Synthesis, characterization, and investigation of the antioxidant activity of some 1,2,4-benzothiadiazine-1,1-dioxides bearing sulfonylthioureas moieties

Article abstract of DOI:10.1139/cjc-2019-0194

A series of 1,2,4-benzothiadiazine-1,1-dioxides bearing a sulfonylthiourea moiety were synthesized, characterized, and screened for their antioxidant activity, using six antioxidant analytical assays comparatively to reference compounds, ascorbic acid and quercetin. The results indicated that several compounds demonstrated strong antioxidant activity in DPPH, ABTS, H₂O₂, and lipid peroxidation assays where some of them were either as active as or more active than reference compounds. However, all compounds were largely less active than references compounds in the reducing power assay. The results indicated that the thiourea moiety probably played a crucial role in the antioxidant activity of the target compounds, as a thiolate ion. The most favorable R₁ groups were the hydrogen atom and methyl group, followed by phenyl and benzyl groups, whereas the most favorable R₂ group was iPr, followed by the phenyl and methyl groups. The combination of benzothiadiazine ring with sulfonylthiourea moieties led to valuable new antioxidants, which could be used in the treatment or the prevention of certain diseases or in the field of cosmetics, which needs further investigations in the future.

Full text of DOI:10.1139/cjc-2019-0194

Page 1 of 47
Synthesis, characterization and investigation of the antioxidant activity of
some 1,2,4-benzothiadiazine 1,1-dioxides bearing sulfonylthioureas moieties
Kamel Harrouche a, Asma Lahouel a, Belghobsi Mebrouk a, Bernard Pirotte b, Smail Khelili *a
a Laboratoire de Phytochimie et de Pharmacologie, Département de Chimie, Faculté des
Sciences Exactes et Informatique, Université Mohamed Seddik Ben Yahia Jijel, B.P. 98
Ouled Aissa, 18000 Jijel, Algeria
b, Laboratoire de Chimie Pharmaceutique, Center for Interdisciplinary Research on Medicines
(CIRM), Université de Liège, 15, Avenue Hippocrate, B-4000 Liège, Belgium
* Corresponding author
Email: skhelili@yahoo.fr
Phone: 00213 671260507
Fax: 00213 34503914
hak_1967@yahoo.fr
asmalahouel@hotmail.fr
meb_mab@yahoo.fr
b.pirotte@ulg.ac.be
1

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Abstract
A series of 1,2,4-benzothiadiazine 1,1-dioxides bearing a sulfonylthiourea moiety were
synthesized, characterized and screened for their antioxidant activity, using six anti-oxidant
analytical assays comparatively to reference compounds, ascorbic acid and quercetin. The
results indicated that a number of compounds demonstrated strong antioxidant activity in
DPPH, ABTS, H2O2 and LP essays where some of them where as active as, or more active
than reference compounds. However, all compounds were largely less active than references
compounds in the reducing power assay. The results indicated that the thiourea moiety
probably played a crucial role in the antioxidant activity of the target compounds, as a thiolate
ion. The most favorable R1 groups were hydrogen atom and methyl, followed by phenyl and
benzyl, while the most favorable R2 groups were iPr, followed by phenyl and methyl. The
combination of benzothiadiazine ring with sulfonylthiourea moieties led to valuable new
antioxidants, which could be used in the treatment or the prevention of certain diseases, or in
the field of cosmetics, which needs further investigations in the future.
Keywords: 1,2,4-benzothiadiazine 1,1-dioxides, sulfonylthiourea moieties, antioxidant
activity, lipid peroxidation, glutathione oxidation prevention.
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Introduction
1,2,4-benzothiadiazine 1,1-dioxides are remarkably important heterocyclic compounds. They
belong to the class of cyclic sulfonamides, which have been widely studied as openers of
ATP-sensitive potassium channels. Such activity makes these compounds attractive for a wide
range of therapeutic applications. For example, diazoxide (1) has been known for a long time
as an antihypertensive agent,1,2 while BPDZ 73 (2) and related compounds have been
described as insulin secretion inhibitors, with possible application in the treatment of
3-6
hyperinsulinism.
Hydrochlorothiazide (3) and polythiazide (4) are used as diuretics in the
treatment of hypertension, cardiac insufficiency and edema.7,8 Furthermore, several
benzothiadiazine dioxides were found to exert antimicrobial activity (5) or behave as potent
hepatitis C virus RNA polymerase inhibitors (6), aldose reductase inhibitors (7) and
antioxidant agents (8) (Fig. 1).9-13
Oxidative stress is a dysregulation of the homeostasis of reactive oxygen species (ROS)
production and their neutralization by the antioxidant system. Oxidative stress is a key event
in the pathogenesis of several diseases such as cancer, diabetes, Alzheimer's disease,
rheumatism, cardiovascular disease and the aging process.14-17 It is important to note that
under normal conditions, ROS play a physiological role mainly in signal transmission.
However, excess in ROS production becomes toxic to the major cell components, like lipids,
proteins and nucleic acids, leading to cell damage and degeneration.18
Antioxidants are substances that delay or prevent the oxidation of oxidizable cellular
substrates. They offer protection against oxidative stress mainly by scavenging the free
radicals, inhibiting the lipid peroxidation and by many other mechanisms.19
Several natural and synthetic compounds such as α-tocopherol, ascorbic acid, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propylgallate (PG), tert
butylhydroquinone (TBHQ), carotenoids, amino acids, peptides, proteins, flavonoids and
3

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other phenolic compounds are used to enrich our diet with antioxidants to protect organism
against harmful diseases.
O
O
O
N
O
N
Cl
S
Cl
S
CH3
N
H
N
H
N
CH3
CH3
H
Diazoxide (1)
BPDZ73 (2)
O
O
O
O
N
H2NO2S
Cl
S
H2NO2S
Cl
S
CH3
NH
S
CF3
N
H
N
H
Hydrochlorothiazide (3)
Polythiazide (4)
O
O
N
O
O
N
S
OH
N
Cl
S
CH3
F
-O
N
H
N+
N
H
O
O
O
(5)
(6)
F
F
O
O
O
N
O
HO
S
Cl
S
N
N
N
OH
F
O
O
(8)
OH
OH
(7)
Fig. 1. Chemical structure of diazoxide, BPDZ 73, hydrochlorothiazide, polythiazide and
some other described 1,2,4-benzothiadiazine-1,1-dioxides, 5, 6, 7, 8.
The development of new antioxidant molecules in the fields of health and cosmetics is
dependent on the advance of knowledge on the role of oxidative stress in the physiopathology
of certain diseases such as Alzheimer's and Parkinson's diseases. All scientific fields are
concerned and must work together, in order to predict maximum effectiveness but also
anticipate side effects. The search for new antioxidants is therefore more than ever a topical
problem. In the present study, the antioxidant activity of a series of 1,2,4-benzothiadiazine-
1,1-dioxides bearing a sulfonylthiourea groups was investigated by in vitro assays. Given the
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complexity of the oxidation process, there is no single method to reflect the antioxidant
profile of a sample. This is why we carried out various tests of measurement of antioxidant
capacity. These assays included the determination of scavenging capacity of 1,1-diphenyl-2-
picrylhydrazyl (DPPH), 2,2’-azino-bis-3-ethyl-benzthiazoline-6-sulphonic acid (ABTS), and
hydrogen peroxide. The evaluation of the reducing power, the measure of inhibiting activity
of lipid peroxidation (LP) in rat liver and the prevention of glutathione oxidation were also
investigated. Since the target molecules have two labile hydrogen atoms, the first one is
located on nitrogen atom linked to both C=O and SO2 groups, while the second one is located
on the heterocyclic N4 atom, the ionization constants (pKa) of active compounds was
determined in order to know the predominant form at the pH values used in the experiments
(Fig. 2).
