Acetylcholinesterase inhibition and antioxidant activity of N-trans-caffeoyldopamine and N-trans-feruloyldopamine

Article abstract of DOI:10.3390/scipharm86020011

Phenolic acids and their derivatives found in nature are well-known for their potential biological activity. In this study, two amides derived from trans-caffeic/ferulic acid and dopamine were synthesized and characterized by Fourier-transform infrared sp

Full text of DOI:10.3390/scipharm86020011

Scientia
Pharmaceutica
Article
Acetylcholinesterase Inhibition and Antioxidant
Activity of N-trans-Caffeoyldopamine and
N-trans-Feruloyldopamine
Muamer Dizdar 1 , Danijela Vidic , Franc Požgan
ID
1
2
ID
, Bogdan Štefane 2
ID
and Milka Maksimovi c´ 1,
*
1
Faculty of Science, University of Sarajevo, Zmaja od Bosne 33-35, 71000 Sarajevo, Bosnia and Herzegovina;
muamer.dizdar@pmf.unsa.ba (M.D.); danijela.vidic@gmail.com (D.V.)
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ve cˇ na pot 113, 1000 Ljubljana,
Slovenia; Franc.Pozgan@fkkt.uni-lj.si (F.P.); Bogdan.Stefane@fkkt.uni-lj.si (B.Š.)
Correspondence: mmaksimo@pmf.unsa.ba; Tel.: +387-33-279-999
2
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 8 February 2018; Accepted: 24 March 2018; Published: 4 April 2018
Abstract: Phenolic acids and their derivatives found in nature are well-known for their
potential biological activity. In this study, two amides derived from trans-caffeic/ferulic acid and
dopamine were synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR),
mass spectrometry, proton and carbon-13 nuclear magnetic resonance spectroscopy. The compounds
were tested for the inhibition of acetylcholinesterase (AChE) from Electrophorus electricus and
•
for antioxidant activity by scavenging 2,2-diphenyl-1-pycrylhydrazyl free radical (DPPH ) and
2
0
•+
,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS ), reducing ferric ions,
and ferrous ions chelation. N-trans-Feruloyldopamine displayed the highest inhibitory effect on
AChE with half-maximal inhibitory concentration (IC ) values of 8.52 M. In addition, an in silico
µ
50
study was done to determine the most favorable AChE cluster with the synthesized compounds.
Further, these clusters were investigated for binding positions at the lowest free binding energy.
Both synthesized hydroxycinnamates were found to be better antioxidants than the parent acids in
in vitro tests applied. N-trans-Caffeoyldopamine showed the best antioxidant activity in the three
•
tested methods—against non-biological stable free radicals IC 5.95
µ
M for DPPH , 0.24
µM for the
50
•
+
ABTS method, and for reducing power (ascorbic acid equivalent (AAE) 822.45
µ
mol/mmol)—while
for chelation activity against Fe2 ions N-trans-feruloyldopamine had slightly better antioxidant
+
activity (IC50 3.17 mM).
Keywords: acetylcholinesterase inhibition; antioxidant activity; N-trans-feruloyldopamine;
N-trans-caffeoyldopamine
1
. Introduction
Phenolic acids constitute a family of natural compounds that can be found in a wide variety of plants
and food [
attention and has been studied by many research groups in recent years. The compounds are known to
have acetylcholinesterase (AChE)-inhibitory [ ], antibacterial [ ], anti-inflammatory [ ], antioxidant [ ],
antiviral [ ], and immunomodulatory [ ] properties. These properties are associated with either their
1]. The biological activity of phenolic acids and other phenolic compounds has attracted much
2
3
4
5,6
7
8
properties as antioxidants and enzyme inhibitors or their binding activity with specific receptors.
Also, derivatives of phenolic acids, which primarily include esters, ethers, glycosides, and amides,
were tested and showed various biological activities [9–11]. Some of these derivatives belong to
clovamide type derivatives that can be found in nature [11,12] but have also been synthesized [13].
Sci. Pharm. 2018, 86, 11; doi:10.3390/scipharm86020011
www.mdpi.com/journal/scipharm

Sci. Pharm. 2018, 86, 11
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This group of derivatives includes N-trans-caffeoyldopamine and N-trans-feruloyldopamine, which has
been shown to have potent protective effects against hepatotoxicity [14], are 2-adrenoceptor
agonists [15], and inhibit platelet–leukocyte interactions and lipid peroxidation [12 16]. All these findings
β
,
make N-trans-caffeoyldopamine and N-trans-feruloyldopamine interesting bioactive compounds for
pharmaceutical and food research as potentially useful ingredients of some medicines. According to
our best knowledge, a comprehensive study of AChE inhibition and the antioxidant activity of these
clovamide type derivatives has not been reported so far.
Acetylcholinesterase is widely distributed in the central and peripheral nervous systems where
it plays a critical role in quickly hydrolyzing the neurotransmitter acetylcholine, which is involved
in the pathogenesis of Alzheimer’s disease (AD). Hydrolysis is the result of nucleophilic attack of
the carbonyl carbon by a serine residue in the enzyme active center and involves charge transfer via
a histidine residue [17]. However, there are several subsites, which can be distinguished in the AChE
active site: esteratic subsite (also called the catalytic triad), oxanion hole, anionic subsite, acyl binding
pocket, peripheral anionic subsite, and other residues of the omega loop [18]. Recognized factors in
AD development include free radicals, inflammation of the brain tissue, and acetylcholine deficiency.
Therefore, acetylcholine breakdown in the brain can be prevented by the inhibition of AChE activity,
leading to an increase in acetylcholine concentration [19]. Today, several AChE inhibitors are used to
treat AD, including tacrine, donepezil, rivastigmine, and carbamates. Also, some natural products
show the ability to inhibit AChE, and the largest number of studies include alkaloids [20], phenolic
compounds [2,21], and terpenes [22].
Free radicals derived from oxygen, nitrogen, and sulfur compounds are highly reactive molecules
because of the presence of an unpaired electron, which is formed in metabolic processes within the
cell. Their role in signaling, apoptosis, gene expression, and ion transport has been proven [23].
In addition, they can attack purine and pyrimidine bases, amino acids, and unsaturated fatty acids,
causing oxidative stress and damage to DNA, RNA, proteins, carbohydrates, and lipids. Many studies
have demonstrated the correlation between these processes and a large number of diseases [24].
Although the human body has a number of enzymatic systems that protect it from oxidative stress,
the ability of these systems decreases with aging, increasing the need to take in antioxidants from
dietary supplements [25]. A large number of compounds from dietary supplements exhibit antioxidant
activity in living systems, among which the most important are L(+)-ascorbic acid, carotenoids, vitamin
E, and phenolic compounds [26].
Following the molecular combination strategy, the aim of this study was to provide a full
analysis of the structure-AChE inhibition/antioxidant activity relationship for two synthesized
trans-hydroxycinnamic amide derivatives. These compounds were investigated for their ability to
inhibit the AChE from Electrophorus electricus by a spectrophotometrically assay based on an Ellman
method [27]. Also, molecular docking studies of AChE were done to estimate the binding positions
and affinity of the complex target/ligand. Their efficiency as radical scavengers was evaluated
•
by their reactivity toward the stable 2,2-diphenyl-1-pycrylhydrazyl free radical (DPPH ) and
2
0
•+
,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS ), while the reducing
power and chelation ability were determined by the ferric-reducing antioxidant power (FRAP) assay
2+
and the inhibition of the Fe –ferrozine complex formation.
2
. Materials and Methods
2
.1. Chemicals and General Tehniques
All chemicals were purchased from commercial sources and when necessary, purified following
the guidelines of Armarego and Chai [28]. The monitoring of the reaction and purity of the obtained
compounds was done by thin-layer chromatography (TLC) on silica gel 60 F254, while the column
chromatography was performed on silica gel (100–200 mesh). The visualization of the chromatograms
was performed either under ultraviolet light and/or with 5% FeCl . The melting points were
3

