assays: DPPH (1,1-diphenyl-2-picrylhydrazyl scavenging capacity test); FRAP
ferric reducing antioxidant power test); and TBARS (thiobarbituric acid reactive substances test). Compounds
[
TBARS
(
1
–4 and 9 in particular are newly synthesized compounds. Also, traditional antioxidants Vitamins E and C and α-
lipoic acid (α-LA) were tested.
The results of DPPH testing: Vitamin C 94.35%, Vitamin E 2.99% and α-LA 1.57%; compounds: 4 33.98%; 2
18.73%; 1 15.62%; 5 6.59%; 3 4.99%; 6–9 demonstrated almost no AOA. The results of TBARS testing (% of LPO
inhibition): Vitamin C 62.32%; Vitamin E 36.29%; α-LA 51.36%; compounds: 1 62.11%; 5 66.71%; 9 60.93%; 4,
6
and 7 demonstrated ∼50%; 3 and 8 displayed ∼38%; 2 23.51%. By FRAP method, Vitamins E and C showed
equal AOA, ∼100%, unlike α-LA (no AOA), and AOA of the tested compounds (expressed as a fraction of the
AOA of Vitamin C) were: 2 and 4–75%; 8, 3 and 1–45%; 5–7 and 9–27%.
Different red-ox reaction principles between these assays dictate different AOA outcomes for a single com-
pound. Vitamin C appeared to be the superior antioxidant out of the traditional antioxidants; and compound 4
was superior to other tested thiazolidinone derivatives. Vitamin C appeared to be the superior antioxidant out of
the traditional antioxidants; and compound 4 was superior to other tested thiazolidinone derivatives. Phenyl-
functionalized benzylidene, amino-carbonyl functional domains and chelating ligand properties of the thiazo-
lidinone derivatives correlated with AOA.
1. Introduction
organism's homeostasis. Disturbances in an organism's red-ox balance
(
the natural consequence of free radical overproduction and/or their
Antioxidant therapy of diseases associated with oxidative stress (OS)
insufficient sequestration by antioxidant defense mechanisms) is asso-
ciated with a number of diseases as well as with aging [2,3]. Under
physiological conditions, the concentration of free radicals – including
reactive oxygen, nitrogen, sulfur and carbon species (ROS, RNS, RSS
and RCS) – is regulated by antioxidant defense systems. The inability of
has been proven to be safe and effective. The intake of antioxidants has
been shown to reduce the risk of cancer as well as neurological and
cardiovascular pathologies among others [1]. The subtle regulation of
free radicals is immensely important to the maintenance of the
∗
Corresponding author.
E-mail address: mirjana.djukic@pharmacy.bg.ac.rs (M. Djukic).
https://doi.org/10.1016/j.cbi.2018.03.013
Received 21 February 2017; Received in revised form 27 February 2018; Accepted 19 March 2018
Available online 21 March 2018
0009-2797/ © 2018 Published by Elsevier B.V.

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
antioxidant defense systems to mediate the concentration of free radi-
cals within the body results in oxidative, nitrosative, thilyl or carbonyl
stress. Free radicals indiscriminately oxidize any substance within
range, causing injury to endogenous as well as exogenous substances,
including all classes of biomolecules (proteins, lipids, DNA), disrupting
normal cell signaling mechanisms, devastating cellular energy supplies,
depleting reducing equivalents (reduced nicotinamide adenine dinu-
cleotide and glutathione) and causing cell death by eventually inducing
apoptosis [3–6].
Compounds with 1,3-thiazole or thiazolidinone structures impose a
broad spectrum of biological activities including the neutralization/
sequestration of ROS and RNS [7–12]. The antioxidant activity (AOA)
of thiazole derivatives has been acknowledged recently [13]. Kavitha
et al. synthesized some novel 4-(4-chlorophenyl)-2-aryl substituted
metheniminothiazoles as possible antioxidant agents [14]. Kachroo
et al. reported the antibacterial and AOA of newly synthesized N-[(4E)-
arylidene-5-oxo-2-phenyl-4,5-dihydro-1H-imidazol-1-yl]-2-(2-methyl-
(Center of Instrumental Analysis of the University of Thessaloniki). The
reactions were monitored by thin layer chromatography (TLC) plates on
F254 silica-gel coated sheets (Merck, Darmstadt, Germany) and each of
the purified compounds showed a single spot. Solvents, unless other-
wise specified, were of analytical reagent grade or of the highest quality
commercially available. Synthetic starting materials, reagents and sol-
vents were purchased from Aldrich Chemie (Steinheimm, Germany).
1,1-Diphenyl 2-picryl hydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine
(TPTZ), Vitamin C (L-ascorbic acid), Vitamin E (α-tocopherol) and α-
lipoic acid (α-LA) were obtained from Sigma Chemical Co. (St. Louis,
USA); thiobarbituric acid (TBA), ferric chloride, ferrous chloride, tri-
chloroacetic acid (TCA) and ethylenediaminetetraacetic acid (EDTA)
were purchased from Merck (Darmstadt, Germany). All reagents were
of analytical grade.
2.2. Chemistry
1,3-thiazol-4-yl) acetamide [15]. Gull et al. synthesized 2- amino-6-
Compounds 5–8 were synthesized and described in our previous
arylbenzothiazoles and investigated, inter alia, their RNS scavenging
activities [16]. Sarkanj et al. synthesized 4-methyl-7-hydroxycoumarin
derivatives (substituting at positions 7 - thiosemicarbazide and 4 -
thiazolidinone) and evaluated their potential AOA [14,17]. Another
interesting core is thiazolidinone with a wide spectrum of biological
activities, including antioxidant [18–20].