S
O
O
R2
S
S
O
O
O
O
N
H
N
R2
S
S
H
N
H
N
N
Combination
H
N
H
R1
O
O
S
N
labile hydrogen atoms
N
H
R1
Fig. 2. Combination of benzothiadiazine-1,1-dioxide ring with sulfonylthiourea moieties.
The introduction of sulfonylthiourea moieties on the benzothiadiazine-1,1-dioxide ring is due
to some reported anterior works which revealed that some thiourea and carbonyl thiourea
derivatives showed strong antioxidant activity.20-27 As examples, Kim et al. reported that
thiourea derivatives prevented decreases in the hepatic glutathione level provoked by CCl4.22
Fox et al. revealed that dimethylthiourea showed a potent preventive activity against
granulocyte-mediated oxidant lung injury in rats via a hydroxyl radical scavenging
mechanism.28 Dong et al. reported that some phenethyl-5-bromo-pyridyl thioureas (PEPT)
effectively inhibited oxidation-induced green fluorescence emission from the free radical-
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sensitive indicator dye, 2’,7’-dichlorodihydrofluorescein diacetate, in CEM human T-cells
and Nalm-6 human B-cells, exposed to hydrogen peroxide.27 We expect the combination of
sulfonylthiourea moieties with the benzothiadiazine-1,1-dioxide ring to provide valuable
antioxidants that can be used to treat or prevent certain diseases, or in the field of cosmetics.
Chemistry
The synthesis of targeted 1,2,4-benzothiadiazine-1,1-dioxides started from 4-amino-6-chloro-
1,3-benzenedisulfonamide 9 by different routes (Scheme 1: paths A, B and C).
In path A, 6-acylamino-4-chloro-1,3-benzenedisulfonamide (10) was prepared by refluxing 9
with a suitable acyl chloride in THF, and then cyclizing the intermediate product by heating in
aqueous basic medium to give 3-aryl(or alkyl)-6-chloro-7-sulfamoyl-4H-1,2,4-
benzothiadiazine 1,1-dioxides 11a-e.29
In path B, either a carboxylic acid was condensed with 4-amino-6-chloro-1,3-
benzenedisulfonamide (9) in the presence of concentrated sulfuric acid as a catalyst, or a
carboxylic anhydride in the corresponding carboxylic acid, which directly led to the
intermediates 11a-e.29
The latter reaction was faster in carboxylic anhydride than in carboxylic acid, due to its
higher reactivity. According to path C, the starting compound 9 was condensed with an
appropriate aldehyde in refluxing DMF to give 3-alkyl-6-chloro-7-sulfamyl-1,2,4-
benzothiadiazine-1,l-dioxides, which were oxidized with diluted KMnO4 in the presence of
NaOH, leading to compounds 11a-e. [7] The latters were converted into the corresponding
sodium salts by the addition of solid NaOH to their solutions in anhydrous DMSO.
Appropriate organic isothiocyanates were added to these solutions and stirred at room
temperature for 24 hours, which led to target compounds, 12a-n (Scheme 1). In an earlier
procedure, NaH was used instead of NaOH.30,31 All compounds were purified by
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1
13
crystallization and characterized by IR, H NMR, C NMR and elemental analysis to obtain
the final materials with the chemical purity required prior to pharmacological evaluation.
H2NO2S
Cl
SO2NH2
NH2
H2NO2S
Cl
SO2NH2
Path A
i
NHCOR1
9
10
Path B
iii
ii
Path C
iv
S
O
O
O
O
O
O
R2
S
v
H2NO2S
S
S
N
N
H
N
N
H
Cl
N
H
R1
Cl
N
H
R1
12a-n
11a-e
Scheme 1. Reagents and conditions: (i) path A: R1COCl, THF reflux; (ii) OH-, H2O; path B:
(iii) R1COOH/H2SO4 or (R1CO)2O/RCOOH ; (iv) path C: 1) R1CHO/DMF, 2)
KMnO4/NaOH; (v) NaOH/DMSO, R2NCS.
Results and discussion
The antioxidant capacity is commonly used as a parameter for the screening of
pharmacologically active compounds. Firstly, we undertook to determine the acidity constants
(pKa) of the most active target compounds, in order to know the predominant form under the
experimental conditions used. The pKa values are depicted in Table 1. Secondly, a variety of
in vitro methods has been set up to assess radical scavenging and reducing ability. In this
study, the antioxidant activity of target compounds, 12a-n, was evaluated and compared to
that of standard antioxidants, like vitamin C (Ascorbic acid) and quercetin. Six model systems
were used, namely 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis-3-ethyl-
benzthiazoline-6-sulphonic acid (ABTS) radical scavenging assays, reducing power assay,
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hydrogen peroxide scavenging activity assay, lipid peroxidation (LP) inhibition essay, and
glutathione (GSH) preventing oxidation assay. The antioxidant properties were expressed as
EC50 values in the first four essays, and as percentage of inhibition of oxidation of lipid and
GSH in the two latter essays. The results are depicted in Table 2.
Determination of constant acidity
Target compounds have two labile hydrogen atoms; one is belonging to NH group linked to
both sulfonyl and carbonyl groups, while the second is linked to N4 heterocyclic nitrogen
atom, and can be exchanged with the N2 heterocyclic nitrogen atom (Scheme 2). Thus, they
can loose these labile hydrogen atoms to give anionic species in aqueous medium (II, III, IV,
V).
S
S
S
O
O
O
O
O
O
O
O
O
O
O
O
R2
S
S
R2
S
S
R2
S
S
N
H
N
N
H
N
N
N
N
N
N
H
H
Cl
I
Cl
II
Cl
N
R1
N
R1
N
R1
H
H
H
III
S
S
O
O
O
O
O
O
O
O
R2
S
S
R2
S
S
N
H
N
H
N
H
N
H
N
N
Cl
N
Cl
V
R1
N
R1
IV
Scheme 2. Ionization equilibriums of target compounds in aqueous media.
The pKa values depicted by Table 1 prove that the predominant form of target compounds
(12a-e and 12i) is anionic at physiological pH (7.4), especially, forms II (and III), where the
pKa values are comprised between 5.61 and 6.95, while those of form IV (and V) are
comprised between 6.36 (and 8.09). For example, the reported pKa value of diazoxide, a
benzothiadiazine-1,1-dioxide, is 8.04 while some reported pKa values of described
sulfonylureas and sulfonylthioureas are comprised between 4.5 and 5.74.31-34 The reported
pKa values of ascorbic acid and quercetin are 4.19 and 7.65 (and 11.8), which means that they
are also ionized in aqueous medium.35,36
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Table 1. pKa values of compounds 12a-e and 12i.
