Synthesis, antioxidant, and antiacetylcholinesterase activities of sulfonamide derivatives of dopamine-related compounds

Article abstract of DOI:10.1002/ardp.201300228

A series of sulfonamides were synthesized from dopamine derivatives. The reactions of amines with methanesulfonyl chloride followed by O-demethylation with BBr₃ afforded phenolic sulfonamides. The antioxidant activities of the synthesized pheno

Full text of DOI:10.1002/ardp.201300228

Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
783
Full Paper
Synthesis, Antioxidant, and Antiacetylcholinesterase Activities
of Sulfonamide Derivatives of Dopamine-Related
Compounds
Hülya Göçer1,2, Akın Akıncıo gꢀ lu1,2, Necla Öztas¸kın , Süleyman Göksu *, and Ilhami Gülçin
2
2
2
_
1
Central Researching Laboratory, Agri Ibrahim Cecen University, Agri, Turkey
Faculty of Science, Department of Chemistry, Atatürk University, Erzurum, Turkey
2
A series of sulfonamides were synthesized from dopamine derivatives. The reactions of amines with
methanesulfonyl chloride followed by O-demethylation with BBr afforded phenolic sulfonamides. The
3
antioxidant activities of the synthesized phenolic sulfonamides were investigated by thiocyanate
0
•þ
method, 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS ), 1,1-diphenyl-2-picryl-hydrazyl
•
•þ
•ꢀ
(
DPPH ), N,N-dimethyl-p-phenylenediamine (DMPD ), and superoxide anion (O2 ) radical scaveng-
2
þ
ing, reducing power, and ferrous ion (Fe ) chelating assays. Sulfonamides 13–16 showed around 75–
5% inhibition on linoleic acid peroxidation. On the other hand, butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), a-tocopherol, and trolox indicated an inhibition of 90.0%, 85.73%,
3.33%, and 85.73% on peroxidation, respectively, in the same system at the same concentration
10 mg/mL). Also, the inhibition effects of the synthesized compounds on acetylcholinesterase (AChE)
8
7
(
activity were evaluated. AChE was effectively inhibited by sulfanomides 13–16, with K values in the
i
range of 33.04 ꢁ 4.3 to 131.68 ꢁ 8.8 nM.
Keywords: Antiacetylcholinesterase activity / Antioxidant activity / Dopamine / Phenolic compound / Radical
scavenging / Sulfonamides
Received: June 17, 2013; Revised: July 27, 2013; Accepted: August 7, 2013
DOI 10.1002/ardp.201300228
Introduction
Antioxidant properties of dopamine (4) and related mono-
amine hormones have previously been reported by Yen and
Hsieh [7] (Fig. 1).
Sulfonamides are prominent biologically active compounds
[
1]. In the last decades, a variety of sulfonamide drugs have
Free radicals, known as reactive oxygen species (ROS) and
•ꢀ
been developed and appeared in the markets. The carbonic
anhydrase inhibitor brinzolamide (1), known as azopt, has
been used to lower intraocular pressure in patients with open-
angle glaucoma or ocular hypertension [2]. Dorzolamide
reactive nitrogen species (RNS), including superoxide (O
2
),
•
•
•
hydroxyl (HO ), peroxyl (ROO ), alkoxyl (RO ), and nitric
•
oxide (NO ) radicals, are highly reactive molecules with
unpaired electrons that can quickly bind to nearby molecules.
ROS are generated as by-products of cellular metabolism,
primarily in the mitochondria. Free radicals are very reactive
molecules; they can attack the molecules in adjacent cells,
and probably the damage caused by them is unavoidable and
is dealt with by repair processes [8–10]. The importance of ROS
has attracted increasing attention over the past decades. In
the presence of low concentrations of free radicals, ROS is
important for normal cellular redox status, tissues function,
and intracellular signaling processes [10]. When cellular
(
trusopt, 2) is used as an anti-glaucoma agent by decreasing
the production of aqueous humor [3]. A sulfonamide drug
sultiame (sulthiame, 3), an anticonvulsant, is reported for
treatment of epilepsy and West syndrome [4]. In addition, the
monoamine hormone dopamine (4) is a neurotransmitter and
plays an important role in the central nervous system-related
disorders such as schizophrenia and Parkinson’s disease [5, 6].
_
Correspondence: Prof. Ilhami Gülçin, Faculty of Science, Department of
Chemistry, Atatürk University, 25240 Erzurum, Turkey.
E-mail: igulcin@atauni.edu.tr
Fax: þ90 442 2360948
Additional correspondence: Prof. Süleyman Göksu,
E-mail: sgoksu@atauni.edu.tr
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H. Göçer et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
HN
N
HN
S
O
O
O
O
O
NH2
S
HO
S
O
S
O
S
NH
2
S
NH
2
N
S NH
2
O
H CO(H C)
S
3
2
3
O
O
O
O
HO
1
Brinzolamide
2 Dorzolamide
3 Sultiame
4 Dopamine
Figure 1. Some sulfonamide drugs (1–3) and dopamine (4).
production of ROS suppresses its antioxidant capacity, there
is damage to cellular macromolecules such as lipids, proteins,
etc. [11, 12]. They may cause DNA damage and, as a result, the
mutation can occur. If ROS are not adequately scavenged by
cellular antioxidant components, they can cause free-radical
chain reactions, subsequently damaging the cellular bio-
molecules such as carbohydrates, proteins, lipids, and nucleic
acids. Finally, they lead to disease conditions [13]. It was
reported that ROS had been implicated in more than 100
diseases, including malaria, acquired immunodeficiency
syndrome, heart disease, stroke, arteriosclerosis, diabetes,
and cancer [14–17]. Chemically, oxidative stress is associated
their chemical structure. Particularly, these properties of
phenols depend on their hydrogen- or electron-donating
ability and, most importantly, their ability to delocalize the
unpaired electron within the aromatic structure [30–33]. They
are known as an integral part of both human and animal
diets. Also, phenolic compounds constitute the majority of
natural antioxidants [34–36].
On the other hand, excess amount of ROS, which causes
oxidative stress, is associated with pathology of many diseases
including Alzheimer’s disease. This disease is a degenerative
disease of the central nervous system characterized especially
by premature senile mental deterioration. It is the most
common form of dementia and a progressive neurologic
disorder characterized by cognitive deficit and behavioral
abnormalities in the patient [37]. It was first described by
German neuropathologist Alois Alzheimer in 1906 and was
named after him [38]. So far, pathogenesis of Alzheimer’s
disease has not been completely clarified. The only known
valid hypothesis being accepted is the lack of enough amount
of acetylcholine (ACh), a neuromediator. Therefore, the
compounds that have acetylcholinesterase (AChE) inhibitory
effects were used for the treatment of Alzheimer’s disease.