In view of the considerable importance of thiazoles and thiazolidi-
nones, which are the core structures in a variety of pharmaceuticals
with a broad spectrum of biological activity, specifically referencing
their ability to prevent ROS formation, the present work is intended to
synthesize new heterocyclic compounds bearing the thiazole moiety
paper [21]. The mechanisms used to synthesize compounds 1–4 are as
portrayed in the generic synthetic Scheme 1, while compound 9 was
synthesized as illustrated in Scheme 2.
2.2.1. Synthesis
of
N-((5-adamantan-1-yl)-1,3,4-thiadiazol-2-yl)-2-
chloroacetamide (Cb)
Anhydrous sodium carbonate (0.0302 mol, 3.2 g) was added with
stirring into a solution of (2-amino-4-adamantane) - 1,3,4-thiadiazole
(
(
Ab) (0.0266 mol, 6.251 g) in anhydrous dimethylformamide (DMF)
64 mL). Afterwards, chloroacetyl chloride (B) (0.0798 mol, 9.02 g) in
anhydrous DMF (34.9 mL) was added dropwise into the mixture. The
reaction was stirred at room temperature for 3 h. The course of the
reaction was controlled by TLC. After the reaction was complete, ice
was added and the precipitate was filtered and washed thoroughly with
water. The solid product (Cb) was recrystallized from ethanol (yield:
[
8]. The substitutions in the chosen derivatives were intended to sta-
bilize the radicals formed from compounds via resonance (as shown in
Figs. 4–8), or to donate labile hydrogens from the 1,3-thiazolidinone
system. These hydrogens could be donated to the DPPH radical to form
stable DPPH molecules. In vivo confirmation of the in vitro documented
AOA for derivatives of thiazole, thiadiazole, or thiazolidinone is the
next step in the preclinical phase of drug development research [9].
The goal of our study was to evaluate the AOA of newly synthesized
compounds (1–4 and 9) as well as that of compounds 5–8 which were
previously synthesized by three in vitro tests [21]. The compounds that
were tested in this study were categorized as follows: group I: 1,3-
thiazole based thiazolidinones (compounds 1 and 2); group II: thia-
diazole-based thiazolidinone (compounds 3 and 4); group III: thiazo-
lidinone derivatives fused to a 1,2,4-triazol heterocyclic system (com-
pounds 5–8); and group IV: an ethylaminothiazole-based chalcone
9
3.4%), m.p.179–180 °C. IR: 1562 (aromatic), 1712 (C=O), 3087 (NH).
1
H NMR: (δ ppm, DMSO-d6, 300 MHz): 1.26 (s, 6H, adamantane),
1.52–1.57 (d, 9H, adamantane), 3.91 (s, 2H, CH2), 7.46 (s, 1H, NHCO).
2.2.2. Synthesis of N-((5-adamantan-1-yl)-1,3,4-thiadiazol-2-yl-imino)-
thiazolidin-4-one (Eb)
While stirring, ammonium thiocyanate (D) (0.1 mol, 7.5 g) was
added into the N-[(5-adamantan-1-yl) -1,3,4-thiadiazol-2-yl] chlor-
oacetamide (Cb) (0.05 mol/16.7 g in 50 mL of 95% ethanol). The mix-
ture was boiled in a water bath and refluxed under stirring for 1 h. The
course of the reaction was controlled by TLC. The product (Eb) re-
mained unchanged overnight and was then filtered, washed with water
and recrystallized from ethanol (yield 61%), m.p. 258–259 °C. IR: 1602
(
compound 9) [21,22] (Fig. 1).
We used three in vitro tests that make use of different principles of
red-ox reactions: a) 1,1-diphenyl-2-picrylhydrazyl scavenging capacity
test, i.e. DPPH radical scavenging capacity (or DPPH) assay [23]; b)
ferric reducing antioxidant power (FRAP) assay [24]; and c) thiobarbituric
acid reactive substances (TBARS) assay [25,26].
(
3
4
aromatic), 1740 (C=O), 3360 (NH). 1H NMR: (δ ppm, DMSO-d6,
00 MHz): 1.75 (s, 6H, adamantane), 1.99–2.06 (d, 9H, adamantane),
.07 (s, 2H, CH2 thiazolidinone), 12.20 (s, 1H, NHCO) [27].
Synthesis: Aryl aldehyde (6 mmol) was added into a well-stirred
solution of 0.8 g of 4/5-substituted (thiazol-2-ylimino) thiazolidin-4-
one (4 mmol) in acetic acid (35 mL) previously buffered with sodium
acetate (8 mmol). The solution was refluxed for 4 h and then poured
into ice-cold water. The precipitate was filtered and washed with water.
The crude product was purified by recrystallization from dioxane [27].
2
. Material and methods
2.1. Chemicals
Melting points were determined with a MELTEMP II capillary ap-
paratus (LAB Devices, Holliston, MA, USA) without correction.
Elemental analyses were performed on a Perkin–Elmer 2400 CHN ele-
mental analyzer. All compounds synthesized were documented to be
within 0.4% of theoretical values. IR spectra were recorded as Nujol
mulls on a Perkin Elmer Spectrum BX. Wave numbers collected from IR
2.2.2.1. Synthesis of (2E,5Z)-((5-adamatane-1-yl)-1,3,4-thiadiazol-2-yl)-
imino)-5-(4-hydroxybenzylidene)-thiazolidin-4-one (3). Yield: 48.9%,
−
1
m.p. 258–259 °C. Rf = 0.71 (toluene-EtOH 7:3). IR (cm , Nujol):
1570 (aromatic), 1708 (C=O), 3089 (NH). 1H NMR (δ ppm, DMSO-
d6, 300 MHz): 1.77 (s, 6H, adamantane), 2.03–2.07 (d, 9H,
adamantane), 6.95–6.98 (d, 2H, C3′-C5′), 7.51–7.53 (d, 2H,C2′- C6′),
7.67 (s, 1H, C=C), 10.22 (s, 1H, -OH), 12.690 (s, 1H, NHCO). Anal.