Compound
pKa1
pKa2
7.18 ± 0.11
12a
6.22 ± 0.05
6.89 ± 0.13
6.09 ± 0.09
6.95 ± 0.05
5.61 ± 0.11
5.93 ± 0.14
4.19*
12b
8.09 ± 0.05
6.49 ± 0.06
7.65 ± 0.10
6.36 ± 0.12
6.95 ± 0.05
11.8*
12c
12d
12e
12i
As Ac
Quercetin
*Ref 35. **Ref 36
7.65**
---------
DPPH radical scavenging activity
Table 2 presents the DPPH scavenging capacity of some 1,2,4-benzothiadiazine 1,1-dioxides
synthesized (12a-n). The result obtained showed an interesting antioxidant activity of these
compounds in particular, 12a-e (0.49 <EC50< 0.98 mM). The most active compound was 12a
(0.49 ± 0.03 mM) which was not far from ascorbic acid (0.31 ± 0.01 mM) but clearly less
active than quercetin (0.18 ± 0.01 mM). Compounds 12b, 12c, 12d and 12i showed similar
DPPH radical scavenging activity (0.70 ± 0.029, 0.77 ± 0.032, 0.73 ± 0.024, 0.72 ± 0.06 mM
respectively, p>0.05) but were less active when compared to reference compounds, ascorbic
acid and quercetin. The most favorable groups for antioxidant activity were hydrogen atom,
CH3, and C6H5, CH2C6H5, as R1 group, and C6H5 and, in particular, isopropyl as R2 group.
Indeed, the most active compound, 12a, has hydrogen atom and isopropyl as R1 and R2
groups respectively. According to pKa values (Table 1), target compounds exist
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predominately in aqueous solution, at least, as thiolate ions. Although the DPPH could
theoretically be neutralized either by direct reduction via electron transfer (ET) or by
hydrogen atom transfer (HAT), it is considered to be mainly based on an ET reaction, and
hydrogen-atom abstraction is a marginal reaction pathway.37-39 The hydrogen atom
abstraction from neutral molecules like phenols (or thiol) by DPPH becomes a marginal
reaction path, because it occurs very slowly in strong hydrogen-bond-accepting solvents, such
as methanol and ethanol.37 In view of these considerations, it can be postulated that the
mechanism for neutralizing the DPPH radical occurred mainly by electron transfer from the
thiolate ion to the DPPH, giving a sulfhydryl radical.
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Table 2. Antioxidant properties of 1,2,4-benzothiadiazine 1,1-dioxides synthesized (EC50) and in vitro % inhibition of lipid peroxidation values
of 12a-n.
% of inhibition
of lipid
EC50 (mM)
% Inhibition of
glutathione oxidation
(200 µM)
H2O2
scavenging
capacity
0.38±0.07a
0.61±0.09b
NE
Reducing
peroxidation
(5.10-4 M)
R1
R2
DPPH
ABTS
power assay
12a
12b
12c
12d
H
iPr
iPr
iPr
iPr
iPr
Me
Me
Me
Ph
0.49±0.03a
0.70±0.03b
0.77±0.03b
0.73±0.03b
0.98±0.05c
1.73±0.13d
NE
0.39±0.03a
0.20±0.02b
0.52±0.04a
1.83±0.11c
1.73±0.07c
3.95±0.17d
NE
1.70±0.07a
2.19±0.15b
2.27±0.08b
1.26±0.11c
2.57±0.15b
8.44±0.38d
1.89±0.05a,b
NE
35.50±3.45
34.92±3.30
14.03±1.50
26.26±3.02
17.61±2.49
23.28±3.02
NE
8,04±1.47
24,80±1.95
17,10±1.94
ND
Me
Ph
Bn
NE
12e 4-OMePh
1.56±0.15c
0.63±0.12b
NE
ND
12f
12g
12h
12i
H
Me
Ph
H
ND
ND
5.71±0.65e
0.72±0.06b
NE
NE
NE
NE
12,47 ±0.54
16,93±1.54
ND
0.40±0.04a
NE
0.30±0.05a
NE
1.05±0.05c
NE
26.27±3.65
NE
12j
Me
Ph
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12k
12l
Ph
H
Ph
5.47±0.84e
NE
NE
NE
NE
NE
5.59±0.26e
8.46±0.73d
NE
NE
NE
ND
ND
C6H11
C6H11
C6H11
------
------
12m
12n
As $
Qr ∞
Me
NE
NE
NE
NE
ND
Ph
NE
NE
NE
NE
NE
ND
-------
-------
0.31±0.01f
0.17±0.01b
5.75±0.89d
0.67±0.03b
0.27±0.04f
0.13±0.02g
-------
33.33±3.23
12,47±0.54
ND
0.18±0.01g 0.032±0.003e
EC50 value: The effective concentration at which the antioxidant activity was 50% for DPPH•, ABTS•+ and H2O2 radicals were scavenged by
50%, respectively, and the absorbance was 0.5 for reducing power. EC50 values were obtained by interpolation from linear regression analysis.
Values were the means of three replicates ± SD. Values with different letters (a, b, c, d, e, f, g) in the same column were significantly different (p
< 0.05). NE: no effect. ND : not determined. * Cpd: Compound. $ As: ascorbic acid. ∞ Qr: quercetin.
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ABTS radical scavenging activity
In the present study, the EC50 value corresponds to the effective concentration at which the
ABTS radicals were scavenged by 50%. According to table 2, the most active compounds in
the DPPH, namely, 12a-e and 12i, also showed the most potent antioxidant activity with the
ABTS essay (0.39 ± 0.03, 0.20 ± 0.02, 0.52 ± 0.04, 1.83 ± 0.11, 1.73 ± 0.07, 0.40 ± 0.04 mM
respectively). In particular, 12b was as active as ascorbic acid (0.17 ± 0.01) while 12a, 12c
and 12i were two folds less active than ascorbic acid. We can see that EC50 values of these
compounds with ABTS assay were lower than those obtained with the DPPH assay, with the
exception of those of 12d and 12e. In contrast, they were all largely less active than the
reference compound quercetin (0.032 ± 0.003 mM). The most favorable groups for
antioxidant activity were hydrogen atom, CH3, and C6H5, as R1 group, while R2 was
preferably C6H5 and isopropyl. Indeed, the most active compound, 12b, has CH3 as R1 and
isopropyl as R2 group. We can give the same remark made above about the effect of the
nature of R1 and R2 groups, on the anti-oxidant activity of target compounds, except the
inversion between the orders of the hydrogen atom and CH3 group (for the R1 side radical)
(12a ˃12b in DPPH assay but 12b ˃12a in ABTS assay). The general order is globally
respected for the others groups. ABTS tests is both hydrogen atom transfer (HAT) and
electron transfer (ET) based assay and the reaction mechanism may shift with pH.38 Since the
target molecules exist mainly as thiolate ions at the experiment pH, the anti-oxidant activity
can be considered working following an electron transfer mechanism.
Reducing power (RP) assay
Reducing power assay is a single electron transfer (SET) based assay. It is a colorimetric
assay of electron transfer, based on the ability of the products tested to reduce iron (the
transition from ferric to ferrous form). The EC50 value of the reducing power is calculated as
the concentration corresponding to 0.5 absorbance of potassium ferrocyanide ferric complex,
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Page 14 of 47
in the medium. According to table 2, the most active compounds in the DPPH and ABTS
essays, namely, 12a-e and 12i, in addition to 12g, which was inactive, showed the most potent
antioxidant activities with the RP assay (1.70 ± 0.07, 2.19 ± 0.15, 2.27 ± 0.08, 1.26 ± 0.11,
2.57 ± 0.15 and 1.05 ± 0.05 mM)). In contrast, they were all largely less active than the
reference compounds ascorbic acid and quercetin (0.27 ± 0.04 and 0.13 ± 0.02 mM
respectively). Furthermore, The EC50 values were largely superior to those obtained with the
DPPH and the ABTS essays. The most active compounds were 12i and 12d bearing H atom
and CH2C6H5 as R1 group, and C6H5 and CH(CH3)2 as R2 group. Since the test was carried
out at pH value equal to 6.6, wich is around the pKa values of target compound, we can
expect that they exist largely as thiolate ion. The thiolate ion could loose one electron and
lead to sulfhydryl radical. Indeed, the direct involvement of the thiol (or thiolate ion) in the
anti-oxidant activity of thiourea derivatives has been reported by Dong et al., who studied the
radical scavenging activity of substituted N-(anilinocarbonothioyl)benzamides (analogues of
sulfonylthioureas) and phenethyl-5-bromo-pyridylthioureas.27 Furthermore, they proved that
the S-alkylation of phenethyl-5-bromo-pyridylthiourea eliminated their antioxidant activity.