However, most of these drugs have undesired side effects.
Thus, the development and utilization of new effective
antioxidants as well as AChE compounds are desired. In a
previous report, it is suggested that the use of antioxidants
may reduce the progression of Alzheimer’s disease and
minimize neuronal degeneration by inhibiting AChE,
which are main enzymes in pathogenesis of Alzheimer’s
disease [39].
On the other hand, ACh acts as an excitatory neurotrans-
mitter for voluntary muscles in the somatic nervous system
and as a preganglionic and a postganglionic transmitter
in the parasympathetic nervous system of vertebrates and
invertebrates [40, 41]. Acetylcholinesterase (AChE. EC 3.1.1.7),
a serine protease, is responsible for ACh hydrolysis and plays a
fundamental role in impulse transmission by terminating
the action of the neurotransmitter ACh at the cholinergic
synapses and neuromuscular junction. It is associated with
the normal neurotransmission by catalyzing the hydrolysis of
ACh to acetate and choline, and acts to remove ACh from the
•ꢀ
with increased production of oxidizing species such as O
2
•
and HO , or a significant decrease in the effectiveness of
antioxidant defenses. Biologically, it was defined as an
imbalance between prooxidant production and the antioxi-
dant capacity of the cell to prevent oxidative injury [13, 18]. In
human metabolism, oxidative stress is thought to be involved
in the development of many chorionic diseases or may
exacerbate their symptoms [19, 20] including atherosclerosis,
heart failure [21], autism [22], cancer [23], myocardial
infarction [24], Parkinson’s and Alzheimer’s diseases [25],
schizophrenia [26], bipolar disorder [27], and chronic fatigue
syndrome [22].
An antioxidant molecule inhibits the oxidation of other
molecules. On the other hand, oxidation process is known as a
chemical reaction that transfers hydrogen atom or electrons
from a substance to an oxidizing agent [28, 29]. The
description of antioxidant is originally related to molecules
that prevent the consumption of oxygen by human tissues. In
other words, an antioxidant is a molecule or species that slows
or prevents the oxidation of another molecule [30]. Physio-
logically there is an appropriate prooxidant–antioxidant
balance in normal cells. This balance can be shifted toward
the prooxidants when levels of antioxidants are diminished or
when the production of ROS is increased greatly. This case is
called as oxidative stress [28, 31].
Phenolic compounds are a class of chemical compounds
containing a hydroxyl group (–OH) bonded directly to an
aromatic hydrocarbon ring. They are esteemed as free radical
scavengers, and their antioxidant properties are related to
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Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
Biological Activities of Dopamine-Related Compounds
785
1
synaptic cleft [42]. It plays a vital role in the CNS, where it
catalyzes the hydrolysis of the neurotransmitter ACh [43].
As sulfonamides and dopamine-related compounds show
useful biological activities, in the present work we focused on
the synthesis of phenolic sulfonamide derivatives of dopa-
mine (4) related compounds 13–16. There are various methods
for evaluation of antioxidant capacity of pure compounds,
which involve different mechanisms [44, 45]. Therefore, in the
present study, we report the synthesis of phenolic compounds
synthesized compounds were characterized by H and
1
3
C NMR spectroscopic techniques. The functional groups
were characterized by IR.
Antioxidant properties
The antioxidant capacity of a phenolic compound is based on
their free radical scavenging ability, donate hydrogen atom or
electron, or chelate metal ions [52, 53]. The structure of a
phenolic compound is a key determinant of their biological
activities such as radical scavenging activity and metal
chelating activity. For example, antioxidant activity of a
phenolic acid depends on the number and position of the
hydroxyl groups (–OH) [54]. Monohydroxy benzoic acids with
the –OH moiety at the ortho- or para- position to the carboxyl
group (–COOH) show no antioxidant activity, though the
same is not true for the meta-hydroxybenzoic acid [18, 54, 55].
The antioxidant properties of phenolic acids or phenolic
compounds increase with increasing degree of hydroxylation.
For example, trihydroxylated gallic acid shows a marked
antioxidant activity. Also, as can be seen in syringic acid,
substitution of the hydroxyl groups at the 3- or 5-position with
methoxyl groups reduces the antioxidant activity [10, 54].
The ferric thiocyanate method is the most effective
antioxidant method and measures the amount of peroxide,
which is the primary product of oxidation produced during
the initial stages of oxidation [56, 57]. Total antioxidant
activity of phenolic sulfonamides 13–16 and standard
compounds was determined by using lipid peroxidation
in the linoleic acid system. As seen in Table 1, phenolic
sulfonamides 13–16 and standard compounds exhibited
effective inhibition on linoleic acid peroxidation. At the
10 mg/mL concentration, percentage inhibition of linoleic
acid peroxidation of phenolic sulfonamides 13–16 and
standards decreased in the following order: butylated
hydroxyanisole (BHA) (90.0%) > butylated hydroxytoluene
(BHT) (85.7%) ¼ trolox (85.7%) ꢂ 15 (85.6%) ꢂ 14 (85.5%) ꢂ 13
(84.0%) > 16 (75.0%) > a-tocopherol (73.3%) and at the control
value reached its maximum absorbance (144 h).
1
3–16 and we determined the antioxidant activity of the
synthesized phenolic compounds by thiocyanate method,
0
•þ
2
1
,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS ),
•
,1-diphenyl-2-picryl-hydrazyl (DPPH ), N,N-dimethyl-p-phe-
•þ
•ꢀ
nylenediamine (DMPD ), and O
2
scavenging activities,
2
þ
reducing power, and ferrous ion (Fe ) chelating activities.
Another main goal of this research was to determine the AChE
enzyme inhibitory effects of phenolic compounds 13–16 as
alternative drugs for the treatment of Alzheimer’s disease.