Sci. Pharm. 2018, 86, 11
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determined using a hot stage apparatus and are uncorrected. Infrared spectra were collected as
−
1
potassium bromide (KBr) pellets in the 4000–400 cm region with a PerkinElmer BX Fourier-transform
infrared spectrometer (FTIR) (PerkinElmer, Waltham, MA, USA). The peak intensities are specified
as strong (s), medium (m), or broad (br). The proton and carbon-13 nuclear magnetic resonance
1
13
(
H- and C-NMR) spectra were recorded using a Bruker AVANCE Ultrashield 500 plus (Bruker,
Billerica, MA, USA), using deuterated dimethyl sulfoxide (DMSO-d ) as the solvent. All shifts
6
are given in ppm (
δ) relative to tetramethylsilane and calibrated by the residual proton signal of
DMSO-d . The coupling constants (J values) are expressed in Hz. High-resolution mass spectra
6
(
MS) were obtained by the Agilent 6224 TOF mass spectrometer (Agilent Technologies, Santa Clara,
CA, USA). All spectrophotometric measurements were made on a PerkinElmer Lambda 25 UV–Vis
spectrophotometer (PerkinElmer, Norwalk, MA, USA).
2
.2. Synthesis
Triethylamine (0.28 mL, 2 mmol) was added to a stirring solution of trans-hydoxycinnamic
acid (2 mmol) dissolved in dimethylformamide (10 mL). The solution was cooled in an ice water
bath and 2 mmol of dopamine hydrochloride was added followed by a solution of 2 mmol
of (benzotriazol-1-yloxa)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) in 5 mL
◦
dichloromethane. The mixture was stirred at 0 C for 30 min and then at room temperature overnight.
Dichloromethane was removed under reduced pressure and the resulting mixture was diluted with
1
00 mL water and extracted with ethyl acetate (EtOAc) (3
were washed with HCl (3 15 mL, 0.5 M), NaHCO (3
and dried (MgSO ) and evaporated under reduced pressure. The mixture was then absorbed onto
×
30 mL). The combined organic layers
×
×
15 mL, 0.5 M), water (3 15 mL),
×
3
4
SiO2 (≈1 g, 100–200 mesh) and purified by column chromatography (EtOAc–hexane, 1:2
→
2:1) to give
the crude products.
2
.3. Acetylcholinesterase Inhibition
2
.3.1. In Vitro Assay
The AChE inhibition of tested compounds was determined by a slightly modified Ellman’s
method [27], in which thiocholine produced by the action of AChE forms a yellow color with Ellman’s
reagent (DTNB). Galantamine was used as a reference compound. The tested compounds were
dissolved in DMSO to provide a final concentration range of 0–50
µM in the mixture solution. Briefly,
0
.3 mL of 100 mM sodium phosphate buffer pH 8, 0.3 mL of sample, and 0.3 mL AChE solution
◦
containing 0.54 U/mL were mixed in a 3 mL cuvette and allowed to incubate for 15 min at 37 C.
Subsequently, 0.3 mL of a solution of acetylcholine iodide (15 mM, dissolved in water) and 1.5 mL of
3
mM DTNB were added. The absorbance at 405 nm was read during the first 5 min of the reaction.
A control reaction, which was considered to have 100% activity, was carried out using the same volume
of DMSO instead of tested solutions. The percentage of AChE inhibition is calculated based on the
absorbance value as follows:
ꢀ
ꢁ
At
%
Inhibition = 1 − A
× 100
(1)
0
where A is the absorbance of the control and A
t
is the absorbance of the tested compound. A linear
0
equation indicating the correlation between the common logarithm of the compound concentration
and the percentage of AChE inhibition was built, from which the IC50 values (the concentration that
inhibits 50% of AChE activity) of the studied compounds were extrapolated.
2
.3.2. Molecular Docking Studies
A molecular docking study was performed in order to suggest a possible binding mode of
synthesized compounds with AChE. A SwissDock server was used for in silico prediction of the lowest