Hydrogen peroxide scavenging capacity (HPSC)
Hydrogen peroxide has a high reduction potential and is therefore a strong oxidizing agent.
However, most of its reactions have high activation energy and are slow. Thiols are among
the few biomolecules (apart from transition metal centers) that react with hydrogen peroxide
directly. The reaction is exclusively with the thiolate and rate constants for low molecular
weight thiols at pH 7.4 increase with decreasing thiol pKa.40
The scavenging ability of benzothiadiazine derivatives on hydrogen peroxide is shown in
Table 2. While the control quercetin showed a strong HPSC, the effect of ascorbic acid was
very week (0.67 ± 0.03 and 5.75 ± 0.89 respectively). The results obtained also showed that
four benzothiadiazine derivatives presented potent HPSC, namely 12i, 12a, 12f and 12b (0.30
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Page 15 of 47
± 0.05, 0.38 ± 0.07, 0.63 ± 0.12, and 0.61 ± 0.09 mM respectively). Indeed, the most active
compounds, 12i and 12a were both two folds more active than quercetin, and 19 and 15 folds
more active than ascorbic acid respectively. Ascorbic acid was almost nine folds less active
than quercetin. Furthermore, compounds 12b and 12f were as active as quercetin, and nine
folds more active than ascorbic acid. The most favourable R1 groups were hydrogen atom and
methyl, while isopropyl, C6H5 and CH3 were most favourable R2 groups.
It is well known that two-electron oxidants, like H2O2, transform thiols to sulfenic acids
(RSOH or RSO-) as the initial product, while one-electron oxidants, like hydroxyl radical,
produce the thiyl radical.41-43 In a detailed experimental study, Luo et al. have clearly shown
that cysteine (CySH) reacts with H2O2 as thiolate ion.44 Curtis et al. studied the reaction of
N,N’-dimethylthiourea (Me2TU) with H2O2 and HO. but only reaction of Me2TU with H2O2
(and not HO.) gave Me2TU dioxide, which led him to hypothesize that measurement of N,N'-
dimethylthiourea dioxide would provide a good indication of the H2O2 scavenging and
protection seen after addition of Me2TU to biological systems.25 As target compounds bear the
thiourea moiety (linked to SO2 group) and are ionized in the experimental conditions of pH
used (7.4), their anti-oxidant activity could follow the second mechanism instead the first one.
This will need additional experimental and theoretical investigations in order to confirm the
actual mechanism.
Inhibition of lipid peroxidation
Lipid peroxidation is a series of free radical-mediated chain reaction processes and is
associated with several types of biological damage. In this study, the lipid peroxidation was
induced by ferric chloride. According to table 2, all compounds showing antioxidant activity
with the four assays previously discussed showed an important inhibition percentage of lipid
peroxidation. Indeed, at 0.5 mM, the most potent compounds, 12a and 12b, showed an anti-
oxidant activity relatively slightly higher than that of quercetin (35.50%, 34.92% and 33.33 ±
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Page 16 of 47
3.23 of inhibition % of lipid peroxidation respectively). The other compounds with relatively
weaker anti-oxidant activity were 12i, 12d, 12f, 12e and 12c (26.27 ± 3.65, 26.26 ± 3.02,
23.28 ± 3.02, 17.61 ± 2.49, and 14.03 ± 1.50 respectively) while 12g, 12h and 12j-n were
completely inactive. The most active compounds, 12a, 12b, bear H, CH3 as R1 group while R2
was isopropyl group. The anti-oxidant mechanism here is probably based on an electron
transfer process, where the thiolate ion could lose one electron and lead to sulfhydryl radical.
Prevention of glutathione oxidation
Thiol group plays a key role in biomolecules function such as enzymes and hormones. The
source of thiol is mainly the cysteine. This group is a subject to oxidation, which leads to
disulfide bonds and provokes an improper folding of the proteins and loss of their
physiological functions. Glutathione (GSH) is a potent non-enzymatic antioxidant in the cells.
Beside its role as an enzyme cofactor, GSH reduces disulfide bonds formed within proteins as
an electron donor. In this process, glutathione is converted to its oxidized form, glutathione
disulfide (GSSG).45 Addressing against the alteration of thiol-redox homeostasis is a current
research area, in this regards many molecules are under investigation for their thiol preventing
effect.
In this assay, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was used as a reagent to measure the
concentration of GSH. Indeed, thiols react with DTNB, cleaving the disulfide bond to give 2-
nitro-5-thiobenzoate (TNB−), which ionizes to the yellow colored TNB2− dianion in water at
alkaline pH, and can be spectrophotometrically assayed at 412 nm. As target compounds are
ionized at alkaline pH as thiolate ion, they would have preventing activity against oxidation of
GSH. According to table 2, the most potent target compounds in the previous assays (12a,
12b, 12c, 12i) were selected to evaluate their eventual preventive effect on GSH oxidation in
alkaline medium (pH 8.5). The results are depicted in table 2. After 24 h incubation, 12i, 12c
and 12b (200 µM), considerably prevented GSH oxidation (16.93 ± 1.54, 17.10 ± 1.94 and
16

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24.80 ± 1.95 % of GSH preventing oxidation respectively) and were markedly more active
than ascorbic acid (12.47 ± 0.54 % of GSH preventing oxidation), while 12a was neatly less
active than ascorbic acid (8.04 ± 1.47).
Comparative discussion between the five assays
We can see that the most potent three compounds in DPPH, ABTS, H2O2 and LP essays were
12a, 12b and 12i, while those in reducing power and GSH preventing oxidation assays were
12a, 12d, 12i and 12b, 12c, 12i respectively. The results obtained showed that, only in the
reducing power essays, the target compounds were largely less active than the reference
compounds, ascorbic acid and quercetin. In the other assays, some compounds were very
active. For example, in DPPH and ABTS essays 12a and 12b were almost as active as
ascorbic acid, while they were more active than quercetin in lipid peroxidation assay. In GSH
preventing oxidation assay, 12b, 12c and 12i were more active than ascorbic acid. In H2O2
scavenging assay, 12a and 12i were more active than quercetin, while 12b and 12f were as
active as this reference compound. On the same latter model 12a, 12b, 12f and 12i were more
active than ascorbic acid.
It is well known that almost all physiological oxidants react with thiols. Most of them show
selectivity between thiols by reacting only with the thiolate anion. Indeed, the most known
biological antioxidant substance is glutathione, which possesses a thiol function and exerts its
antioxidant activity through the thiolate anion.43 By referring to the pKa values and
antioxidant activity, depicted in tables 1 and 2 respectively, target compounds are probably
largely ionized at pH values used. This fact explains the strong antioxidant activity showed by
some target compounds. Globally, the most favorable R1 groups were hydrogen atom and
methyl, followed by phenyl and benzyl, while the most favorable R2 groups were iPr,
followed by Phenyl and Methyl.