Results and discussion
Chemistry
Our synthesis commences from commercially available
methoxylated dopamine derivatives 5–8. The reactions of
free amines with methanesulfonyl chloride (MsCl) in the
presence of Et
following this procedure, the reactions of amines 5–8 with
MsCl in the presence of Et N at 0–25°C for 15 h afforded novel
3
N have previously been reported [46]. By
3
sulfonamides 9, 10, and known compounds 11 [47] and 12 in
moderate yields. Cleavage of arylmethyl ethers is an integral
functional group transformation for the synthesis of biologi-
cally or synthetically important phenols. O-Demethylation
of arylmethyl ethers can be achieved by HBr [48, 49] or
BBr
3
[50, 51]. Because of the solubility problems of compounds
9
–12, we know very well from our earlier studies that the
latter procedure is the most convenient one for O-demethyla-
tion of 9–12. Therefore, the reactions of compounds 9–12 with
BBr
3 2 2
in CH Cl at 0–25°C for 20 h gave novel phenolic
sulfonamide derivatives of dopamines 13–15 and a known
compound 16 in high yields (Scheme 1). The structures of all
The reducing ability of a biologically active compound may
serve as a significant indicator of its potential antioxidant
R1
R1
O
S
O
R1
O
S
O
H
N
H
N
R2
R3
NH2
i
R2
R3
ii
R2
R3
R
4
R
4
R
4
5
6
7
8
R =R =OMe, R =R =H
9
R =R =OMe, R =R =H, 77%
13 R =R =OH, R =R =H, 84%
1 2 3 4
1
2
3
4
1
2
3
4
R =R =OMe, R =R =H
10 R =R =OMe, R =R =H, 75%
14 R =R =OH, R =R =H, 81%
2 4 1 3
2
4
1
3
2
4
1
3
R =R =OMe, R =R =H
11 R =R =OMe, R =R =H, 73%
12 R =OMe, R =R =R =H, 78%
15 R =R =OH, R =R =H, 77%
2 3 1 4
2
3
1
4
2
3
1
4
R =OMe, R =R =R =H
16 R =OH, R =R =R =H, 82%
1 2 3 4
1
2
3
4
1
2
3
4
Scheme 1. Synthesis of sulfonamides. (i) MeSO
2
Cl/NEt
3 2 2 2 2
, CH Cl , 0–25°C, 15 h; (ii) BBr3, CH Cl , 0–25°C, 20 h.
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H. Göçer et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
2þ
3þ
2þ
Table 1. The percentage inhibition of lipid peroxidation, Fe –Fe reductive potential, FRAP assay, and cupric ions (Cu ) reducing
ability of phenolic sulfonamides (13–16) and reference antioxidants at the same concentrations (10 mg/mL)
3
þ
2þ
Fe reducing
FRAP
assay
Cu reducing
Inhibition of lipid
peroxidation
a)
a)
a)
b)
Antioxidants
ability
ability
BHA
BHT
a-Tocopherol
Trolox
1.632 ꢁ 0.279
1.407 ꢁ 0.112
0.727 ꢁ 0.078
0.675 ꢁ 0.167
0.305 ꢁ 0.106
2.411 ꢁ 0.225
0.652 ꢁ 0.212
2.454 ꢁ 0.345
2.143 ꢁ 0.110
1.023 ꢁ 0.217
1.238 ꢁ 0.267
0.856 ꢁ 0.040
0.444 ꢁ 0.036
1.569 ꢁ 0.095
0.413 ꢁ 0.013
2.418 ꢁ 0.059
1.173 ꢁ 0.066
1.140 ꢁ 0.098
0.330 ꢁ 0.061
0.268 ꢁ 0.027
0.203 ꢁ 0.106
1.901 ꢁ 0.037
0.255 ꢁ 0.039
2.617 ꢁ 0.019
90.0
85.7
73.3
85.7
84.0
85.5
85.6
75.0
13
14
15
16
BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene.
a)
The values were expressed as absorbance. High absorbance indicates high reducing ability.
The values were expressed as percentage of inhibition of linoleic acid emulsion peroxidation.
b)
capacity. An antioxidant compound can donate electrons to
decreased in the following order: 16 (2.418 ꢁ 0.059) > BHA
(2.143 ꢁ 0.110) > 14 (1.569 ꢁ 0.095) > a-tocopherol (1.238 ꢁ
0.267) > BHT (1.023 ꢁ 0.217) > trolox (0.856 ꢁ 0.040) > 13
(0.444 ꢁ 0.036) ꢂ 15 (0.413 ꢁ 0.013). In the FRAP assay, higher
reactive radicals or ROS. It can reduce them into more stable
and unreactive species [58]. The reducing power of phenolic
3
þ
2þ
sulfonamides 13–16 was investigated by Fe –Fe transfor-
mation, FRAP, and CUPRAC assays.
3
þ
absorbance values indicate higher Fe -TPTZ reducing ability.
Also, of the used reduction methods, the highest reduction
ability was observed for the phenolic sulfonamide 16. Benzie
and Strain reported that the FRAP assay offers a well-known
index of antioxidant, or reducing, potential of samples or
As shown in Table 1, phenolic sulfonamides 13–16 had
2þ
powerful ferric ions (Fe ) reducing ability. In this study, it
was found that reducing powers of phenolic sulfonamide
compounds 16 (2.454 ꢁ 0.345) and 14 (2.411 ꢁ 0.225) were
higher than those of all used standard compounds such
as BHA (1.632 ꢁ 0.279), BHT (1.407 ꢁ 0.112), a-tocopherol
3
þ
pure compounds. At low pH values, a Fe –TPTZ complex is
3þ
2þ
reduced to the ferrous (Fe ) form (Fe –TPTZ), which has an
intense blue color and maximum absorption at 593 nm [10].
Ferrous ion chelating activities of phenolic sulfonamides
13–16, and standard antioxidants are presented in Table 2. As
a priority, ferrozine constitutes a complex with unbounded
(
0.727 ꢁ 0.078), and trolox (0.675 ꢁ 0.167). The reducing
power of the phenolic sulfonamides 13–16 and standard
antioxidants decreased in the following order: 16 ꢂ 14 > BHA
>
BHT > a-tocopherol > trolox ꢂ 15 > 13. It was reported that
3 2þ
þ
2þ
2þ
Fe –Fe reducing ability is frequently used as a marker of
electron-donating ability. This bioanalytical method is an
important mechanism of phenolic antioxidants [10].
Fe . Thus, a decrease occurs in the amount of ferrozine–Fe
complex [61]. The data obtained from Table 2 clearly
demonstrate that phenolic sulfonamides 13–16 possess a
marked capacity for iron binding. In this assay, phenolic
sulfonamides 13–16 interfered with the formation of the
ferrous–ferrozine complex. According to the results, phenolic
sulfonamides 13–16 have effective metal chelating abilities.
IC50 value for metal chelating activity of phenolic sulfona-
mides 13–16 was in the range of 5.5–11.50 mg/mL (Table 2).