Calc. for C22H22N4O2S2 (MW 438): C: 60.25%; H: 5.06%; N: 12.78%.
Found: C: 60.13%; H: 5.11%; N: 12.75%.
−1
1
13
spectra are given in cm
synthesized compounds in DMSO-d6 or CDCl
.
H NMR. C NMR spectra of the newly
solution were recorded
3
on a Bruker AC 300 instrument (Bruker, Karlsruhe, Germany) at 298 K.
Chemical shifts are reported as δ (ppm) relative to TMS which was used
as an internal standard. Coupling constants (J) are expressed in Hertz
120

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Fig. 1. Structures of nine tested thiazolidinones.
2.2.2.2. Synthesis of (2E,5Z)-((5-adamatane-1-yl)-1,3,4-thiadiazol-2-yl)-
2.3.1. DPPH assay
imino)-5-(4-hydroxy-3-methoxybenzylidene)-thiazolidin-4-one (4). Yield:
The reducing properties of the examined compounds were eval-
uated by their ability to transform violet 1,1-diphenyl-2-picrylhydrazyl
radical (DPP , i.e. DPPH radical) into its pale, yellow reduced form
(DPPH). The decrease in absorbance is related to the AOA of the
compound in question (the loss of violet color was measured at 517 nm)
[23].
−
1
6
2.3%, m.p. 264–266 °C. IR (cm , Nujol): 1600 (aromatic), 1711
1
•
(
C=O), 3089 (NH). H NMR (δ ppm, DMSO-d6, 300 MHz): 1.77 (s, 6H,
adamantane), 2.03–2.08 (d, 9H, adamantane), 3.85 (s, 3H, -OCH3),
7
1
.00–7.03 (d, 1H, C5′), 7.15–7.17 (d, 1H, C6′), 7.29 (s, 1H, C2′), 7.72 (s,
H, C=O), 9.95 (s, 1H, -OH), 12.69 (s, 1H, NHCO). Anal. Calc. for
C23H24N4O3S2 (MW 468): C: 58.95%; H: 5.16%; N: 11.96%. Found: C:
In brief, 100 μL of the stock solution was diluted to 2 mL with
ethanol (0.1 mg/2 mL) and mixed with 0.5 mL of DPPH radical solution
59.03%; H: 5.21%; N: 12.05%.
(
0.5 mM), resulting in a final concentration of the tested compound of
2
.2.3. Synthesis of 1-(2-ethylamino)4-methylthiazol-5-yl)-ethanone (J)
An acetone solution of 3-chloracetylacetone (2.26 mL, 0.02 mol)
0.04 mg/mL according to Das et al. [30]. The solutions were incubated
for 30 min in a dark place at room temperature. The absorbance was
measured at 517 nm. The mixture of ethanol and 0.5 mM DPPH radical
solution (1:4, v/v) was used as the control. The capacity of the tested
substance to reduce DPPH radical to DPPH corresponds to its AOA and
was expressed as a percentage of DPPH radical inhibition [I (%)] ac-
cording to the following equation:
(
5 mL) was added dropwise into an acetone solution of 1-methyl-
thiourea/ethylthiourea (1.8 g, 0.02 mol) (50 mL). The mixture was re-
fluxed for 1.5 h and the solid product was filtered and recrystallized
from ethanol [28,29].
2
5
.2.3.1. (E)-3-(2,6-dimethoxyphenyl)-1-(2-(ethylamino-4-methylthiazol-
Ac − Aa
-yl)-prop-2-en-1-one (9). Yield: 47.9%, m.p. 157–159 °C, Rf: 0.58
I (%) =
× 100
Ac
−1
(
CHCl3:MeOH, 9.5:0.5). IR (cm , Nujol): 3199 (NH), 3072 (C-H
1
vinyl.), 1632 (C=O), 1596 (C=C), 1560 (C-H arom). H NMR: (δ
ppm, DMSO-d6, 300 MHz): 1.18 (t, 3H, CH3-C-N), 2.55 (s, 3H, thiazole-
Legend: inhibition of DPPH radical - I; absorbance of the control
(Ac) and tested samples (Aa).
4
3
′-CH3), 3.25–3.28 (q, 2H, C-CH2-N), 3.77 (s, 3H, Ar-5′-′CH3), 3.84 (s,
H, Ar-2′-OCH3), 6.97–7.05 (m, 2H, Ar. 3′,4′), 7.27 (s, 1H, Ar. 6′), 7.37
2.3.2. FRAP assay
(
d, J = 15 Hz, 1H, CO-CH), 7.73 (d, J = 15 Hz, 1H, Ar-CH), 8.51 (s, 1H,
The principle of FRAP assays is to assess a reactant's ability to re-
NH). Anal. Calc. for C17H20N2O3S: C: 61.42%; H: 6.06%; N: 8.43%.
Found: C: 61.45%; H: 6.06%; N: 8.46%.
duce ferric tripyridyl triazine [Fe(III)-TPTZ] [Fe3+ complexed with
2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene
chloride]
into blue colored Fe(II)-TPTZ, measurable at 593 nm, at pH 3.6 (to
2.3. Evaluation of compounds' antioxidant activity
maintain iron solubility by the action of electron-donating antioxidants)
[
24,31–35].
Stock solutions of the tested thiazolidinones were prepared in
FRAP reagent was freshly prepared by mixing acetate buffer
ethanol (1 mg/mL). Measurements were repeated five times for chosen
concentrations of the test compounds for all three assays as is consistent
with the literature.