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Conclusion
The combination of sulfonylthiourea moieties to the 7-position of 1,2,4-benzothiadiazine 1,1-
dioxide ring led to new hybrid compounds. The structures of the latters were confirmed by
1
13
elemental analysis, IR, H NMR and C NMR spectrometry, and evaluated for antioxidant
activity. The results showed that some compounds (12a, 12b, 12c, 12d, 12e, 12f and 12i)
have a strong activity in five assays (DPPH, ABTS, and H2O2 scavenging capacities,
inhibition of lipid peroxidation, inhibition of glutathione oxidation), but were weakly active in
reducing power assay. The most important antioxidant activity was obtained with 12a, 12b
and 12i in the five tests cited above, by comparison with the two reference substances,
ascorbic acid and quercetin. The study of the structure-activity relationship indicates that, the
most favorable R1 groups were hydrogen atom and methyl, followed by phenyl and benzyl,
while the most favorable R2 groups were iPr, followed by phenyl and methyl. An antioxidant
may, in some cases, act by multiple mechanisms in a single system or by a different single
mechanism depending on the reaction system. Furthermore, antioxidants may respond in a
different manner to different radical or oxidant sources, because the latters do not have the
same pro-oxidant reactivity. Thus, further experimental and theoretical studies are required to
determine the exact predominant mechanism of action of these molecules.
Experimental section
Chemistry
Melting points were determined with a Büchi B-540 capillary apparatus and are uncorrected.
IR spectra were recorded as KBr pellets on a FTIR-8400 S, CE (SHIMADSU)
spectrophotometer. The NMR spectra were recorded on a Bruker Avance (500 MHz for 1H
NMR and 125 MHz for 13C NMR APT) instrument using DMSO-d6 as the solvent. Chemical
shifts (δ) are given in parts per million relative to that of internal standard TMS. The
abbreviations s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet, are used
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throughout. Elemental analysis (C, H, N, S) were realized on a Thermo Scientific Flash EA
1112-elemental analyzer and were within 0.4 % of the theoretical values. All reactions were
routinely checked by TLC on silica gel (Merck 60 F254).
General method for the synthesis of 1,2,4-benzothiadiazine 1,1-dioxides (12a-n)
29
1 mmol of 11a-e was dissolved in a minimum of DMSO, to which 0.08 g of NaOH was
added with stirring until complete dissolution of the base, followed by the addition of 1 mmol
of a suitable isothiocyanate. The reaction mixture was stirred for 24 hours at room
temperature. The solution obtained was poured into 20 ml of water and filtered. The filtrate
was acidified with 2N HCl solution. The resulted precipitate was filtered, washed with water
and dried.
6-chloro-4H-7-(isopropylaminothiocarbonylaminosulfonyl)-1,2,4-benzothiadiazine 1,1-
dioxide (12a). 80 % yield; mp 231-232°C. IR (KBr) (ʋmax/cm-1): 3336, 3261, 3080, 2975,
1
2867, 1311, 1165. H NMR (DMSO) (δ/ppm): 1.12 (d, J = 6.5 Hz, 6H, CH(CH3)2), 4.13 (m,
1H, CH(CH3)2), 7.53 (s, 1H, CHar), 8.14 (s, 1H, CHar), 8.22 (d, J = 7.2 Hz, 1H,
NHCH(CH3)2), 8.35 (s, 1H, CHar), 11.50 (s, 1H, NH), 12.70 (s, 1H, NH). 13C NMR (DMSO)
(δ/ppm): (CH3) : 21.03 ; (CH) : 46.01 ; (CHar) : 120.21, 129.43, 148.80 (C = N) ; (Cq) :
120.29, 134.29, 136.41, 138.85, 175.99 (C = S). Anal. (C11H13ClN4O4S3) theoretical: 33.29 %
C, 3.30 % H, 14.12 % N, 24.24 % S; found: 33.11 % C, 3.33 % H, 13.85 % N, 24.50 % S.
6-chloro-4H-3-methyl-7-(isopropylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12b). 85 % yield; mp 250-252°C. IR (KBr) (ʋmax/cm-1): 3334,
1
3274, 3076, 2972, 1388, 1175. H NMR (DMSO) (δ/ppm): 1.11 (d, J = 6.5 Hz, 6H,
CH(CH3)2), 2.35 (s, 3H, CH3), 4.15 (m, 1H, CH(CH3)2), 7.46 (s, 1H, CHar), 8.22 (d, J = 7.2
Hz, 1H, NHCH(CH3)2), 8.33 (s, 1H, CHar), 11.54 (s, 1H, NH), 12.70 (s, 1H, NH). 13C NMR
(DMSO) (δ/ppm): (CH3) : 21.03, 22.99 ; (CH) : 46.01; (CHar) : 119.79, 129.22 ; (Cq) :
118.80, 133.64, 134.21, 139.35, 158.53(C = N), 175.99 (C = S). ). Anal. (C12H15ClN4O4S3)
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theoretical: 35.07 % C, 3.68 % H, 13.63 % N, 23.44 S; found: 34.89 % C, 3.78 % H, 13.26 %
N, 23.40 % S.
6-chloro-4H-3-phenyl-7-(isopropylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12c). 75 % yield; mp 217-220°C. IR (KBr) (ʋmax/cm-1): 3348,
3053, 2987, 1394, 1170. 1H NMR (DMSO) (δ/ppm): 1.12 (d, J = 6.4 Hz, 6H, CH(CH3)2), 4.15
(m, 1H, CH(CH3)2), 7.66 (t, J = 7.5 Hz, 2H, CHar), 7.74 (t, J = 7.5 Hz, 1H, CHar), 7.84 (s,
1H, CHar), 8.06 (d, J = 7.5 Hz, 2H, CHar), 8.25 (d, J = 7.3 Hz, 1H, NHCH(CH3)2), 8.40 (s,
1H, CHar), 11.53 (s, 1H, NH), 12.53 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm): (CH3) : 21.04 ;
(CH) : 46.02 ; (CHar) : 121.10, 128.47, 128.99, 129.02, 133.41 ; (Cq) : 119.29, 131.22,
133.79, 134.24, 139.69, 155.63 (C = N), 175.57 (C = S). Anal. (C17H17ClN4O4S3) theoretical:
43.17 % C, 3.62 % H, 11.85 % N, 20.34 % S; found: 43.46 % C, 3.82 % H, 11.77 % N, 20.35
% S.
3-benzyl-6-chloro-4H-7-(isopropylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12d). 68 % yield; mp 164-166°C. IR (KBr) (ʋmax/cm-1): 3458,
3284, 3056, 1367, 1170. 1H NMR (DMSO) (δ/ppm): 1.10 (d, J = 6.4 Hz, 6H, CH(CH3)2), 3.94
(s, 2H, CH2), 4.13 (m, 1H, CH(CH3)2), 7.23-7.39 (m, 5H, CHar), 7.53 (s, 1H, CHar), 8.21 (d,
J = 7.3 Hz, 1H, NHCH(CH3)2), 8.32 (s, 1H, CHar), 11.54 (s, 1H, NH), 12.68 (s, 1H, NH). 13C
NMR (DMSO) (δ/ppm): (CH3) : 21.03 ; (CH2) : 45.99 ; (CH) : 41.59 ; (CHar) : 120.16,
127.30 ; 128.62, 129.19, 129.46 ; (Cq) : 118.88, 134.06, 134.27, 134.37, 139.27, 159.79 (C =
N), 176.03 (C = S). Anal. (C18H19ClN4O4S3) theoretical: 44.39 % C, 3.93 % H, 11.50 % N,
19.75 % S; found: 44.11 % C, 4.10 % H, 11.16 % N, 19.85 % S.