On the other hand, these values were found similar to those
of standard compounds such as BHA (9.90 mg/mL),
BHT (6.30 mg/mL), a-tocopherol (12.50 mg/mL), and trolox
(12.35 mg/mL). Accordingly, lower IC50 values indicate high
metal binding ability. It suggests that phenolic sulfonamides
13–16 have chelating effects and are able to capture ferrous
ion before ferrozine agent.
In the present study, we use the CUPRAC assay, which is
2
þ
þ
based on reduction of Cu to Cu by phenolic sulfonamides
3–16. The CUPRAC method has been used to determine
1
the reducing power of antioxidant compounds, especially
phenolic compounds [59]. As seen in Table 1, cupric ion
2þ
(
Cu ) reducing power of phenolic sulfonamides 13–16 and
standard compounds decreased in the following order:
6 (2.617 ꢁ 0.019) > 14 (1.901 ꢁ 0.037) > BHA (1.173 ꢁ 0.066)
ꢂ BHT (1.140 ꢁ 0.098) > a-tocopherol (0.330 ꢁ 0.061) > trolox
0.268 ꢁ 0.027) ꢂ 15 (0.255 ꢁ 0.039) > 13 (0.203 ꢁ 0.106). In the
CUPRAC assay, higher absorbance values indicate higher
1
(
2þ
cupric ions (Cu ) reducing ability.
3þ
2þ
The FRAP assay measures the Fe –Fe reducing ability of
the substance and was initially proposed for the measurement
of total antioxidant capacity [60]. According to results
obtained from FRAP assay (Table 1), reducing power of
phenolic sulfonamides 13–16 and standard compounds
•
•þ
•þ
2•ꢀ
DPPH , ABTS , DMPD , and O scavenging assays are
frequently used for the determination of the radical
scavenging ability of various plant samples or pure
compounds [59]. In the present study, the radical scavenging
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Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
Biological Activities of Dopamine-Related Compounds
787
2
þ
•
•þ
Table 2. Concentration required for 50% scavenging (IC50) of ferrous ions (Fe ) chelating activity, DPPH scavenging activity, ABTS
scavenging activity, DMPD , and O
•þ
•ꢀ
2
scavenging activity of phenolic sulfonamides (13–16) and standard radical scavengers such as
BHA, BHT, a-tocopherol, and trolox
•
•þ
•þ
2•ꢀ
O scavenging
Metal chelating
DPPH scavenging
ABTS scavenging
DMPD scavenging
2
2
2
2
2
Antioxidants
IC50
r
IC50
r
IC50
r
IC50
r
IC50
r
BHA
BHT
a-Tocopherol
Trolox
9.90
6.30
12.50
12.35
11.50
5.77
0.858
0.938
0.904
0.827
0.926
0.896
0.971
0.971
8.35
25.67
7.62
7.79
12.38
7.79
0.957
0.965
0.950
0.876
0.913
0.875
0.951
0.943
4.82
4.92
4.91
6.36
4.88
5.09
4.74
4.20
0.926
0.945
0.954
0.935
0.907
0.935
0.916
0.914
18.72
–
0.922
0.985
0.917
0.913
0.922
0.935
0.930
0.926
36.47
30.13
20.38
43.32
9.90
10.83
57.75
12.60
0.908
0.927
0.913
0.949
0.937
0.982
0.949
0.947
–
18.73
25.67
15.40
16.12
13.32
1
1
1
1
3
4
5
6
11.50
5.51
11.75
5.50
•
•þ
0
•þ
DPPH , 1,1-diphenyl-2-picryl-hydrazyl free radicals; ABTS , 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid radicals; DMPD , N,N-
•
ꢀ
dimethyl-p-phenylenediamine dihydrochloride radicals, O
2
, superoxide anion radicals; BHA, butylated hydroxyanisole; BHT,
butylated hydroxytoluene.
ability of phenolic sulfonamides 13–16 was evaluated by
In the presence of a suitable oxidant solution, DMPD can
•
•þ
•þ
•ꢀ
•þ
DPPH , ABTS , DMPD , and O2 scavenging assays.
form a stable and colored radical cation (DMPD ) at acidic
•
•þ
DPPH assay is esteemed as a distinguished method for
pH [64]. As seen in Table 2, DMPD
radical inhibition by
the evaluation of radical scavenging activity of an antioxidant
compound. A radical compound is stable and does not have
to be generated as in other radical scavenging assays [62].
Concentration required for 50% DPPH free radical
scavenging (IC ) for phenolic sulfonamides 13–16 and
phenolic sulfonamides 13–16 and standard antioxidants (BHA
and trolox) increased in the following order: 16 (13.32 mg/mL,
2
2
2
r : 0.926) < 14 (15.40 mg/mL, r : 0.935) ꢃ 15 (16.12 mg/mL, r :
2
2
0.930) < BHA (18.72 mg/mL, r : 0.922) ꢂ trolox (18.73 mg/mL, r :
50
2
0.913) < 13 (25.67 mg/mL, r : 0.922). Lower IC values indicate
5
0
•
þ
•þ
standards increased in the following order: 16 (5.50 mg/mL,
high DMPD radical scavenging ability. DMPD method is a
sensitive method. Its reproducibility dramatically decreased
when hydrophobic antioxidants such as a-tocopherol or BHT
were used. For this reason, both standard antioxidants were
not used in this assay [65].
2
2
r : 0.943) < a-tocopherol (7.62 mg/mL, r : 0.950) < 14
2
2
(
(
(
7.79 mg/mL, r : 0.875) ¼ trolox (7.79 mg/mL, r : 0.876) < BHA
2
2
8.35 mg/mL, r : 0.957) < 15 (11.75 mg/mL, r : 0.951) < 13
2
2
12.38 mg/mL, r : 0.913) < BHT (25.67 mg/mL, r : 0.965). These
results clearly showed that all phenolic sulfonamides 13–16
had effective scavenging effect on DPPH radicals. Lower IC50
values indicate high DPPH free radical scavenging ability.
Phenolic groups easily stabilize a radical formed on phenolic
carbon with their resonance structure. In phenolic sulfon-
amides 13–15, phenolic groups also have two hydroxyl units.
On the other hand, phenolic sulfonamide 16 has one hydroxyl
unit. Abstracting hydrogen atoms from phenolic hydroxyl
groups may occur easily.