(300 mM, pH 3.6), TPTZ solution (10 mM TPTZ in 40 mM HCl) and
20 mM FeCl water solution (20 mM) in the ratio 10:1:1. 100 μL of the
stock solution was mixed with FRAP reagent up to 3 mL (final
3
121

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Scheme 1. A. Synthesis of the compounds 1-4.
Scheme 2. A. Synthesis of the compound 9.
concentration was 0.033 mg/mL) and incubated for 30 min at 37 °C.
The absorbance was measured at 593 nm, using FRAP working solution
as a blank.
malondialdehyde (MDA) and other aldehydes] and TBA reagents (in
ratio 1:2), measureable at 532 nm. The reaction takes place at a low pH
and at an elevated temperature [25].
Different substrates (as a source of MDA) can be used to mimic cell
membranes in this test, such as lecithin liposomes, free fatty acids, low
density lipoproteins, etc. In this case, lecithin liposomes were used as a
surrogate for a cell membrane's phospholipid bilayer. Lecithin water
solution (in final concentration of 3%) was prepared from 10% lecithin
2
.3.3. TBARS assay (inhibition of lipid peroxidation)
The principle of the method is based on the formation of a pink-
colored complex between carbonyls [decomposition products of oxi-
datively damaged poly unsaturated fatty acids (PUFA), including
122

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
commercial product (“Lipotech 10”) and kept it in an ultrasonic bath
for 30 min to obtained liposomes [26]. The reaction mixtures (total
3.3. Antioxidant activity of thiazolidinone derivatives obtained by TBARS
assay
volume of 4 mL) containing 3.9 mL of 0.075 M FeSO
FeSO × 7H O in 5 mL of water), 50 μL of liposome suspension, 10 μL
of the ethanol stock solutions (0.1 mg of the tested compound), 20 μL of
.1 M L-ascorbic acid (0.0882 g in 5 mL of water) and 20 μL of phos-
phate buffer (pH 7.4, I = 0.1) were incubated at 37 °C for 1 h. Then,
.2 mL of 0.1 M EDTA and 1.5 mL of TBA reagent (0.375 g TBA, 15 g
TCA and 2.1 mL HClO in 100 mL of distilled water) were added into
4
(0.1043 g
4
2
According to the equation, the % of LPO inhibition reached by the
conventional antioxidants Vitamins C and E and α-LA was 62.32%,
36.29% and 51.36% respectively. Compound 5 demonstrated the
highest observed AOA (66.71%), even higher than Vitamin C (62.32%).
Compounds 9 (60.93%) and 1 (62.11%) showed almost identical AOA
as Vitamin C. Slightly less AOA was recorded from compounds 6, 4 and
7, around 50%, (p < 0.0001), with α-LA and compounds 8 and 3
demonstrating around 38% inhibition (p < 0.0001), similar to that of
Vitamin E (36%). Compound 2 demonstrated the lowest AOA (23.51%)
(p < 0.0001) (Graph 3).
0
0
4
the reaction mixtures and heated at 100 °C for additional 15 min. The
final concentration of the tested compounds equals 1.75 μg/mL. The
control reaction mixture contained 10 μL of ethanol in place of a test
4
compound. The blank mixture did not contain FeSO and L-ascorbic
acid. Further on, samples were cooled on ice, centrifuged at 3000 rpm
for 10 min and the absorbance of supernatants was measured at
The data regarding the AOA of the synthesized compounds and the
conventional antioxidants collected via the three in vitro methods ap-
plied are summarized in Table 1.
532 nm. The results were expressed as a percentage of lipid peroxida-
tion (LPO) inhibition [I (%)] and calculated according to the following
equation:
The AOA of the tested compounds, expressed as the fraction of the
AOA of conventional antioxidants, were quantitatively categorized into
five categories (0–21%, 22–43%, 44–65%, 66–87%, 88–109%) within
the assays. Overall, out of the traditional antioxidants, Vitamin C had
the greatest antioxidant effect across all three tests. Vitamin E showed
no AOA in DPPH testing, moderate effects in TBARS and maximal ef-
fects in FRAP. By contrast, α-LA displayed any meaningful antioxidant
effects in the TBARS assay. Results regarding the AOA of the tested
compounds are mixed and will be covered under Discussions.
Spearman correlation analysis showed that FRAP and TBARS assays
Ac − Aa
I (%) =
× 100
Ac
Legend: inhibition of LPO - I; absorbance of the control (Ac) and
tested samples (Aa).
2.4. Statistics
2
were perfectly correlated (Slope = 1, r = 1) for each individually
tested compound and reference standards except for the DPPH assay.
Statistical software SPSS version 7.0 was used for the statistical
analysis. Descriptive statistical analysis (x, STDEV, med, SEM) and
Gaussian distribution of data were performed as well as a one-way
ANOVA test to compare differences between AOA of the tested com-
pounds between the three methods. Afterwards, a post-hoc Tukey test
was used to analyze the associations between the tested compounds.
The results were presented as an average ± STDEV. A Spearman cor-
relation was tested for using Graph Pad Prism Version 5.0 in order to
measure the associations between the results of the different methods
according to individual compounds.
4
. Discussion
The interpretation and the evaluation of AOA for the tested com-
pounds (1–9) was done with respect to Vitamins C and E and α-LA,
their chemical structures and metal chelating potency and the princi-
ples and thermodynamics of the underlying test reactions [36,37].