6-chloro-3-(4-methoxyphenyl)-4H-7-isopropylaminothiocarbonylaminosulfonyl-1,2,4-
benzothiadiazine 1,1-dioxide (12e). 44 % yield; mp 226-228°C. IR (KBr) (ʋmax/cm-1): 3323,
1
3271, 3082, 2975, 1373, 1168. H NMR (DMSO) (δ/ppm): 1.11 (d, J = 6.4 Hz, 6H,
CH(CH3)2), 3.89 (s, 3H, OCH3), 4.15 (m, 1H, CH(CH3)2), 7.20 (d, J = 8.8 Hz, 2H, CHar),
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7.86 (s, 1H, CHar), 8.08 (d, J = 8.8 Hz, 2H, CHar), 8.26 (d, J = 7.3 Hz, 1H, NHCH(CH3)2),
8.38 (s, 1H, CHar), 11.53 (s, 1H, NH), 12.37 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm): (CH3)
: 22.65, 55.34 ; (CH) : 45.84 ; (CHar) : 115.17, 121.31 ; 127.47, 128.51 ; (Cq) : 123.15,
125.99, 131.81, 135.52, 145.61, 157.12, 161.43, 171.89 (C = S). Anal. (C18H19ClN4O5S3)
theoretical: 42.98 % C, 3.81 % H, 11.14 % N, 19.12 % S; found: 42.85 % C, 4.08 % H, 10.92
% N, 19.34 % S.
6-chloro-4H-7-(methylaminothiocarbonylaminosulfonyl)-1,2,4-benzothiadiazine1,1-
dioxide (12f). 70 % yield; mp 277-279°C. IR (KBr) (ʋmax/cm-1): 3481, 3284, 3053, 2941,
1311, 1168. 1H NMR (DMSO) (δ/ppm): 2.84, (s, 3H, CH3), 7.52 (s, 1H, CHar), 8.13 (s, 1H,
HC(=N)NH), 8.21 (s, 1H, NH), 8.37 (s, 1H, CHar), 11.50 (s, 1H, NH), 12.70 (s, 1H, NH). 13C
NMR (DMSO) (δ/ppm): (CH3) : 31.95 ; (CHar) : 120.72, 129.93, 149.29 (CH = N), (Cq) :
120.29, 134.82, 136.41, 139.32, 178.55 (C = S). Anal. (C9H9ClN4O4S3) theoretical: 29.31 %
C, 2.46 % H, 15.19 % N, 26.08 % S; found: 29.16 % C, 2.14 % H, 14.83 % N, 25.85 % S.
6-chloro-4H-3-methyl-7-(methylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12g). 75 % yield; mp 231-233°C. IR (KBr) (ʋmax/cm-1): 3257,
3064, 2960, 1368, 1166. 1H NMR (DMSO) (δ/ppm): 2.33 (s, 3H, CH3), 2.83 (s, 3H, CH3),
7.45 (s, 1H, CHar), 7.87 (s, 1H, CHar), 8.27 (s, 1H, NH), 12.47 (s, 1H, NH), 12.55 (s, 1H,
NH). 13C NMR (DMSO) (δ/ppm): (CH3) : 22.85, 30.01 ; (CHar) : 119.88, 124.85 ; (Cq) :
118.70, 133.19, 134.37, 139.46, 157.84 (C=N), 168.20 (C=S). Anal. (C10H11ClN4O4S3)
theoretical: 31.37 % C, 2.90 % H, 14.63 % N, 25.12 % S; found: 31.41 % C, 2.88 % H, 14.42
% N, 24.73 %S.
6-chloro-4H-3-phenyl-7-(methylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12h). 80 % yield; mp 264-266°C. IR (KBr) (ʋmax/cm-1): 3406,
3280, 3085, 1375, 1164. 1H NMR (DMSO) (δ/ppm): 2.86 (s, 3H, CH3), 7.65 (t, J = 7.5 Hz,
2H, CHar), 7.72 (t, J = 7.5 Hz, 1H, CHar), 7.83 (s,1H, CHar), 8.05 (d, J = 7.5 Hz, 2H, CHar),
21

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13
8.40 (s, 1H, NH), 8.43 (s, 1H, CHar), 11.55 (s, 1H, NH), 12.58 (s, 1H, NH). C NMR
(DMSO) (δ/ppm): (CH3) : 31.47, (CHar) : 121.07, 124.66, 128.47, 129.03, 133.91, (Cq) :
119.22, 131.20, 134.17, 138.66, 139.65, 155.63 (C=N), 178.16 (C=S). Anal.
(C15H13ClN4O4S3) theoretical: 40.49 % C, 2.94 % H, 12.59 % N, 21.62 % S; found: 40.86
% C, 2.78 % H, 12.64 % N, 21.85 % S.
6-chloro-4H-7-(phenylaminothiocarbonylaminosulfonyl)-1,2,4-benzothiadiazine1,1-
dioxide (12i). 65 % yield ; mp 217°C. IR (KBr) (ʋmax/cm-1): 3280, 3250, 1310, 1180. 1H NMR
(DMSO) (δ/ppm): 7.29 (m, 2H, CHar), 7.32 (m, 1H, CHar), 7.56 (m, 2H, CHar), 7.90 (s, 1H,
CHar), 8.12 (s, 1H, CHar), 8.28 (s, 1H NH), 8.36 (s, 1H, CHar), 9.84 (s, 1H, NH), 12.64 (s,
1H, NH). 13C NMR (DMSO) (δ/ppm): (CHar) : 119.04, 120.31, 125.06, 128.28, 128.88,
148.80 (CH = N) ; (Cq) : 120.17, 129.77, 134.25, 137.56, 138.88, 178,06 (C = S). Anal.
(C14H11ClN4O4S3) theoretical: 39.02 % C, 2.57 % H, 13.00 % N, 22.32 % S; found: 39.39 %
C, 2.61 % H, 12.78 % N, 21.93 % S.
6-chloro-4H-3-methyl-7-(phenylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12j). 55 % yield; mp 241-243°C. IR (KBr) (ʋmax/cm-1): 3326,
3224, 3059, 2926, 2846, 1332, 1166. 1H NMR (DMSO) (δ/ppm): 2.36 (s, 3H, CH3), 7.28 (m,
1H, CHar), 7.46 (m, 3H, CHar), 7.62 (m, 3H, CHar), 8.19 (s, 1H, NH), 11.60 (s, 1H, NH),
12.53 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm): (CH3): 22.87; (CHar): 117.17, 120.27,
121.98, 126.07, 129.17; (Cq): 120.65, 129.20, 134.05, 137.52, 139.42, 158.54 (C = N), 179.57
(C=S). Anal. (C15H13ClN4O4S3) theoretical: 40.49 % C, 2.94 % H, 12.59 % N, 21.62 % S;
found: 40.71 % C, 2.99 % H, 12.36 % N, 21.43 % S.