ABTS radical cation decolorization assay is another effective
method for the measurement of radical scavenging activity of
pure molecules [63]. IC50 values belonging to scavenging of
ABTS radical by phenolic sulfonamides 13–16 and standard
antioxidants increased in the following order: 16 (4.20 mg/mL,
Superoxide anion radicals are also highly reactive radicals
formed in biological systems such as mitochondrial respira-
tion and NADPH oxidase. They have been implicated as two
highly damaging species in free radical pathology, capable of
damaging almost every molecule found in living cells [33]. As
it can be seen in Table 2, the inhibition of superoxide radical
formation by phenolic sulfonamides 13–16 and standard
antioxidants increased in the following order: 13 (9.90 mg/mL,
2
2
2
r : 0.937) < 14 (10.83 mg/mL, r : 0.982) < 16 (12.60 mg/mL, r :
2
0.947) < a-tocopherol (20.38 mg/mL, r : 0.913) < BHT (30.13 mg/
2
2
mL, r : 0.927) < BHA (36.47 mg/mL, r : 0.908) < trolox
2
2
(43.32 mg/mL, r : 0.949) < 15 (57.75 mg/mL, r : 0.949). Lower
IC50 values indicate high superoxide radical scavenging
ability.
2
2
2
r : 0.914) ꢃ 15 (4.74 mg/mL, r : 0.916) ꢃ BHA (4.82 mg/mL, r :
2
0
.926) ꢃ 13 (4.88 mg/mL, r : 0.907) ꢃ a-tocopherol (4.91 mg/mL,
Acetylcholinesterase inhibition
2
2
2
r : 0.954) ꢃ BHT (4.92 mg/mL, r : 0.945) ꢃ 14 (5.09 mg/mL, r :
Inhibition of AChE enzyme was determined on commercially
available purified AChE (E.C. 3.1.1.7, Product no: C0663-50 UN,
Sigma–Aldrich) from human erythrocyte based on the
method of Ellman et al. [65]. It was reported that the
2
0.935) ꢃ trolox (6.36 mg/mL, r : 0.935). At the same manner,
lower IC values indicate high ABTS radical scavenging
5
0
ability (Table 2).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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7
88
H. Göçer et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
Table 3. Acetylcholinesterase enzyme inhibition parameters of
phenolic sulfonamides (13–16)
e
2
Sulfonamides
IC50 (nM)
K
i
(nM)
r
d
1
1
1
3
4
5
442.8 ꢁ 33.01
368.4 ꢁ 50.04
452.9 ꢁ 43.11
771.3 ꢁ 131.68
55.0
33.04 ꢁ 4.3
59.05 ꢁ 5.6
43.11 ꢁ 2.8
131.68 ꢁ 8.8
–
0.9771
0.9542
0.9709
0.9705
–
O
c
N
16
C
Donepezil hydrochloride
a
O
OH
Ser
b
All of these studies clearly showed that an investigation into
the mechanism of action of AChE may lead to the design
of the mechanism-based inhibitors, which could be of future
therapeutic use. Different types of AChE inhibitors have been
studied for the treatment of Alzheimer’s disease. Some of
the AChE inhibitors differ in their mechanism of action,
metabolism, and brain selectivity.
Figure 2. The interaction between acetylcholine and active sites of
acetylcholinesterase enzyme.
AChE was very effectively inhibited by phenolic sulfon-
amides 13–16, with K
131.68 ꢁ 8.8 nM (Table 3). The K
amides 13–16 were similar to each other. The differences
between the highest and the lowest K values of phenolic
i
s in the range of 33.04 ꢁ 4.3 to
inhibition of AChE has been used for the determination of
neurotoxic properties of chemicals capable of interfering
with normal neurotransmission of the parasympathetic and
sympathetic nervous system. It is well known that some
chemical compounds like organophosphate and carbamate
pesticides [40, 66–68], enantiomeric inhibitors [43], metals,
and non-pesticide contaminants have also been reported to
inhibit AChE enzymatic activity [42, 69–72]. Also, it was
demonstrated that the main AChE inhibitory effects was
primarily associated with aromatic compounds and, to lesser
degree, with aliphatic compounds [42].
i
values of phenolic sulfon-
i
sulfonamides 13–16 were only fourfold. The most active one
was phenolic sulfonamide 13 demonstrating a 33.04 ꢁ 4.3 nM
i
K value. On the other hand, donepezil hydrochloride,
which is used for the treatment of mild-to-moderate
Alzheimer’s disease and various other memory impairments,
had been shown to lower AChE inhibition activity (IC50
:
55 nM) [73]. Donepezil hydrochloride had N-benzylpiperidine
and an indanone moiety that shows longer and more
selective action.
In an important study, the three-dimensional structure of
AChE from the Pacific electric ray (Torpedo californica) electric
organ has been determined by X-ray analysis. The active Conclusions
site of AChE also consists of at least five major binding sites
(
Fig. 2):
In conclusion, starting from commercially available methoxy-
lated dopamine-related compounds 5–8, three new phenolic
sulfonamides 13–15 and a known sulfonamide 16 were
synthesized. In our study, phenolic sulfonamides 13–16
demonstrated effective antioxidant properties in the follow-
ing assays: total antioxidant activity in linoleic acid emulsion,
a. Firstly an oxyanion hole stabilizes the tetrahedral
intermediate.
b. Then an esteratic site, which is comprised of the active site
serine residue, attacks the esteratic carbonyl group (C–O).
3þ
2þ
•
•þ
c. An anionic substrate binding site that contains a small
number of negative charges but many aromatic residues,
where the quaternary ammonium pole of ACh molecule
and of various active site ligands binds through a
preferential interaction of quaternary N atoms by Z
electrons of aromatic groups.
d. An active site-selective aromatic binding site that is
contiguous with or near the esteratic and anionic loci
and that is important in binding aryl substrates and active
site ligands.
Fe
2•ꢀ
DMPD , and O scavenging and metal chelating activities
and Cu
reducing capabilities, DPPH , ABTS
,
•
þ
when they are compared to standard antioxidant compounds.
Based on results obtained from this study, phenolic
sulfonamides 13–16 had powerful antioxidant activity and
can be used for minimizing or preventing lipid oxidation in
food and pharmaceutical products and for prolonging the
shelf life of pharmaceuticals and nutritional quality of foods.
Also, phenolic sulfonamides 13–16 had effective AChE
inhibition properties and they can be good candidates for
the treatment of mild-to-moderate Alzheimer’s disease and
various other memory impairments.
e. A peripheral anionic binding site that may bind to the
hydrophobic part of the leaving group from the active site.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
Biological Activities of Dopamine-Related Compounds
789
Experimental
1317, 1263, 1237, 1193, 1147, 1077, 1026. Anal. calcd. for
S): C, 50.95; H, 6.61; N, 5.40; S 12.36. Found C, 50.88; H,
.60; N, 5.45; S, 12.40.