Vitamin C's AOA was evaluated through a two-step reduction and
Vitamin E's through a one-step reduction [38,39]. Stable semi-dehy-
droascorbic acid radical (Asc2 /Asc ) (Scheme 3) spontaneously re-
−
•-
duces compounds with a standard reducing potential (ΔE°´) higher than
+
282 mV and +174 mV, which corresponds to ascorbic monoanion/
3
. Results
•
+
−
•-
2−
radical and dianion/radical (AscH ,
pH = 7) respectively [40].
H
/AscH
and Asc /Asc
)
(
3.1. Antioxidant activity of thiazolidinone derivatives obtained by DPPH
Vitamin E (TOH) can reduce compounds with ΔE°´ > +500 mV,
assay
•
+
which corresponds to the couple tocopheroxyl (TO ), H /TOH, by a
one-step reduction mechanism (Scheme 4). The phylyl side chain of
Vitamin E makes it liposoluble [40,41].
According to the equation for the percent of DPPH radical inhibi-
tion, Vitamin C reached an inhibition of 94.35%, Vitamin E 2.99% and
The reduced form of α-LA, dihydrolipoic acid (DHLA), is a potent
reducing agent (α-LA/DHLA, ΔE°´ = −320 mV) with amphiphilic
properties (Scheme 5). Additionally, DHLA is capable of forming a
complex with Fe3+ [36].
α-LA 1.57%. Compounds 4, 2 and 1 resulted in 33.98%, 18.73% and
1
6
5.62% inhibition respectively; compounds 5 and 3 demonstrated
.59% and 4.99% inhibition, while the rest of the derivatives showed
almost no detectable AOA (Graph 1). Overall, the tested thiazolidinones
showed lower AOA compared to Vitamin C (p < 0.0001) via the DPPH
assay.
4.1. DPPH assay
•
•
Transfer of H from the tested compound to the N of DPPH radical
3
.2. Antioxidant activity of thiazolidinone derivatives obtained by FRAP
reagent follows SET and/or HAT types of antioxidant mechanisms
(Scheme 6) [23].
assay
The extremely high AOA of hydrophilic Vitamin C (confirmed by all
three in vitro assays, Graphs 1-3) can be attributed to the two OH groups
on adjacent carbon atoms of the gamma-lactone ring, responsible for
the two-step transfer of H+ and e . Vitamin E did not achieve AOA by
According to the FRAP method, Vitamins E and C showed maximal
2
+
2+
and almost equal AOA (812.40 μM Fe
vs. 868.40 μM Fe , respec-
−
tively), though α-LA achieved no AOA (Graph 2). The AOA of the tested
compounds was lower compared to Vitamins C and E (p < 0.0001) and
higher when compared to α-LA (p < 0.0001). Expressed as a percen-
tage of Vitamin C's AOA reached by this method, the AOA of the de-
rivatives was classified in three categories as follows: compounds 2 and
•
DPPH assay, as the reduction of DPP by Vitamin E is thermo-
•
+
•
dynamically unfeasible (TO , H /TOH: ΔE°´ = +500 mV; DPP /DPPH:
ΔE°´∼+250 mV) [42]. Dihydrolipoic acid is a strong reducing agent
(DHLA/α-LA ΔE°´ = −320 mV), thus the lack of AOA can be explained
by the presence of its oxidized form, α-LA, in the reaction mixture, and
α-LA is not capable of donating H to DPP• [36]. Also, the overall
4
(72–78%); compounds 8, 3 and 1 (43–46%); and compounds 5, 6, 9
and 7 (25–29%) (Graph 2).
•
123

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Graph 1. Antioxidative activity of 1,3-thiazolone and
1,3-thiazole derivatives and reference standards as-
sessed by DPPH assay. Number of measurements:
n = 5. Final concentrations of the tested compounds
equal 0.04 mg/mL. Values are expressed as
•
mean ± STDEV. Values are expressed as a % of DPP
inhibition (i.e. its sequestration by tested com-
pounds). Legend: ¥-compared with vitamin C,
$
-compared with vitamin E, ¤- compared with α-li-
poic acid. Levels of significances were graduated as: *
(
(
p < 0.05); ** (p < 0.02); *** (p < 0.004); ****
p < 0.003); ***** (p < 0.0001).
Graph 2. Antioxidative activity of 1,3-thiazolone and
,3-thiazole derivatives and reference standards as-
1
sessed by FRAP assay. Number of measurements:
n = 5. Final concentrations of the tested compounds
equal 0.033 mg/mL. Values are expressed as
mean ± STDEV. Values are expressed as a FRAP
2
+
units, i.e. μM Fe . Legend: ¥-compared with vitamin
C, $-compared with vitamin E, ¤- compared with α-
lipoic acid. Level of significance was *****
(p < 0.0001).
Graph 3. Antioxidative activity of 1,3-thiazolone and
,3-thiazole derivatives and reference standards as-
1
sessed by TBARS method. Number of measurements:
n = 5. Final concentrations of the tested compounds
equal 1.75 μg/mL Values are expressed as
mean ± STDEV. Values are expressed as a % of LPO.
Legend: ¥-compared with vitamin C, $-compared
with vitamin E, ¤- compared with α-lipoic acid.
Levels of significances were graduated as:
*
(
(
p < 0.05); ** (p < 0.02); *** (p < 0.004); ****
p < 0.003); ***** (p < 0.0001).
reaction would require the generation of an intermediate between DPP•
and α-LA which just so happens to be stereochemically unfeasible
1) likely attributable to the donation of an H from the amino group
(NH) and N-radical intermediate stabilization which enables the reac-
tion (Fig. 2).
•
(
Scheme 7).