6-chloro-4H-3-phenyl-7-(phenylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12k). 60 % yield; mp 253°C. IR (KBr) (ʋmax/cm-1): 3380,
3257, 3031, 1340, 1174. 1H NMR (DMSO) (δ/ppm): 7.13 (t, J = 7.4 Hz, 1H, CHar), 7.33 (t, J
= 7.4 Hz, 2H, CHar), 7.48 (d, J = 7.4 Hz, 2H, CHar), 7.66 (t, J = 7.3 Hz, 2H, CHar), 7.73 (t, J
22

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= 7.3 Hz, 1H, CHar), 7.83 (s, 1H, CHar), 7.92 (s, 1H, CHar), 8.06 (d, J = 7.3 Hz, 2H, CHar),
8,33 (s, 1H, NH), 9.77 (s, 1H, NH), 12.48 (s,1H, NH). 13C NMR (DMSO) (δ/ppm): (CHar) :
121.25, 123.48, 123.63, 124.38, 124.68, 128.41, 128.99, 133.34 ; (Cq) : 119.26, 131.29,
134.28, 138.79, 139.30, 139.42, 155.45 (C=N), 179.58 (C=S). Anal. (C20H15ClN4O4S3)
theoretical: 47.38 % C, 2.98 % H, 11.05 % N, 18.97 % S; found: 47.65 % C, 2.93 % H,
11.33 % N, 18.60 % S.
6-chloro-4H-7-(cyclohexylaminothiocarbonylaminosulfonyl)-1,2,4-benzothiadiazine1,1-
dioxide (12l). 75 % yield; mp 254-256°C. IR (KBr) (ʋmax/cm-1): 3385, 3322, 3068, 2939,
2846, 1358, 1178. 1H NMR (DMSO) (δ/ppm): 1.1-1.9 (m, 10H, (CH2)5), 3.83 (m, 1H, CH),
7.07 (d, J = 6.9 Hz 1H, NH(C6H11)), 7.57 (s, 1H, CHar), 8.11 (s, 1H, CHar), 8.15 (s, 1H,
CHar), 9.75 (s, 1H, NH), 12.67 (s, 1H, NH). 13C NMR (DMSO) (δ/ppm): (CH2) : 24.26,
25.18, 31.37 ; (CH) : 53.04 ; (CHar) : 117.31, 120.09, 148.75; (Cq) : 121.72, 130.58, 134.21,
135.87, 160.56 (C = S). Anal. (C14H17ClN4O4S3) theoretical: 38.48 % C, 3.92 % H, 12.82 %
N, 22.01 % S; found: 38.72 % C, 4.22 % H, 12.98 % N, 21.84 % S.
6-chloro-4H-3-methyl-7-(cycohexylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12m). 70 % yield; mp 262-263°C. IR (KBr) (ʋmax/cm-1): 3375,
1
3321, 3068, 2939, 2846, 1358, 1173. H NMR (DMSO) (δ/ppm): 1.1-1.9 (m, 10H, (CH2)5),
2.35 (s, 3H, CH3), 3.9 (m, 1H, CH), 7,10 (m, 1H, NH), 7.55 (s, 1H, CHar), 8.21 (s, 1H,
13
CHar), 9,80 (s, 1H, NH), 12,50 (1s, 1H, NH). C NMR (DMSO) (δ/ppm): (CH3) : 21.64;
(CH2) : 24.75, 25.31, 32.56 ; (CH) : 52.20 ; (CHar) : 120.86, 123.75; (Cq) : 120.18, 127.58,
131.43, 140.80, 150.21, 162.76 (C = S). Anal. (C15H19ClN4O4S3) theoretical: 39.95 % C, 4.25
% H, 12.42 % N, 21.33 % S; found: 40.12 % C, 3.97 % H, 12.34 % N, 20.92 % S.
6-chloro-4H-3-phenyl-7-(cyclohexylaminothiocarbonylaminosulfonyl)-1,2,4-
benzothiadiazine 1,1-dioxide (12n). 65 % yield; mp 245-247°C. IR (KBr) (ʋmax/cm-1): 3373,
3264, 3072, 3078, 1334, 1167. 1H NMR (DMSO) (δ/ppm): 1.1-1.9 (m, 10H, (CH2)5), 3.9 (m,
23

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1H, CH), 7.60-8.20 (m, 7H, CHar), 8.40 (s, 1H, NH), 9.80 (s, 1H, NH), 12.60 (1s, 1H, NH).
13C NMR (DMSO) (δ/ppm): (CH2): 24.79, 25.35, 32.47; (CH): 52.28; (CHar): 121.32,
123.15, 126.10, 127.35, 132.56; (Cq): 121.72, 126.65, 127.41, 131.09, 140.12, 150.88, 162.90
(C = S). Anal. (C20H21ClN4O4S3) theoretical: 46.82 % C, 4.13 % H, 10.92 % N, 18.75 % S;
found: 46.68 % C, 3.98 % H, 10.80 % N, 18.73 % S.
pKa determination
Determination of the pKa value of actives compounds was performed by a spectrophotometric
method.46 UV–visible spectra were recorded on a Hitachi U 3010 spectrophotometer at 20 ±
2°C. Compounds were dissolved in methanol to provide mother solutions (2.5.10-3 M). A
series of buffer solutions, ranging from pH 1 to pH 12, were prepared with distilled water and
adjusted with HCl, KH2PO4, Na2HPO4 and NaOH solutions. The experimental solutions were
obtained by mixing 0.5 mL of the mother solution with 5 mL of the appropriate buffer and the
exact pH of the solution was measured by means of a pH meter. All spectra were measured
against a blank consisting of a buffer used as the solvent for the compound. Two wavelengths
were selected at the maximum absorbance of band and the pKa1 and pKa2 values were
calculated according to the method of Yamazaki et al.47
In vitro evaluation of antioxidant activity
DPPH radical scavenging activity
The DPPH radical scavenging activity of the benzothiadiazine derivatives was measured
according to the procedure described by Brand-Williams et al.48 DPPH is a stable nitrogen-
centered free radical commonly used for testing radical scavenging activity of molecules. The
assay is based on the decrease of light absorption of purple-colored DPPH• when it is reduced
to DPPH (yellow colored) in the presence of an antioxidant. Briefly, A volume of 50µl of
each target compounds (0.2–10 mM) and references compounds, ascorbic acid and quercetin
(0.06 – 1.2 mM), dissolved in methanol were added to 1950 µL of 60 µM methanolic solution
24

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of DPPH. A control was prepared as well containing methanol only, instead of the tested
molecule. The mixture was shaken vigorously and allowed to stand in the dark at room
temperature (20 ± 2 °C) for 30 min. The decrease in absorbance of the resulting solution was
then measured spectrophotometrically at 515 nm against methanol as a blank. All
measurements were made in triplicates and averaged. The ability to scavenge DPPH radical
was calculated by the following equation (Eq. 1):
퐑퐚퐝퐢퐜퐚퐥 퐬퐜퐚퐯퐞퐧퐠퐢퐧퐠 퐚퐜퐭퐢퐯퐢퐭퐲 (%) = ퟏ ― 퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐬퐚퐦퐩퐥퐞 퐚퐭 ퟓퟏퟓ 퐧퐦 .ퟏퟎퟎ
Eq. 1
[
]
퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐜퐨퐧퐭퐫퐨퐥 퐚퐭 ퟓퟏퟓ 퐧퐦
ABTS radical scavenging activity
The radical antioxidant potential of benzothiadiazine derivatives was measured using ABTS
radical cation decolorization assay as described by Re et al. with minor modifications.49
ABTS•+ cation radical was produced by the reaction between 7 mM ABTS in water and 2.45
mM potassium persulfate (1:1), stored in the dark at room temperature for 12-16 h before use.
ABTS•+ solution was then diluted with ethanol to obtain an absorbance of 0.700 at 734 nm.