Sci. Pharm. 2018, 86, 11
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free binding energy using EADock DSS software [29
,
30]. The crystal structure of the target AChE from
E. electricus (1C2B) [31] was taken in protein data bank (PDB) format and analyzed in mol2 format as
required for calculation. UCSF Chimera 1.12 software was used for the visualization of the results and
creating three-dimensional (3D) images [32].
2
.4. Antioxidant Activity
2
.4.1. 2,2-Diphenyl-1-Pycrylhydrazyl Free Radical Scavenging Assay
•
The 2,2-diphenyl-1-pycrylhydrazyl free radical (DPPH ) scavenging method is widely used to
evaluate the free radical scavenging ability of natural antioxidants [33]. 2,2-Diphenyl-1-pycrylhydrazyl
is a stable nitrogen centered free radical, which has a violet color that changes to yellow after reduction
by the process of either hydrogen atom or single electron transfer mechanism. Compounds that are
able to execute this reaction can be considered antioxidants and therefore radical scavengers [1]. In the
radical form, the DPPH molecule has an absorbance of 517 nm, which disappears after the acceptance
of an electron or hydrogen radical from an antioxidant compound, to become a stable diamagnetic
•
molecule [34]. The DPPH scavenging activities of the tested compounds were estimated using the
•
method of [35] with slight modification. To 1 mL methanolic solution of DPPH (0.08 mM), 0.1 mL of
the tested compounds were added. The resulting mixture was shaken thoroughly and allowed to stand
at room temperature in the dark for 30 min, after which the absorbance of the solution was measured
•
at 517 nm. L(+)-Ascorbic acid was used as the positive control. The negative control contained DPPH
and methanol. The ability to scavenge DPPH radical was calculated using the equation:
ꢀ
ꢁ
At
%
DPPH radical scavenge = 1 − A
× 100
(2)
0
•
where A is the absorbance of the control and A
t
is the absorbance of the tested compound. The DPPH
0
absorbance decreases with an increase in the DPPH radical scavenging activity. The results were
expressed as IC50 concentration, where 50% inhibition of the DPPH radical was obtained.
0
2
.4.2. 2,2 -Azinobis(3-Ethylbenzothiazoline-6-Sulphonic Acid) Radical Cation Scavenging Assay
0
•+
The 2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS ), which absorbs
at 734 nm, is formed by oxidation of the commercially available neutral species. This oxidation can be
done by potassium persulphate, manganese (IV) oxide, or electrochemical methods. In the presence of
a hydrogen-donating antioxidant, the nitrogen quenches the hydrogen atom, which is followed by
•
+
decolorization [36,37]. In this study, the ABTS was produced by mixing 7 mM ABTS stock solution
with 2.45 mM potassium persulfate and allowing the mixture to stand in a dark at room temperature
for 16 h before use. Different concentrations of the tested compounds (0.1 mL) were added to 1 mL
•
+
ABTS solution. The reaction mixtures were mixed thoroughly, incubated at room temperature for
min, and the absorbance was recorded at 734 nm. The ability to scavenge the ABTS radical cation
was calculated using the following equation:
7
ꢀ
ꢁ
At
%
ABTS radical cation scavenge = 1 − A
× 100
(3)
0
is the absorbance of the tested compound. The ABTS +
•
where A is the absorbance of the control and A
t
0
solution without any sample was used as a control, prepared by using the same procedure. From the
percentage of the scavenging activity at different tested compounds concentrations, IC50 values were
calculated and compared with those of standard L(+)-ascorbic acid solution.

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2
.4.3. Ferric-Reducing Antioxidant Power Assay
The FRAP assay is a typical electron transfer-based method that measures the reduction of
Fe )-ligand complex to the intensely blue-colored ferrous (Fe )-ligand complex by antioxidants [38].
3+
2+
(
The FRAP assay is carried out under acidic pH conditions in order to maintain iron solubility and
more importantly, drive electron transfer. This will increase the redox potential, causing a shift in the
dominant reaction mechanism [39]. The FRAP assay was done according to the method described by
Benzie and Strain [40] with slightly modifications. The stock solutions included 300 mM acetate buffer
(
pH 3.6), 10 mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) solution in ethanol, and 20 mM FeCl solution
3
in 20 mM HCl. To prepare the FRAP working solution, acetate buffer, TPTZ, and FeCl solutions were
3
◦
mixed in this order at a volume ratio of 10:1:1 and then warmed at 37 C before using. An aliquot of
0
0
.1 mL of the tested compound was allowed to react with 3.0 mL of the FRAP working solution and
.3 mL of water. All readings where then taken at 593 nm against a reagent blank at the end of 6 min.
The results are expressed as L(+)-ascorbic acid equivalent (AAE).
2
.4.4. Fe(II)-Chelating Assay
Among the transition metals, Fe2+ ions are well-known for their significant role as a prooxidants
in the lipid oxidation process and the formation of hydroxyl radicals in Fenton-type reactions [41].
However, effective chelating agents can reduce the damage resulting from these reactions. The most
important spectrophotometric method for studying chelation activity is the inhibition of the
2+
2+
Fe –ferrozine complex formation, during which ferrozine reacts quantitatively with Fe ions in
the presence of a chelating agent and complex formation is disrupted. Measurement of color reduction
therefore allows estimation of the metal chelating activity of the coexisting chelator. The chelating
abilities on Fe2 by the tested compounds were estimated by the method as described by Dinis [42].
+
Briefly, 0.4 mL of the tested compound was added to 0.05 mL of FeCl (2 mM) solution. The reaction
2
was initiated by the addition of ferrozine (0.2 mL, 5 mM). The total volume of the systems was adjusted
to 4 mL with ethanol. Then, the mixture was shaken vigorously and left at room temperature for 10 min.
The absorbance of the solution was then measured spectrophotometrically at 562 nm. The percentage
of chelating effect was calculated by using the equation below:
ꢀ
ꢁ
At
%
Chelating effect = 1 − A
× 100
(4)
0
where A is the absorbance of the control and A
t
is the absorbance of the tested compound. The results
0
were expressed as IC50 concentration, where 50% inhibition of the Fe2+–ferrozine complex was obtained
and compared with those of the standard disodium ethylenediaminetetraacetate (Na EDTA) solution.
2
2
.5. Statistical Analysis
All data on AChE inhibition/antioxidant activity are the average of triplicate analyses (n = 3) and
the data were recorded as mean
the dose-response relation and IC50 values were obtained from the equations y = a ± bx.
±
standard deviation. Regression analysis was performed to calculate
3
. Results and Discussion
3
.1. Synthesis
The synthesis of amides via reactive esters is one of the most common synthetic methods used in
the chemicals of natural products and peptides. These methods are usually simple, require minimal
purification, and give high yields [43]. For the synthesis of amide derivatives, a coupling method with
BOP was used (Figure 1) [44].