Group I (derivatives 1 and 2) accomplished moderate AOA (Graph
The only difference between compounds 1 and 2 relates to the
124

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Table 1
In vitro determined AOA of title compounds, vitamins C and E and α-lipoic acid. Number of measurements: n = 5. Concentration of ethanol stock solution of the
tested compound was 1 mg/mL (Final concentrations in AOA, in vitro tests were: DPPH: 0.04 mg/mL; FRAP: 0.033 mg/mL; and TBARS; 1.75 μg/mL). Values are
expressed as mean ± STDEV.
Compounds
Name
Structure
Antioxidative potential
DPPH
FRAP
TBARS
1
,3-Thiazole based thiazolidinone
derivatives
1
2
Ethyl 2-{[(5Z)-5-(2-methoxybenzylidene)-4-
oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}-1,3-
thiazol-4-yl-acetate
15.62 ± 4.91
365.40 ± 134.54 62.11 ± 20.79
639.40 ± 233.33 23.51 ± 9.04
(
Group I)
Ethyl 2-{[(5Z)-5-(3-methoxybenzylidene)-4-
oxo-4,5-dihydro-1,3-thiazol-2-yl]amino}-1,3-
thiazol-4-yl-acetate
18.73 ± 5.98
Thiadiazole-based thiazolidinone
derivatives
3
4
5-(4-Hydroxybenzylidene)-2-[(5- adamantyl-
1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-
4-one
4.99 ± 2.24
33.98 ± 9.65
364.40 ± 145.89 39.64 ± 4.76
592.40 ± 240.12 47.80 ± 9.35
(
Group II)
5-(4-Hydroxy-3-methoxybenzylidene)-2-[(5-
adamantyl-1,3,4-thiadiazol-2-yl)imino]-1,3-
thiazolidin-4-one
Thiazolidinone derivatives fused
to 1,2,4-triazol heterocyclic
system
5
6
5-(4-Hydroxy-3-methoxybenzylidene)-2-
adamantyl-1,3-thiazolo [3,2-b]1,2,4-triazol-
6(5H)-one
6.59 ± 3.84
1.36 ± 1.29
217.40 ± 59.90
231.40 ± 64.46
205.40 ± 70.01
66.71 ± 20.69
53.33 ± 16.01
53.8 ± 21.51
(
Group III)
5-(4-Methoxybenzylidene)-2-adamantyl-1,3-
thiazolo [3,2-b]1,2,4-triazol-6(5H)-one
7
8
5-(3-Hydroxybenzylidene)-2 adamantyl-1,3-
thiazolo [3,2-b]1,2,4-triazol-6(5H)-one
2.62 ± 1.88
3.80 ± 1.69
5-(4-Hydroxy-3,5-dimetoxybenzylidene)-2
adamantyl-1,3-thiazolo [3,2-b]1,2,4-triazol-
379.40 ± 114.48 37.10 ± 14.51
6
(5H)-one
Ethylaminothiazole-based
9
3-(2,6-Dimethoxyphenyl)-1-[2-(ethylamino)-
4-methyl-1,3-thiazol-5-yl]prop-2-en-1-one
0.15 ± 1.20
238.40 ± 82.90
60.93 ± 21.67
chalcone (Group IV)
Reference standards
Vitamin C
94.35 ± 32.05 812.40 ± 321.85 62.32 ± 20.63
Vitamin E
2.99 ± 2.04
1.57 ± 1.48
868.40 ± 345.87 36.29 ± 9.26
α-Lipoic acid
0.16 ± 1.42
51.36 ± 17.78
position of a methoxy group (OMe) (orto OMe in compound 1 and meta
OMe in compound 2), which could be the reason for the higher ob-
served AOA of compound 2. Namely, stabilization of the N by delo-
Phenol groups are stronger electron-donating groups (EDG) than
NH, thus an electron delocalization toward C=O orchestrated by para
•
•
OH probably exceeds the N intermediate stabilization in compound 3.
calization of electrons towards carbonyl group (C=O) (electron with-
drawing group, or EWG) is more pronounced in the case of the meta
OMe derivative (compound 2), as electron delocalization occurs only
inside the benzylidene ring and does not extend towards C=O (Fig. 3).
From group II, compound 4 accomplished the highest AOA out of all
tested compounds, though the observed AOA was moderate in com-
parison to Vitamin C (Graph 1). By combining the properties of the
phenol (PhOH) and NH functional groups, group II compounds can
employ concomitant phenoxy/amino radical reaction mechanisms,
contributing to its higher AOA, comparing to the other derivatives
The presence of meta OMe in compound 4 which enhances the stabi-
lization of the O-radical intermediate is the only differentiating factor
between the two compounds and the most likely cause of its six-fold
higher AOA [43].
Group III compounds showed poor AOA as compared to Vitamin C
•
by DPPH assay. The only possible source for H donation refers to the
PhOH of the benzylidene ring as there is no NH available in group III
derivatives. The benzylidene moiety of compound 5 closely resembles
that of compound 4 (para OH and meta OMe) and is probably the reason
for the fact that a higher AOA was recorded for compound 5 compared
to the other group III derivatives (Fig. 6) [43].
(
Figs. 4 and 5).
125

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Scheme 3. Two steps reduction mechanism of vitamin C.
Scheme 4. One step reduction mechanism of vitamin E.
Scheme 5. Two steps reduction mechanism of α-lipoic
acid.
9
, probably because its N • intermediate is less stabilized due to the
presence of the ethyl group instead of an aromatic ring (Fig. 8).
To conclude, the compounds labeled 1–9 accomplished weak AOA
by DPPH assay as compared to Vitamin C. It is possible that con-
comitant resonant stabilization of the N-radical and the boosted stabi-
lization of the O-radical intermediate of the para OH and meta OMe
groups in the benzylidene ring contributed to the enhanced AOA of
compound 4. The somewhat lower AOA of compounds 1 and 2 are
likely attributable to the presence of the NH group and OMe substituent
in the orto and meta position of the benzylidene ring respectively. The
negligible AOA of compound 5, though higher than that of other group
III derivatives, is probably due to the presence of the benzylidene
moiety (as in compound 4). Compound 9 did not accomplish AOA,
Scheme 6. SET and HAT AO mechanisms.
perhaps due to poor N • intermediate radical stabilization.