11 4
(C H17NO
6
All chemicals and solvents are commercially available and were
used after distillation or treatment with drying agents. Melting
points were determined on a capillary melting apparatus (BUCHI
N-(2-Methoxyphenethyl)methanesulfonamide (12)
5
30) and are uncorrected. IR spectra were obtained from solutions
1
Yellowish oil. H NMR (400 MHz, CDCl
J ¼ 1.6 and 7.7 Hz), 7.15 (dd, 1H, Ar–H, J ¼ 1.3 and 7.3 Hz), 6.93–
.86 (m, 2H, Ar–H), 4.52 (bs, 1H, NH), 3.84 (s, 3H, OCH ), 3.38
q, 2H, CH
, J ¼ 6.6 Hz), 2.76 (s, 3H,
) d 157.7 (C), 131.1 (CH), 128.6 (CH),
3
) d 7.24 (dt, 1H, Ar–H,
1
in 0.1 mm cells with a Perkin-Elmer spectrophotometer. The H
1
3
and C NMR spectra were recorded on a 400 (100)-MHz Varian
and 400 (100)-MHz Bruker spectrometer; d in ppm, Me Si as the
6
(
CH
3
4
2
–N, J ¼ 6.6 Hz), 2.88 (t, 2H, CH
2
internal standard. Elemental analyses were performed on a Leco
CHNS-932 apparatus. All column chromatography was per-
formed on silica gel (60-mesh, Merck). PLC is preparative thick-
layer chromatography: 1 mm of silica gel 60 PF (Merck) on glass
plates.
13
3
). C NMR (100 MHz, CDCl
3
1
26.5 (C), 121.1 (CH), 110.8 (CH), 55.6 (OCH
3
), 43.6 (CH
3
), 40.2
ꢀ1
(CH
2
–N), 31.3 (CH
2
). IR (CH
2
Cl
2
, cm ): 3565, 3291, 3010, 2935,
2
1
838, 2637, 2483, 2293, 2051, 1905, 1601, 1588, 1495, 1465, 1439,
410, 1317, 1244, 1198, 1149, 1120, 1076, 1053, 1034. Anal. calcd.
3
for (C10H15NO S): C, 52.38; H, 6.59; N, 6.11; S 13.98. Found C,
Synthesis
52.41; H, 6.61; N, 6.08; S, 13.93.
Standard procedure for the synthesis of sulfonamides
2 2
Amine 5 (1.30 g, 7.17 mmol) was dissolved in CH Cl (30 mL) and
Standard procedure for the synthesis of phenolic
sulfonamides
this solution was cooled to 0°C. To this solution were added Et
3
N
(
0.87 g, 8.61 mmol) and MeSO Cl (0.99 g, 8.61 mmol). The reaction
2
A solution of sulfonamide 5 (1 g, 3,86 mmol) in CH
cooled to 0°C and then a solution of BBr (7.36 g, 29.36 mmol) in
CH Cl (5 mL) was added dropwise under N (g) over 5 min. After
the cold bath was removed, the mixture was stirred at RT under
(g) for 1 day. The mixture was added to ice-water (40 mL) and
2 2
Cl (25 mL) was
mixture was stirred at room temp. for 15 h. After the solvent was
evaporated, the residue was chromatographed on silica gel (30 g)
column with 20% EtOAc–hexane. Sulfonamide 9 was synthesized
as yellowish oil (1.44 g, 77% yield).
Sulfonamides 10–12 were also synthesized by this procedure
with yields of 75%, 73%, and 78%, respectively.
3
2
2
2
N
2
then the organic phase was extracted. The water phase was
extracted with EtOAc (2 ꢄ 30 mL). The combined organic phases
2 4
were dried over Na SO and solvent was evaporated. The phenolic
N-(2,3-Dimethoxyphenethyl)methanesulfonamide (9)
sulfonamide 13 (0.75 g, 84%) was obtained.
1
Yellowish oil. H NMR (400 MHz, CDCl
3
) d 7.01 (t, 1H, Ar–H,
J ¼ 7.9 Hz), 6.84 (dd, 1H, Ar–H, J ¼ 1.3 and 8.2 Hz), 6.78 (dd, 1H,
N-(2,3-Dihydroxyphenethyl)methanesulfonamide (13)
Ar–H, J ¼ 1.3 and 7.6 Hz), 4.68 (t, 1H, NH, J ¼ 5.2 Hz), 3.86 (s, 3H,
1
Brown oil. H NMR (400 MHz, acetone-d
6
) d 8.37 (bs, 1H, OH), 7.19
OCH
t, 2H, CH
d 152.8 (C), 147.2 (C), 131.8 (C), 124.4 (CH), 122.4 (CH), 111.4 (CH),
0.6 (OCH ), 55.7 (OCH ), 44.0 (CH ), 39.9 (CH –N), 30.9 (CH ).
IR (CH Cl , cm ): 3287, 2952, 2924, 2854, 1738, 1717, 1619, 1586,
479, 1465, 1362, 1315, 1268, 1219, 1186, 1150, 1080. Anal. calcd.
for (C11 S): C, 50.95; H, 6.61; N, 5.40; S 12.36. Found C,
0.96; H, 6.65; N, 5.38; S, 12.32.
3
), 3.85 (s, 3H, OCH
3
), 3.38 (q, 2H, CH
2
–N, J ¼ 6.7 Hz), 2.89
1
3
(bs, 1H, OH), 6.61 (dd, 1H, Ar–H, J ¼ 1.6 and 7.6 Hz), 6.54–6.46
(
2
, J ¼ 6.7 Hz), 2.78 (s, 3H, CH
3 3
). C NMR (100 MHz, CDCl )
(
2
m, 2H, Ar–H), 5.86 (bs, 1H, NH), 3.21 (q, 2H, CH
2
–N, J ¼ 7.2 Hz),
1
3
.74 (t, 2H, CH
2
, J ¼ 7.2 Hz), 2.71 (s, 3H, CH
3
). C NMR (100 MHz,
6
3
3
3
2
2
ꢀ1
6
acetone-d ) d 145.4 (C), 144.4 (C), 126.3 (C), 122.6 (CH), 120.3 (CH),
2
2
ꢀ1
1
3
1
14.5 (CH), 44.1 (CH
428, 3305, 3305, 3019, 2929, 2851, 1620, 1594, 1502, 1477, 1437,
406, 1308, 1278, 1193, 1144, 1078. Anal. calcd. for (C S):
3 2 2 2 2
), 39.8 (CH –N), 31.9 (CH ). IR (CH Cl , cm ):
1
H
17NO
4
9 4
H13NO
5
C, 46.74; H, 5.67; N, 6.06; S 13.86. Found C, 46.76; H, 5.71; N, 5.97;
S, 13.91.