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Figure 1. Synthesis of amide derivatives. R = H and CH , respectively, for trans-caffeic and
3
ferulic acid. BOP: (benzotriazol-1-yloxa)tris(dimethylamino)phosphonium hexafluorophosphate;
DMF: dimethylformamide; DCM: dichloromethane; Et N: triethylamine.
3
Both synthesized derivatives are characterized by several instrumental methods and the obtained
1
13
results were related to the proposed structures. All corresponding spectra (MS, H and C-NMR are
given in the Supplementary Materials.
3
.1.1. N-trans-Caffeoyldopamine
Yield: 258 mg, 41%; Beige solid; R 0.24 (EtOAc–hexane, 1:1); Melting point 182–184 C;
◦
−1
/cm
ν
f
1
(
(
(
(
(
KBr): 3450–3250 (br, s), 1653 (s), 1600 (s), 1521 (s), 1443 (s), 1372 (m), 1283 (s), 1195 (s), 1113 (s); H-NMR
500 MHz, DMSO-d6) H: 9.34 (s, 1H), 9.11 (s, 1H), 8.75 (s, 1H), 8.64 (s, 1H), 8.01 (t, J = 5.7 Hz, 1H), 7.23
δ
d, J = 15.7 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 6.83 (dd, J = 8.2, 2.1 Hz, 1H), 6.74 (d, J = 8.1 Hz, 1H), 6.64
d, J = 7.9 Hz, 1H), 6.60 (d, J = 2.1 Hz, 1H), 6.46 (dd, J = 8.1, 2.1 Hz, 1H), 6.32 (d, J = 15.7 Hz, 1H), 3.30
q, J = 7.0 Hz, 2H), 2.57 (t, J = 7.5 Hz, 2H); 13C-NMR (125 MHz, DMSO-d6)
δ
C: 165.75, 147.71, 145.99,
1
45.52, 144.00, 139.40, 130.74, 126.89, 120.83, 119.68, 119.08, 116.45, 116.21, 115.96, 114.26, 41.19, 35.23;
+
HRMS (ESI+, m/z): 316.1179 [M + H] (C H NO requires 316.1177).
17
18
5
3
.1.2. N-trans-Feruloyldopamine
◦
Yield: 349 mg, 53%; Pale yellow solid; R 0.32 (EtOAc–hexane, 1:1); Melting point 144–145 C;
f
−
ν
/cm 1 (KBr): 3450–3250 (br, s), 1654 (s), 1594 (s), 1516 (s), 1448 (m), 1374 (m), 1272 (s), 1210 (m),
1
1
160 (m), 1123 (m); H-NMR (500 MHz, DMSO-d )
δ
H: 9.41 (s, 1H), 8.75 (s, 1H), 8.64 (s, 1H), 7.97
6
(t, J = 5.7 Hz, 1H), 7.31 (d, J = 15.6 Hz, 1H), 7.11 (d, J = 2.0 Hz, 1H), 6.98 (dd, J = 8.2, 2.0 Hz, 1H), 6.79
(d, J = 8.1 Hz, 1H), 6.64 (d, J = 7.9 Hz, 1H), 6.60 (d, J = 2.1 Hz, 1H), 6.46 (dd, J = 8.0, 2.1 Hz, 1H), 6.43
(
d, J = 15.7 Hz, 1H), 3.80 (s, 3H), 3.31 (q, J = 6.9 Hz, 2H), 2.58 (t, J = 7.4 Hz, 2H); 13C-NMR (125 MHz,
DMSO-d6)
δ
: 165.74, 148.68, 148.28, 145.52, 143.99, 139.30, 130.71, 126.91, 121.96, 119.67, 119.54,
C
+
1
16.44, 116.11, 115.96, 111.20, 55.98, 41.15, 35.18; HRMS (ESI+, m/z): 330.1336 [M + H] (C H NO
18
20
5
requires 330.1340).
3
.2. Acetylcholinesterase Inhibition
Acetylcholinesterase inhibitors have been widely used in the treatment of neurodegenerative
disease. Both synthesized derivatives and phenolic acids were tested to evaluate their ability to inhibit
AChE from E. electricus. The results are reported in Table 1.
Table 1. In vitro inhibition of acetylcholinesterase (AChE).
Compound
AChE Inhibition IC50 (µM)
trans-Caffeic acid
trans-Ferulic acid
N-trans-Caffeoyldopamine
N-trans-Feruloyldopamine
Galantamine
42.81 ± 1.79
20.57 ± 0.65
19.12 ± 0.83
8.52 ± 0.27
3.89 ± 0.10