4.2. FRAP assay
−
The antioxidant mechanism of FRAP test concerns only e transfer.
The reaction between Vitamins C and/or E and Fe3+ occurs sponta-
neously at low pH since their reducing potentials are lower than for
3+
2+
Fe /Fe (ΔE°´ = +770 mV) [39,41,44]. α-Lipoic acid did not react
with Fe3+ in spite of the strong reducing potential of α-LA/α-DHLA
(
ΔE°´ = −320), probably because the potential of DHLA to form a
3+
complex with Fe
dominated [39].
•
Compounds 2 and 4 exhibited the highest AOA by the FRAP method
around 75% of the Vitamins C and/or E) compared to other thiazoli-
Scheme 7. Stereochemically unfeasible reaction between DPP and α-LA.
(
dinone derivatives (Graph 2). Both compounds exhibit chelating ligand
activities that could potentially replace bidentate heterocyclic ligands
The negligible AOA of other group III derivatives can be explained
by the lack of any H (6); sterically hindered resonance between para
•
of FRAP reagent, whereas
a SET mechanism would occur in-
OH in the benzylidene moiety and the C=O group (para OH is sur-
rounded by two OMe at positions 3 and 5) (8); and the meta OH fixed
electron delocalization within the benzylidene ring and not toward the
EWG C=O (7) (Fig. 7).
tramolecularly. The formation and the stability of –S→Fe←S– and –S→
Fe←N– types of coordinate bonds, associated with intramolecular cy-
clisation forming 5-membered rings in compounds from groups I and II,
are responsible for their higher AOA, while the weaker –O→Fe←S–
coordinate bond and the formation of a 4-member ring contributed to
•
The donation of H from NH did not result in any AOA of compound
126

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Fig. 2. a,b. Resonant stabilization of N-radical in group I derivative, 1 (a) and 2 (b).
theory of electron transfer (Reaction 1). Autoxidation of the AscH‾ is
6
−1 −
much slower, with k∼10 M
s at a pH of 7.4 [37]. The production of
hydrogen peroxide (H ) (Reaction 3) precedes the production of the
2 2
O
•
most potent of the hydroxyl radicals, HO , which forms either by
homolytic cleavage or through a Fenton reaction (Reaction 4). The
•
-
•
2
protonated form of O , the perhydroxyl radical (HO , pKa = 4.7) or
•
+
HO can initiate LPO by the abstraction of bis-allylic H from PUFA
Reactions 2, 5-10) [40]. The thermodynamic reactions described
(
below could possibly promote LPO (initiation, propagation and termi-
nation) and precede MDA formation [39,40].
The propagation of LPO is comprised of the formation of PUFA ra-
•
•
•
dical (PUFA ), PUFA peroxyl (PUFA-OO ), PUFA alkoxyl (PUFA-O ) and
PUFA hydroperoxides (PUFA-OOH), which undergo β-scission reac-
tions, or intramolecular cyclisation, followed by decomposition into
carbonyls (including MDA) in the termination phase of LPO [26].
The LPO inhibition accomplished by Vitamin C, α-LA and Vitamin E
Fig. 3. Resonant stabilization of 1 and 2 O-radicals.
(
62.32%, 51.36% and 36.29%, respectively), decreases in accordance
the lower level of AOA of compound 9.
with their reducing potentials and the chelating properties of DHLA
(Graph 3). The strong LPO inhibition achieved by Vitamin C is attri-
butable to its two-step reduction capability and low standard reducing
potentials. Vitamin E terminates LPO through its reaction with PUFA-
OO (reaction 12) that prevails thermodynamically and kinetically over
reactions of LPO propagation (reaction 9, with a low rate constant)
[40,41,44].
Within group III, chelating ligand activities are diminished in con-
densed heterocyclic rings. Compound 8 (in which a para OH is sur-
rounded by two meta OMe in a benzylidene moiety) demonstrated the
highest level of AOA in this group, suggesting a type of reaction in-
volving ligand exchange occurred.
However, the presence of meta OMe in the benzylidene rings of
compounds 2 and 4 (which accomplished the highest AOA of all com-
pounds by this method) indicated the importance of NH and meta OMe
in benzylidene ring above that of the enhanced O-radical intermediate
•
The strong reducing potential of α-LA, in combination with DHLA's
chelating affinity with Fe3+, contributes to LPO inhibition.
The tested compounds can inhibit LPO at initialization (assuming
the reactions with Fe2+, O2 and HO ), propagation and termination
(reactions with lipid peroxy radicals) [25]. The LPO inhibitory effect
was observed, from highest to lowest, to be: compounds 1, 5 and 9
•-
•
(
which was suggested to be the crucial factor in achieving high levels of
AOA in the DPPH assay).
(
between 61 and 67%); compounds 4, 6, 7 (∼50%), equal to that of α-
LA; compound 8 (∼38%), equal to that of Vitamin E; and compound 2
23.51%). The meta position of the OMe group within compound 2
4
.3. TBARS assay
(
The inhibition of LPO (i.e. prevention of MDA production) in the
decreases its lipophilicity as compared to the ortho position of the OMe
group in compound 1, possibly hindering its AOA in the TBARS assay
TBARS assay evaluates the flow of reducing equivalents between
Asc /Asc , molecular oxygen (O
termediates on the one hand and the tested compounds on the other
2−
•-
3+
2+
2
), Fe /Fe , and PUFA radical in-
[
46].