N-(3,5-Dimethoxyphenethyl)methanesulfonamide (10)
1
White solid. m.p. 83–85°C. H NMR (400 MHz, CDCl
Ar–H), 4.35 (t, 1H, NH, J ¼ 5.5 Hz), 3.78 (s, 6H, 2OCH
CH ), 2.82 (t, 2H, CH
–N, J ¼ 6.7 Hz), 2.87 (s, 3H, CH
C NMR (100 MHz, CDCl ) d 161.2 (2C), 140.1 (C), 106.9 (2CH), 98.7
CH), 55.3 (2OCH ), 44.2 (CH ), 40.4 (CH –N), 36.7 (CH ). IR (CH Cl
cm ): 3478, 3289, 3053, 2931, 2845, 1736, 1597, 1547, 1463,
431, 1342, 1314, 1265, 1206, 1149, 1071. Anal. calcd. for
S): C, 50.95; H, 6.61; N, 5.40; S 12.36. Found C, 50.93; H,
.58; N, 5.46; S, 12.32.
3
) d 6.35 (s, 3H,
), 3.39 (q, 2H,
, J ¼ 6.7 Hz).
N-(3,5-Dihydroxyphenethyl)methanesulfonamide (14)
Standard procedure described above for 13 was applied to
3
2
3
2
1
3
sulfonamide 10 to give 14 (81%). Brown solid. m.p. 84–86°C.
3
1
(
3
3
2
2
2
2
,
6
H NMR (400 MHz, acetone-d ) d 8.13 (bs, 2H, 2OH), 6.15 (d, 2H,
ꢀ
1
Ar–H, J ¼ 2.1 Hz), 6.13 (t, 1H, Ar–H, J ¼ 2.1 Hz), 5.90 (t, 1H, NH,
J ¼ 5.4 Hz), 3.20 (q, 2H, CH –N, J ¼ 7.7 Hz), 2.77 (s, 3H, CH ), 2.62
, J ¼ 7.7 Hz). C NMR (100 MHz, acetone-d ) d 159.3 (2C),
142.2 (C), 108.4 (2CH), 101.8 (CH), 45.3 (CH ), 40.0 (CH –N),
, cm ): 3261, 3014, 2957, 2924, 2851, 1711,
602, 1456, 1362, 1307, 1145, 1076. Anal. calcd. for (C S):
1
(
6
2
3
1
3
(t, 2H, CH
2
6
11 4
C H17NO
3
2
ꢀ1
2 2 2
37.2 (CH ). IR (CH Cl
1
9 4
H13NO
N-(3,4-Dimethoxyphenethyl)methanesulfonamide (11)
C, 46.74; H, 5.67; N, 6.06; S 13.86. Found C, 46.70; H, 5.61; N, 6.10;
S, 13.90.
1
White solid. m.p. 78–80°C. (lit. [47] m.p. 72°C). H NMR (400 MHz,
CDCl
3
) d 6.81 (d, 1H, Ar–H, J ¼ 8.1 Hz), 6.75–6.71 (m, 2H, Ar–H),
.37 (t, 1H, NH, J ¼ 6.5 Hz), 3.87 (s, 3H, OCH ), 3.86 (s, 3H, OCH ),
.37 (q, 2H, CH ), 2.79 (t, 2H, CH
–N, J ¼ 6.7 Hz), 2.83 (s, 3H, CH
) d 149.4 (C), 148.2 (C), 130.4
C), 121.1 (CH), 112.2 (CH), 111.7 (CH), 56.18 (OCH ), 56.16 (OCH ),
, cm ): 3564, 3280,
008, 2935, 2838, 2283, 2052, 1731, 1608, 1592, 1517, 1465, 1420,
4
3
3
3
,
N-(3,4-Dihydroxyphenethyl)methanesulfonamide (15)
2
3
2
1
3
J ¼ 6.7 Hz). C NMR (100 MHz, CDCl
3
Compound 15 was synthesized from 11 (77%). Brown solid. m.p.
1
(
3
ꢀ
3
88–90°C. H NMR (400 MHz, acetone-d
6
) d 7.64 (bs, 1H, OH), 7.60
1
4
4.8 (CH
3
), 40.6 (CH
2
–N), 36.3 (CH
2
). IR (CH
2
Cl
2
(bs, 1H, OH), 6.62–6.60 (m, 2H, Ar–H), 6.45 (dd, 1H, Ar–H, J ¼ 1.9
3
and 8.1 Hz), 5.79 (bs, 1H, NH), 3.14 (q, 2H, CH
2
–N, J ¼ 7.4 Hz), 2.71
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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7
90
H. Göçer et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
1
3
2þ
(
s, 3H, CH
3
), 2.59 (t, 2H, CH
2
, J ¼ 7.4 Hz). C NMR (100 MHz,
Ferrous ion (Fe ) chelating activity
2
þ
acetone-d ) d 145.9 (C), 144.5 (C), 131.4 (C), 121.0 (CH), 116.8 (CH),
6
Ferrozine forms complexes with Fe , but in presence of ion
chelating agents, the complex formation is disrupted, resulting
in a decrease in the red color of the complexes [79]. Fe chelating
activity of phenolic sulfonamides 13–16 and standard com-
pounds was measured by inhibiting the formation of Fe
ferrozine complex after treatment of test material with Fe . Fe
chelating ability of phenolic sulfonamides 13–16 was determined
by the absorbance of the Fe –ferrozine complex at 562 nm [58].
ꢀ1
1
3
1
16.1 (CH), 45.7 (CH
522, 3299, 3053, 2926, 2851, 1725, 1626, 1605, 1527, 1443, 1362,
309, 1265, 1191, 1144, 1075. Anal. calcd. for (C S):
3 2 2 2 2
), 39.9 (CH –N), 36.7 (CH ). IR (CH Cl , cm ):
2þ
9
H
13NO
4
2
þ
2þ
C, 46.74; H, 5.67; N, 6.06; S 13.86. Found C, 46.75; H, 5.62; N, 6.01;
S, 13.88.