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Among the tested compounds, N-trans-feruloyldopamine showed the best ability to inhibit
AChE with IC50 8.52 M, while the weakest inhibitory activity was found in the case of caffeic
µ
acid (IC50 42.81 µM). The reported data clearly indicate that two main features influence the
enzyme inhibition:
•
•
the nature of the R substituent at the aromatic ring, which plays a key role in the inhibitory
potency, and
the presence of the dopamine moiety via the amide-coupled bond.
To support the experimental results obtained by enzymatic inhibition, the interaction between
AChE and tested compounds was also predicted by molecular docking using SwissDock (freely
available on Swiss Institute of Bioinformatics Website) [29]. The 3D crystal structure of AChE was
obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank
(
PDB ID: 1C2B) [31]. The protein preparation and the molecular docking results visualization were
carried out using the UCSF Chimera 1.12 package. The docking results predicted that the lowest
binding energy ( G) was equal to 34.12 kJ/mol for N-trans-caffeoyldopamine and 36.13 kJ/mol for
N-trans-feruloyldopamine. The more negative value of G indicated good binding affinity of the target
molecule with the ligand and was correlated with the number of hydrogen bonds in the resulting
cluster. In our case, the estimated G corresponded to the interaction with the peripheral anionic,
∆
−
−
∆
∆
esteratic subunit (catalytic triad), and anionic subsite (Figure 2).
For N-trans-caffeoyldopamine, the predicting binding positions included a hydrogen bond
341
between the m-phenolic group from the caffeoyl moiety and the O-Tyr (1.835 Å) and a hydrogen
bond between the H-Ser2 with the m-phenolic group (2.574 Å) on the dopamine moiety. In the
lowest binding energy for N-trans-feruloyldopamine, a total of four hydrogen bonds were predicted,
03
as follows: H-Gln3 and the carbonyl group (2.508 Å), O-Glu via the m- and p-phenolic group
from the dopamine moiety (1.805 and 1.833 Å), and H-Asn2 and the p-phenolic group from the
dopamine moiety (1.939 Å). Also, in our case, a great correlation was obtained between experimentally
69
313
33
obtained values of IC50 and the estimated
∆G for the binding positions. Most of the predicted binding
sites were already proven places of excellent inhibition with donepezil, huperzine A, tacrine and its
hybrids (Gln3 , Glu , and Asn ) [45
,46], organophosphorus compounds (Ser ) [47], coumarins,
69
313
233
203
341
and phenolic acids (Tyr ) [2].
Figure 2. The complex target–ligand as viewed in UCSF Chimera 1.12. Possible interaction of AChE
from E. electricus with: (a) N-trans-caffeoyldopamine; (b) N-trans-feruloyldopamine.
3
.3. Antioxidant Activity
Phenolic acids and other phenolic compounds belong to natural compounds with potent
antioxidant activity [6]. This activity is the consequence of their redox properties that allow them to
scavenge free radicals, decompose peroxides, reduce transition metal ions, and stop chain reactions.
This study investigated antioxidant potential based on the ability to scavenge non-biological
•
•+
stable free radicals (DPPH and ABTS ) and reduce ferric and chelate ferrous ions. By the mechanism

Sci. Pharm. 2018, 86, 11
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•
•+
of antioxidant action, DPPH , ABTS , and FRAP assays belong to the reduction tests, which result
in the transfer of hydrogen atom and/or electron from the antioxidant molecule [38]. The results
obtained by these methods are presented in the Table 2.
Table 2. Antioxidant activity of tested compounds.
•
•+
DPPH Scavenge IC
ABTS Scavenge IC50
FRAP AAE
(µmol/mmol)
5
0
Compound
(
µM)
(µM)
trans-Caffeic acid
trans-Ferulic acid
N-trans-Caffeoyldopamine
N-trans-Feruloyldopamine
L(+)-Ascorbic acid
•
18.86 ± 0.22
19.93 ± 0.18
5.95 ± 0.12
12.29 ± 0.04
1.14 ± 0.03
1.19 ± 0.02
1.62 ± 0.01
0.24 ± 0.00
0.74 ± 0.00
0.11 ± 0.00
526.05 ± 12.87
486.80 ± 11.75
822.45 ± 13.53
661.53 ± 13.51
-
0
DPPH :
2,2-diphenyl-1-pycrylhydrazyl; ABTS: 2,2 -azinobis(3-ethylbenzothiazoline-6-sulphonic acid);
FRAP: ferric-reducing antioxidant power.
The antioxidant activity of tested compounds, against non-biological free radicals and ferric ion
reduction, decreased in the following order: N-trans-caffeoyldopamine > N-trans-feruloyldopamine >
trans-caffeic acid > trans-ferulic acid. This sequence indicates that the antioxidant activity of the test
compounds is due to their hydrogen-donating ability and the number of phenolic groups. For these
assays, the correlation of the number of phenolic groups and the resulting antioxidant activity was
2
good (r > 0.932). The second explanation lies in the fact of proven excellent antioxidative activity of
phenolic compounds with catechol arrangement (as in the case of caffeic acid and dopamine) through
the stabilization of the semiquinone radical by intramolecular hydrogen bonds [48,49]. An effect that
also works in favor of this is that the electron donor o-hydroxyl group on the aromatic ring reduces
O–H bond dissociation enthalpy and thus improves antiradical activity [50]. The o-methoxy group has
a similar effect, but not to the same extent, which is a possible explanation for why feruloyl analogs
show lower activity than the corresponding caffeoyl. In addition, the acid proton appears to have little
impact on the antioxidant activity, while the allylic group enhances the resonance stability of phenoxyl
radical and improves the antioxidant activity [51].
The direct reaction of antioxidants with free radicals is not the only mechanism by which the free
radical species are inactivated. There is another mechanism, by which antioxidants do not convert
free radicals into less reactive species, but rather slow down the rate of the oxidation reaction through
several mechanisms [52]. The most important mechanism of secondary antioxidants is the chelation
of pro-active metal ions [53]. Iron, copper, and other transition metals promote oxidation reactions
by catalyzing free radical reactions. These redox active metals carry out the transfer of one electron
by changing the oxidation state. By chelation, their pro-oxidative effect decreases by reducing redox
potential and stabilizing their oxidation state. These chelating compounds can also sterically hinder the
formation of hydroperoxide metal complexes. This effect was investigated by the method of inhibiting
the formation of the purple complex between ferrous ions and ferrozine. The obtained results are
presented in Figure 3.
Figure 3. Chelation ability of tested compounds.