Compounds with PhOH/NH showed better AOA, particularly in
cases when N-/O-radicals are stabilized by electron delocalization [44].
2−
[
39,40,45]. The product of Asc
autoxidation with molecular oxygen
•-
(
O
2
) is the superoxide anion radical (O ) according to the Marcus
2
127

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Fig. 4. a,b. Resonant stabilization of N- and O- radical intermediate of 3.
Fig. 5. a,b. Resonant stabilization of N- and O- radical intermediate of 4.
It is possible that the chelating ability of the tested compounds con-
tributed to the AOA profile generated. Accordingly, compounds 1 and 9
probably combine both N-radical stabilization and chelation of Fe2+
transfer mechanisms as well as to coordinate binding of Fe by the tested
compounds (Fe is present in the reaction mixtures, in both methods).
Different red-ox reaction principles of the applied assays dictate dif-
ferent AOA outcomes for a single compound (Table 2) [47]. Out of the
tested compounds, 4 achieved the maximum AOA by DPPH, closely
followed by compounds 2 and 1, with all other compounds showing
negligible AOA. Compounds 2 and 4 reached the highest AOA by FRAP
assay, with 2 achieving a greater AOA than 4. Results gathered from
testing the other compounds produced either moderate or low AOA,
with α-LA demonstrating no AOA. Almost all the compounds
within their mechanisms, while the stabilization of the phenoxy radical
•
and its reaction with O
2
‾ dictated strong LPO inhibitory effect of
compound 5.
The FRAP and TBARS methods turned out to be the most nuanced
tests for AOA measurement, evidenced by the gradation in results re-
2
corded. A perfect correlation (Slope = 1, r = 1) between FRAP and
TBARS methods was observed which can be attributed to one-electron
128

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Fig. 6. Resonant stabilization of 5 O- radical.
Fig. 7. Profile of electron delocalization directed by meta PhOL in 7 derivatives.
Fig. 8. Proposed resonance stabilization for 9 that requires considerable deal of energy.
demonstrated relatively high values of AOA according to the TBARS
method except for compound 2.
carbonyl functional domains [–NH–C=N–C(O)–] and the chelating li-
gand properties of the tested compounds determined their primary AOA
mechanisms. Of the compounds being tested, compound 4 accom-
plished the highest overall AOA with results from other compounds
being mixed. Accordingly, in vitro testing of the thiazolidinone deriva-
tive, compound 4, can be accepted as a model for future in vivo ex-
periments within the preclinical phase of drug development research.
We have confirmed through our results that compounds with
PhOH/NH show better AOA according to FRAP and TBARS, especially
in cases where N-/O-radicals were stabilized by electron delocalization.
Accordingly, compounds 1 and 9 probably undergo both N-radical
2
+
stabilization and chelation of Fe
which increases their AOA, while
•
-
stabilization of the phenoxy radical and its reaction with O
2
de-
termined the strong LPO inhibitory effect of compound 5. In regard to
conventional antioxidants, Vitamin C was the superior of the three
traditional antioxidants (ascertained by all three tests), unlike Vitamin
E and α-LA which gained maximum of AOA only by FRAP and TBARS
assays, respectively.
Reactions 1-12
•-
was generated in the reaction of autoxidation between Asc2−
O
2
and O
2
, based on Marcus theory of electron transfer (Reaction 1). Au-
6
−1 −
toxidation of AscH‾ is much slower, k∼10 M
s , at pH 7.4 [30]. The
production of H (Reaction 3) precedes the production of the most
2 2
O
•
potent HO by homolytic cleavage or Fenton reaction (Reaction 4). The
5
. Conclusion
+
initiation of LPO starts by the abstraction of bis-allylic H from PUFA,
•
-
•
protonated form of O , perhydroxyl radical (HO
2
2
, pKa = 4.7) (Reaction
) or HO (Reactions 5-10) [33]. Reaction 12 relates to AOA of vitamin
E [40].
In this study we evaluated the synthesis and AOA of thiazolidinone
•
derivatives of 1,3-thiazole and 1,3,4-thiadiazole, by three in vitro tests:
DPPH, FRAP and TBARS. The FRAP and TBARS methods appeared to be
the most sophisticated assays for AOA measurement. Different red-ox
reaction principles of the applied assays predicted different AOA out-
comes for the individual compounds (demonstrated by the traditional
and tested compounds). The presence of an –OH group in the benzyli-
dene ring, the position of the OMe in the benzylidene ring, amino-
-
→ Asc•- + O
•-
2
−1 −1
Asc
2
+ O
2
2
k = 10 M
s
(Reaction 1)
(Reaction 2)
(Reaction 3)
O2• + H+ → HO •
-
2
•-
+ 2H+ → H
O
2 2 2
O
129

M. Djukic et al.
Chemico-Biological Interactions 286 (2018) 119–131
Table 2
Antioxidant activity of the tested compounds across the in vitro assays. Percentage range of mea-
sured outcomes for each test: 0–21%, 22–43%, 44–65%, 66–87%, 88–109%). In vitro
2
+
tests: DPPH (% DPPH radical neutralization, used concentration 0.04 mg/mL); FRAP: (μM Fe
,
used concentration 0.033 mg/mL); and TBARS (% LPO inhibition, used concentration 1.75 μg/mL).
Number of repetitions for one concentration of the tested compounds: n = 5.
Fe2+ + H
→ Fe3+ + HO + HO ΔE°' = 320–110 = 210 mV (*)
•
−
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O
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HO
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ΔE°' = 1060–600 = 460 mV (*)
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(
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[
[
[
[
[
•
Legend: * Fenton reaction, **preferred site for HO
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2
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