After the addition of 100 μl of sample or (ascorbic acid or quercetin as positive control) at
different concentrations to 900 µl of diluted ABTS•+ solution, the absorbance was measured at
734 nm after 10 min of incubation at room temperature (20 ± 2 °C). All determinations were
carried out in triplicates. The ability to scavenge ABTS•+ radical was calculated by the
following equation (Eq. 2):
퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐬퐚퐦퐩퐥퐞 퐚퐭 ퟕퟑퟒ 퐧퐦
퐑퐚퐝퐢퐜퐚퐥 퐬퐜퐚퐯퐞퐧퐠퐢퐧퐠 퐚퐜퐭퐢퐯퐢퐭퐲 (%) = ퟏ ―
.ퟏퟎퟎ
(퐄퐪. ퟐ)
[
]
퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐜퐨퐧퐭퐫퐨퐥 퐚퐭 ퟕퟑퟒ 퐧퐦
Reducing power assay
The reducing power of the benzothiadiazine derivatives was determined according to the
method described by Chung et al.50 A 0.1 mL aliquot of each benzothiadiazine derivative (0.2
– 10 mM), ascorbic acid or quercetin (0.06 –1.2 mM) was mixed with an equal volume of 0.2
M phosphate buffer (pH 6.6) and 1% potassium ferricyanide, and then incubated at 50 °C for
20 min. 0.25 mL of 1% trichloroacetic acid was added to the mixture to stop the reaction, and
25

Page 26 of 47
then the mixture was centrifuged at 2790 g for 10 min. The supernatant (0.25 mL) was mixed
with 0.25 mL of distilled water and 0.1% of FeCl3 (0.5 mL) and then the absorbance was
measured at 700 nm. The intensity of the reducing power is proportional with the absorbance
increase provoked by the formation of a Perl’s Prussian blue potassium ferrocyanide-ferric
complex. The EC50 represents the concentration of the tested compound corresponding to 0.5
absorbance at 700 nm.
Hydrogen peroxide scavenging capacity
The ability of the synthetic molecules to scavenge H2O2 was determined according to the
method of Ruch et al. with minor modifications.51 Briefly, a solution of H2O2 (40 mM) was
freshly prepared in phosphate buffer (50 mM, pH 7.4). Neutralisation of 900 µl of H2O2 in the
presence of 100 µl of the tested molecules at different concentrations was determined by
following the decrease in absorbance after 10 min incubation (at 20°C) at 230 nm comparing
to control. The test was effectuated against a blank containing only phosphate buffer and the
tested molecule. Ascorbic acid and quercetin were used as standards molecules. The
scavenging activity was calculated according the following equation (Eq. 3):
퐒퐜퐚퐯퐞퐧퐠퐢퐧퐠 퐚퐜퐭퐢퐯퐢퐭퐲 (%) = ퟏ ― 퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐬퐚퐦퐩퐥퐞 퐚퐭 ퟐퟑퟎ 퐧퐦 .ퟏퟎퟎ
(퐄퐪. ퟑ)
[
]
퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐜퐨퐧퐭퐫퐨퐥 퐚퐭 ퟐퟑퟎ 퐧퐦
Lipid peroxidation inhibition Assay
Lipid peroxidation was induced in vitro in rat liver homogenate treated with FeCl3 in the
presence or absence of tested molecules at different concentrations.52 Liver homogenate was
prepared by homogenizing liver in 10 volumes of phosphate buffer (50 mM, KCl 1.5 % , pH
7.4). The supernatant was used freshly after centrifugation at 3000 rpm for 20 min. The
reaction mixture consisting of 0.3 ml of liver homogenate and 0.15 ml of FeCl3 (20 mM) was
incubated at 37 °C with agitation for 1 hour in the presence of 50µl of the tested molecules at
different concentrations or in the absence of tested molecules as control. A blank was
prepared as well containing only the same phosphate buffer used above, FeCl3 and methanol
26

Page 27 of 47
instead of the tested molecules. After incubation, 1 ml of thiobarbituric acid (TBA) (0.67 %)
prepared in 20% of acetic acid was added to the mixture and incubated at 95 °C for 1hr. the
degree of lipid peroxidation in the mixture was deduced by quantifying MDA levels
spectrophotometrically at 532 nm according to Ohkawa’s method.53 The percent inhibition of
lipid peroxidation was calculated the following equation (Eq. 4):
퐋퐢퐩퐢퐝 퐩퐞퐫퐨퐱퐢퐝퐚퐭퐢퐨퐧 퐢퐧퐡퐢퐛퐢퐭퐨퐫퐲 퐚퐜퐭퐢퐯퐢퐭퐲 (%) = ퟏ ― 퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐬퐚퐦퐩퐥퐞 퐚퐭 ퟓퟑퟐ 퐧퐦 .ퟏퟎퟎ
(Eq. 4)
[
]
퐀퐛퐬퐨퐫퐛퐚퐧퐜퐞 퐨퐟 퐜퐨퐧퐭퐫퐨퐥 퐚퐭 ퟓퟑퟐ 퐧퐦
Glutathione oxidation prevention
Glutathione (GSH) is a tripeptide consisted of glycine, glutamate and cysteine who confers to
the molecule a thiol function. GSH is easily oxidized to GSSG in an alkaline medium.54 In
this test, we evaluated whether the synthesized molecules are able to prevent GSH oxidation.
For that, a 500µM solution of GSH was prepared in phosphate buffer (100mM, pH 8.5). 100
µl of the tested molecules at concentration of 200µM and 900 µl of freshly prepared GSH
were incubated for one day, four days and seven days in room temperature. A control
containing only GSH in the absence of the tested molecules and a blank containing the tested
molecules alone in phosphate buffer were also prepared and incubated in the same conditions.
At the end of the incubation time, the concentration of the remaining reduced GSH was
evaluated by spectrophotometry according to Ellman method.55 Briefly, 20 µl of 20 µM
solution of 5,5'-disulfanediylbis(2-nitrobenzoic acid) (DTNB), or Ellman's reagent, were
added to 1ml of GSH mixture and incubated for 20 min. The concentration of reduced GSH
was evaluated spectrophotometrically at 412 nm according to a standard curve performed in
the same conditions and using pure GSH as a standard. The inhibition effect was calculated
according the following equation (Eq.5):
(퐢퐧퐜퐮퐛퐚퐭퐞퐝 퐰퐢퐭퐡 퐭퐡퐞 퐭퐞퐬퐭퐬 퐦퐨퐥퐞퐜퐮퐥퐞) ― 퐆퐒퐇 퐜퐨퐧퐜퐞퐧퐭퐫퐚퐭퐢퐨퐧(퐜퐨퐧퐭퐫퐨퐥)
퐼 = (퐆퐒퐇 퐜퐨퐧퐜퐞퐧퐭퐫퐚퐭퐢퐨퐧
).100
(Eq.5)
퐆퐒퐇 퐜퐨퐧퐜퐞퐧퐭퐫퐚퐭퐢퐨퐧(퐜퐨퐧퐭퐫퐨퐥)
27

Page 28 of 47
Statistical analysis
The EC50 values were calculated using GraphPad Prism software with one parameter model
[Y = aX + b]. Results were expressed as the mean ± the standard deviation (SD) of triplicate
analysis. Statistical comparisons were performed using the Student’s t-test. Differences were
considered significant at p < 0.05.
Acknowledgements
This work was supported by the Algerian Ministry of Higher Education and Scientific
Research and Laboratory of Phytochemistry and Pharmacology (University of Mohamed
Seddik Ben Yahia, Jijel). The authors gratefully acknowledge the technical assistance of M.
Aibech Riad.
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