Sci. Pharm. 2018, 86, 11
9 of 12
The iron binding ability of the chelators could be related to the type of functional group used
in iron chelation [54]. As both N-trans-caffeoyldopamine (3.48 M) and N-trans-feruloyldopamine
3.17 M) have lower IC50 values than their parent molecules, it has been shown that an additional
µ
(
µ
catechol group via dopamine or the presence of the amide group increases the ability to chelate
ferrous ions. But the results also suggest a low activity of all investigated compounds compared to the
2+
hexadentate Na EDTA, which was used as a positive control. Because Fe behaves as a borderline
2
Lewis acid, it does not bind as strongly to the hard oxygen atoms of phenol ligands and the low activity
of the test compounds can be attributed to the low stability of the complexes and their oxidation in the
presence of O [55].
2
4
. Conclusions
The results of this study indicate that N-trans-caffeoyldopamine and N-trans-feruloyldopamine
fulfill the requirements to be considered potent antioxidants because they not only inhibit the
radical-mediated process through reactions involving hydrogen atom and electron transfer, but also
through chelating transition metals. Moreover, introducing the dopamine moiety in hydroxycinnamic
acids resulted in a significant increase in AChE inhibition, which suggesting these compounds could
be used as potential active ingredients of some medicines.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-0532/86/2/11/s1.
Figure S1: MS spectra of N-trans-caffeoyldopamine, Figure S2: MS spectra of N-trans-feruloyldopamine, Figure S3:
1
1
1
H-NMR spectra of N-trans-caffeoyldopamine, Figure S4: H-NMR spectra of N-trans-feruloyldopamine, Figure S5:
3
13
C-NMR spectra of N-trans-caffeoyldopamine, Figure S6: C-NMR spectra of N-trans-feruloyldopamine.
Acknowledgments: This work was supported by the Federal Ministry of Education and Science of Bosnia and
Herzegovina (Grant: 0101-7552-20/15).
Author Contributions: M.D. and M.M. conceived and designed the experiments. M.D. and D.V. performed the
1
13
synthesis and tested the inhibition of AChE and antioxidant activity. Mass analysis, H, and C-NMR were
recorded by F.P. and B.Š., while all authors have participated in writing the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Chen, Y.; Xiao, H.; Zheng, J.; Liang, G. Structure-thermodynamics-antioxidant activity relationships of
selected natural phenolic acids and derivatives: An experimental and theoretical evaluation. PLoS ONE
2
015, 10, e0121276. [CrossRef] [PubMed]
Szwajgier, D. Anticholinesterase activity of phenolic acids and their derivatives. Z. Naturforsch. C 2013, 68,
25–132. [CrossRef] [PubMed]
2
.
.
1
3
Sanchez-Maldonado, A.F.; Schieber, A.; Ganzle, M.G. Structure-function relationships of the antibacterial
activity of phenolic acids and their metabolism by lactic acid bacteria. J. Appl. Microbiol. 2011, 111, 1176–1184.
[CrossRef] [PubMed]
4
.
Taofiq, O.; Calhelha, R.C.; Heleno, S.; Barros, L.; Martins, A.; Santos-Buelga, C.; Queiroz, M.; Ferreira, I.
The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic
extracts, individual parent molecules and synthesized glucuronated and methylated derivatives. Food Res.
Int. 2015, 76, 821–827. [CrossRef] [PubMed]
5
6
.
.
Graf, E. Antioxidant potential of ferulic acid. Free Radic. Biol. Med. 1992, 13, 435–448. [CrossRef]
Piazzon, A.; Vrhovsek, U.; Masuero, D.; Mattivi, F.; Mandoj, F.; Nardini, M. Antioxidant activity of phenolic
acids and their metabolites: Synthesis and antioxidant properties of the sulfate derivatives of ferulic and
caffeic acids and of the acyl glucuronide of ferulic acid. J. Agric. Food Chem. 2012, 60, 12312–12323. [CrossRef]
[PubMed]
7
.
Chavez, J.H.; Leal, P.C.; Yunes, R.A.; Nunes, R.J.; Barardi, C.R.; Pinto, A.R.; Simoes, C.M.; Zanetti, C.R.
Evaluation of antiviral activity of phenolic compounds and derivatives against rabies virus. Vet. Microbiol.
2
006, 116, 53–59. [CrossRef] [PubMed]