–
2
þ
2
þ
N-(2-Hydroxyphenethyl)methanesulfonamide (16)
Compound 16 was synthesized from 12 (82%). Brown solid. m.p.
1
7
6
2–74°C. H NMR (400 MHz, CDCl
3
) d 7.09–7.06 (m, 2H, Ar–H),
Radical scavenging activities
The DPPH free radical scavenging activity of phenolic sulfon-
amides 13–16 was evaluated by the method of Blois [80]. The
radical scavenging activity of phenolic sulfonamides
13–16 was performed according to the method of Re et al. [81].
has a characteristic absorbance at 734 nm. The DMPD
radical scavenging ability of phenolic sulfonamides 13–16 was
.85–6.80 (m, 2H, Ar–H), 6.56 (bs, 1H, OH), 5.68 (t, 1H, NH,
–N, J ¼ 6.7 Hz), 2.85 (t, 2H, CH
J ¼ 5.7 Hz), 3.36 (q, 2H, CH
2
3
2
,
1
•þ
J ¼ 6.7 Hz), 2.75 (s, 3H, CH
3
). C NMR (100 MHz, CDCl
3
) d 154.2 (C),
),
, cm ): 3428, 3305, 3064,
025, 2918, 2849, 1608, 1595, 1505, 1490, 1456, 1408, 1311, 1264,
234, 1174, 1145, 1107, 1075. Anal. calcd. for (C S):
ABTS
1
3
3
1
31.1 (CH), 128.3 (CH), 124.7 (C), 120.9 (CH), 115.7 (CH), 43.8 (CH
3
ꢀ
1
•þ
9.8 (CH
2
–N), 30.9 (CH
2
). IR (CH
2
Cl
2
ABTS
9
H
13NO
3
determined by the method of Gülçin [59]. This assay is based on
•
þ
C, 50.21; H, 6.09; N, 6.51; S 14.90. Found C, 50.19; H, 6.06; N, 6.55;
S, 14.89.
the capacity of the extract to inhibit DMPD
cation radical
scavenging activity of phenolic sulfonamides
3–16 was determined by the riboflavin/methionine/illuminate
•
ꢀ
formation. O2
1
assay. This assay is based on the capability of antioxidant
compounds to inhibit the photochemical reduction of NBT [79].
The percentage of above-mentioned metal chelating and radical
scavenging capabilities of phenolic sulfonamides 13–16 and
standards was calculated using the following equation:
Biochemistry
General experimental procedures
Materials, methods, and instrumentations were previously
reported [10, 18]. In the present study, the following experimental
procedures were applied to evaluation of antioxidant and radical
scavenging activities of phenolic sulfonamides 13–16.
Metal chelating or radical scavenging ð%Þ
ꢀ
ꢁ
lC
¼
1 ꢀ lS ꢄ 100
Total antioxidant activity – Ferric thiocyanate method
The antioxidant activity of phenolic sulfonamides 13–16 and
standard antioxidants was determined according to the ferric
thiocyanate method [74]. This method was described previously
C S
where l is the absorbance of control and l is the absorbance
in the presence of phenolic sulfonamides 13–16 or standard
compounds [82].
[
57]. Peroxides are formed during the linoleic acid oxidation.
2þ
3þ
These peroxides lead to oxidation of Fe to Fe . Cupric ions
3þ
ꢀ
(
Fe ) form a complex with SCN . The resulting complex
Determination of anticholinesterase activity
has maximum absorbance at 500 nm. High absorbance indicates
high linoleic acid emulsion oxidation. The percentage inhibition
of lipid peroxidation in linoleic acid was calculated by the
following equation:
The inhibitory effects of phenolic sulfonamides 13–16 on AChE
activities were measured by slightly modifying the spectrophoto-
metric method of Ellman et al. [65]. Acetylthiocholine iodide was
0
used as substrate of the reaction. DTNB (5,5 -dithio-bis(2-nitro-
ꢀ
ꢁ
benzoic)acid (product no: D8130-1G, Sigma–Aldrich)) was used for
the measurement of the AChE activity. Briefly, 100 mL of Tris/HCl
buffer (1 M, pH 8.0), 10 mL of sample solution dissolved in
lC
lS
ILP ð%Þ ¼ 100 ꢀ
ꢄ 100
deionized water at different concentrations and 50 mL AChE
where ILP is the inhibition of lipid peroxidation, l
absorbance of control reaction, and l is the absorbance in
the presence of phenolic sulfonamides 13–16 or standard
C
is the
ꢀ3
(
2
5.32 ꢄ 10 U) solution were mixed and incubated for 10 min at
S
5°C. Then 50 mL of DTNB (0.5 mM) was added. The reaction was
then initiated by the addition of 50 mL of acetylthiocholine iodide
10 mM, product no: 01480-1G, Sigma–Aldrich). The hydrolysis of
compounds [75].
(
these substrates was monitored spectrophotometrically by the
formation of yellow 5-thio-2-nitrobenzoate anion as the result of
the reaction of DTNB with thiocholine, released by the enzymatic
hydrolysis of acetylthiocholine iodide, at a wavelength of
412 nm [83].
In order to determine the effect of phenolic sulfonamides 13–
16 on AChE, different phenolic sulfonamides 13–16 concen-
trations were added into the reaction medium. The enzyme
activity was measured, and an experiment in the absence of drug
was used as control. The IC50 values were obtained from activity
(%) versus phenolic sulfonamides 13–16 concentration plots.
Reducing power abilities
3þ
2þ
Fe –Fe reducing power of phenolic sulfonamides 13–16 was
3
þ
2þ
measured according to the method of Gülçin [75]. The Fe –Fe
reducing ability is spectrophotometrically measured by absor-
bance of Perl’s Prussian blue complex at 700 nm. Cu reducing
power of phenolic sulfonamides 13–16 was determined accord-
ing to the method of Apak et al. [76]. The second used reducing
power assay is ferric reducing antioxidant power, which is
2
þ
3
þ
based upon reduction of the Fe –TPTZ complex under acidic
conditions [77]. Increased absorbance of blue-colored ferrous
2
þ
form (Fe –TPTZ complex) is spectrophotometrically measured
To determine the K constants in the media with phenolic
i
at 593 nm [78].
sulfonamides 13–16 as inhibitor, the different substrate (ACh)
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 783–792
Biological Activities of Dopamine-Related Compounds
791
concentrations were used. Inhibitor solution was added into the
reaction medium, resulting in three different fixed concentra-
tions of inhibitors. Lineweaver–Burk graphs [84] were drawn
using 1/V versus 1/[S] values. K constants were calculated from
i
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