Sci. Pharm. 2018, 86, 11
10 of 12
8
.
Kilani-Jaziri, S.; Mokdad-Bzeouich, I.; Krifa, M.; Nasr, N.; Ghedira, K.; Chekir-Ghedira, L.
Immunomodulatory and cellular anti-oxidant activities of caffeic, ferulic, and p-coumaric phenolic acids: A
structure–activity relationship study. Drug Chem. Toxicol. 2017, 40, 416–424. [CrossRef] [PubMed]
da Cunha, F.M.; Duma, D.; Assreuy, J.; Buzzi, F.C.; Niero, R.; Campos, M.M.; Calixto, J.B. Caffeic acid derivatives:
In vitro and in vivo anti-inflammatory properties. Free Radic. Res. 2004, 38, 1241–1253. [CrossRef] [PubMed]
9
1
.
0. Fiuza, S.M.; Gomes, C.; Teixeira, L.J.; Girao da Cruz, M.T.; Cordeiro, M.N.; Milhazes, N.; Borges, F.; Marques, M.P.
Phenolic acid derivatives with potential anticancer properties-a structure-activity relationship study. Part 1:
Methyl, propyl and octyl esters of caffeic and gallic acids. Bioorg. Med. Chem. 2004, 12, 3581–3589. [CrossRef]
[PubMed]
1
1
1
1
1. Pedersen, H.A.; Steffensen, S.K.; Christophersen, C.; Mortensen, A.G.; Jorgensen, L.N.; Niveyro, S.; de
Troiani, R.M.; Rodriguez-Enriquez, R.J.; Barba-de la Rosa, A.P.; Fomsgaard, I.S. Synthesis and quantitation of
six phenolic amides in Amaranthus spp. J. Agric. Food Chem. 2010, 58, 6306–6311. [CrossRef] [PubMed]
2. Park, J.B.; Schoene, N. Clovamide-type phenylpropenoic acid amides, N-coumaroyldopamine and
N-caffeoyldopamine, inhibit platelet-leukocyte interactions via suppressing P-selectin expression. J. Pharmacol.
Exp. Ther. 2006, 317, 813–819. [CrossRef] [PubMed]
3. Shimada, C.; Uesawa, Y.; Ishihara, M.; Kagaya, H.; Kanamoto, T.; Terakubo, S.; Nakashima, H.; Takao, K.;
Saito, T.; Sugita, Y.; Sakagami, H. Quantitative structure–cytotoxicity relationship of phenylpropanoid
amides. Anticancer Res. 2014, 34, 3543–3548. [PubMed]
4. Wu, Z.R.; Bai, Z.T.; Sun, Y.; Chen, P.; Yang, Z.G.; Zhi, D.J.; Li, Y.; Wang, X.; Du, J.J.; Yang, R.; Cui, P.; Zhang, Y.;
Li, H.Y. Protective effects of the bioactive natural product N-trans-Caffeoyldopamine on hepatotoxicity
induced by isoniazid and rifampicin. Bioorg. Med. Chem. Lett. 2015, 25, 5424–5426. [CrossRef] [PubMed]
5. Park, J.B. N-coumaroyldopamine and N-caffeoyldopamine increase cAMP via beta 2-adrenoceptors in
myelocytic U937 cells. FASEB J. 2005, 19, 497–502. [CrossRef] [PubMed]
1
1
6. Locatelli, M.; Travaglia, F.; Giovannelli, L.; Coïsson, J.D.; Bordiga, M.; Pattarino, F.; Arlorio, M. Clovamide
and phenolics from cocoa beans (Theobroma cacao L.) inhibit lipid peroxidation in liposomal systems. Food Res.
Int. 2013, 50, 129–134. [CrossRef]
1
7. Zhou, Y.; Wang, S.; Zhang, Y. Catalytic reaction mechanism of acetylcholinesterase determined by
Born-Oppenheimer ab initio QM/MM molecular dynamics simulations. J. Phys. Chem. B 2010, 114, 8817–8825.
[CrossRef] [PubMed]
1
1
2
2
8. Wiesner, J.; Kriz, Z.; Kuca, K.; Jun, D.; Koca, J. Acetylcholinesterases—The structural similarities and
differences. J. Enzyme Inhib. Med. Chem. 2007, 22, 417–424. [CrossRef] [PubMed]
9. Retz, W.; Gsell, W.; Munch, G.; Rosler, M.; Riederer, P. Free radicals in Alzheimer’s disease. J. Neural Transm.
Suppl. 1998, 54, 221–236. [PubMed]
0. Konrath, E.L.; Passos Cdos, S.; Klein, L.C., Jr.; Henriques, A.T. Alkaloids as a source of potential anticholinesterase
inhibitors for the treatment of Alzheimer’s disease. J. Pharm. Pharmacol. 2013, 65, 1701–1725. [CrossRef] [PubMed]
1. Pinho, B.R.; Ferreres, F.; Valentao, P.; Andrade, P.B. Nature as a source of metabolites with cholinesterase-
inhibitory activity: An approach to Alzheimer’s disease treatment. J. Pharm. Pharmacol. 2013, 65, 1681–1700.
[CrossRef] [PubMed]
2
2
2
2
2
2. Miyazawa, M.; Watanabe, H.; Kameoka, H. Inhibition of acetylcholinesterase activity by monoterpenoids
with a p-menthane skeleton. J. Agric. Food Chem. 1997, 45, 677–679. [CrossRef]
3. Cadenas, E. Mitochondrial free radical production and cell signaling. Mol. Aspects Med. 2004, 25, 17–26.
[CrossRef] [PubMed]
4. Phaniendra, A.; Jestadi, D.B.; Periyasamy, L. Free radicals: Properties, sources, targets, and their implication
in various diseases. Indian J. Clin. Biochem. 2015, 30, 11–26. [CrossRef] [PubMed]
5. Zhang, H.; Davies, K.J.A.; Forman, H.J. Oxidative stress response and Nrf2 signaling in aging. Free Radic.
Biol. Med. 2015, 88, 314–336. [CrossRef] [PubMed]
6. Carocho, M.; Ferreira, I.C. A review on antioxidants, prooxidants and related controversy: Natural and
synthetic compounds, screening and analysis methodologies and future perspectives. Food Chem. Toxicol.
2
013, 51, 15–25. [CrossRef] [PubMed]
2
7. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Feather-Stone, R.M. A new and rapid colorimetric determination
of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [CrossRef]
2
8. Armarego, W.L.F.; Chai, C. Chapter 4–Purification of organic chemicals. In Purification of Laboratory Chemicals;
Butterworth-Heinemann: Boston, MA, USA, 2013; pp. 103–554, ISBN 978-0-12-382161-4.

Sci. Pharm. 2018, 86, 11
11 of 12
2
3
3
3
9. Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a protein-small molecule docking web service based on
EADock DSS. Nucleic Acids Res. 2011, 39, 270–277. [CrossRef] [PubMed]
0. Grosdidier, A.; Zoete, V.; Michielin, O. Fast docking using the CHARMM force field with EADock DSS.
J. Comput. Chem. 2011, 32, 2149–2159. [CrossRef] [PubMed]
1. Bourne, Y.; Grassi, J.; Bougis, P.E.; Marchot, P. Conformational flexibility of the acetylcholinesterase tetramer
suggested by x-ray crystallography. J. Biol. Chem. 1999, 274, 30370–30376. [CrossRef] [PubMed]
2. Pettersen, E.F.; Goddard, T.D.; Huang, C.C.; Couch, G.S.; Greenblatt, D.M.; Meng, E.C.; Ferrin, T.E. UCSF
Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25, 1605–1612.
[CrossRef] [PubMed]
3
3
3
3. Xie, J.; Schaich, K.M. Re-evaluation of the 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay for
antioxidant activity. J. Agric. Food Chem. 2014, 62, 4251–4260. [CrossRef] [PubMed]
4. Kedare, S.B.; Singh, R.P. Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol.
2
011, 48, 412–422. [CrossRef] [PubMed]
5. Yang, H.L.; Cai, P.; Liu, Q.H.; Yang, X.L.; Fang, S.Q.; Tang, Y.W.; Wang, C.; Wang, X.B.; Kong, L.Y. Design,
synthesis, and evaluation of salicyladimine derivatives as multitarget-directed ligands against Alzheimer’s
disease. Bioorg. Med. Chem. 2017, 25, 5917–5928. [CrossRef] [PubMed]
3
3
3
6. Tomasina, F.; Carabio, C.; Celano, L.; Thomson, L. Analysis of two methods to evaluate antioxidants. Biochem.
Mol. Biol. Educ. 2012, 40, 266–270. [CrossRef] [PubMed]
7. Miller, N.J.; Rice-Evans, C.A. Spectrophotometric determination of antioxidant activity. Redox Rep. 1996, 2,
1
61–171. [CrossRef] [PubMed]
8. Apak, R.; Ozyurek, M.; Guclu, K.; Capanoglu, E. Antioxidant activity/capacity measurement. 1.
Classification, physicochemical principles, mechanisms, and electron transfer (ET)-based assays. J. Agric.
Food Chem. 2016, 64, 997–1027. [CrossRef] [PubMed]
3
9. Hagerman, A.E.; Riedl, K.M.; Jones, G.A.; Sovik, K.N.; Ritchard, N.T.; Hartzfeld, P.W.; Riechel, T.L. High
molecular weight plant polyphenolics (tannins) as biological antioxidants. J. Agric. Food Chem. 1998, 46,
1
887–1892. [CrossRef] [PubMed]
4
4
4
0. Benzie, I.F.; Strain, J.J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”:
The FRAP assay. Anal. Biochem. 1996, 239, 70–76. [CrossRef] [PubMed]
1. Enami, S.; Sakamoto, Y.; Colussi, A.J. Fenton chemistry at aqueous interfaces. Proc. Natl. Acad. Sci. USA
2
014, 111, 623–628. [CrossRef] [PubMed]
2. Dinis, T.C.; Maderia, V.M.; Almeida, L.M. Action of phenolic derivatives (acetaminophen, salicylate,
and 5-aminosalicylate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers.
Arch. Biochem. Biophys. 1994, 315, 161–169. [CrossRef] [PubMed]
4
3. El-Faham, A.; Albericio, F. Peptide coupling reagents, more than a letter soup. Chem. Rev. 2011, 111, 6557–6602.
[
CrossRef] [PubMed]
0
4
4. Castro, B.; Dormoy, J.R.; Evin, G.; Selve, C. Reactifs de couplage peptidique I (1) - l hexafluorophosphate
de benzotriazolyl N-oxytrisdimethylamino phosphonium (B.O.P.). Tetrahedron Lett. 1975, 16, 1219–1222.
[CrossRef]
4
4
5. Chierrito, T.P.C.; Pedersoli-Mantoani, S.; Roca, C.; Requena, C.; Sebastian-Perez, V.; Castillo, W.O.;
Moreira, N.C.S.; Perez, C.; Sakamoto-Hojo, E.T.; Takahashi, C.S.; et al. From dual binding site
acetylcholinesterase inhibitors to allosteric modulators: A new avenue for disease-modifying drugs in
Alzheimer’s disease. Eur. J. Med. Chem. 2017, 139, 773–791. [CrossRef] [PubMed]
6. Sheeja Malar, D.; Beema Shafreen, R.; Karutha Pandian, S.; Pandima Devi, K. Cholinesterase inhibitory,
anti-amyloidogenic and neuroprotective effect of the medicinal plant Grewia tiliaefolia—An in vitro and in
silico study. Pharm. Biol. 2017, 55, 381–393. [CrossRef] [PubMed]
4
7. Rosenfeld, C.A.; Sultatos, L.G. Concentration-dependent kinetics of acetylcholinesterase inhibition by the
organophosphate paraoxon. Toxicol. Sci. 2006, 90, 460–469. [CrossRef] [PubMed]
4
8. Farhoosh, R.; Johnny, S.; Asnaashari, M.; Molaahmadibahraseman, N.; Sharif, A. Structure-antioxidant
activity relationships of o-hydroxyl, o-methoxy, and alkyl ester derivatives of p-hydroxybenzoic acid.
Food Chem. 2016, 194, 128–134. [CrossRef] [PubMed]
4
9. Brewer, M.S. Natural antioxidants: Sources, compounds, mechanisms of mction, and potential applications.
Compr. Rev. Food Sci. Food Saf. 2011, 10, 221–247. [CrossRef]

Sci. Pharm. 2018, 86, 11
12 of 12
5
0. Guitard, R.; Nardello-Rataj, V.; Aubry, J.M. Theoretical and kinetic tools for selecting effective antioxidants:
Application to the protection of omega-3 oils with natural and synthetic phenols. Int. J. Mol. Sci. 2016, 17,
220. [CrossRef] [PubMed]
1
5
5
5
5
5
1. Rice-Evans, C.A.; Miller, N.J.; Paganga, G. Structure-antioxidant activity relationships of flavonoids and
phenolic acids. Free Radic. Biol. Med. 1996, 20, 933–956. [CrossRef]
2. Koncic, M.Z.; Barbaric, M.; Perkovic, I.; Zorc, B. Antiradical, chelating and antioxidant activities of
hydroxamic acids and hydroxyureas. Molecules 2011, 16, 6232–6242. [CrossRef] [PubMed]
3. Galano, A.; Mazzone, G.; Alvarez-Diduk, R.; Marino, T.; Alvarez-Idaboy, J.R.; Russo, N. Food antioxidants:
Chemical insights at the molecular level. Annu. Rev. Food. Sci. Technol. 2016, 7, 335–352. [CrossRef] [PubMed]
4. Adjimani, J.P.; Asare, P. Antioxidant and free radical scavenging activity of iron chelators. Toxicol. Rep. 2015
,
2
, 721–728. [CrossRef] [PubMed]
5. Perron, N.R.; Brumaghim, J.L. A review of the antioxidant mechanisms of polyphenol compounds related to
iron binding. Cell Biochem. Biophys. 2009, 53, 75–100. [CrossRef] [PubMed]
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