Synthesis of Some Novel 2-Substitutedbenzyl-(4)7-phenyl-1H-benzo[d]imidazoles in Mild Conditions as Potent Anti-Tyrosinase and Antioxidant Agents

Article abstract of DOI:10.1002/ardp.201600224

Novel 2-substitutedbenzyl-4(7)-phenyl-1H-benzo[d]imidazole compounds were synthesized and characterized. Although 2a and 2b were reported previously in the literature, 11 compounds were synthesized (nine of them were newly synthesized) and the tyrosinase inhibitory effects and antioxidant activities of these compounds were studied for the first time. All of the synthesized compounds displayed certain inhibitory effects on tyrosinase, with IC₅₀ values ranging from 37.86 ± 0.24 to 75.81 ± 2.49 μM. Among the compounds, 2j exhibited similar tyrosinase inhibitory effect (IC₅₀ = 37.86 ± 0.24 μM) to the positive control, kojic acid (IC₅₀ = 21.93 ± 0.11 μM). Kinetic studies revealed it to act as non-competitive tyrosinase inhibitor with a K_i value of 50.2 μM. The antioxidant activities of the compounds were investigated by using in vitro antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). All of these results indicated that the compounds might have potential application as tyrosinase inhibitors.

Full text of DOI:10.1002/ardp.201600224

RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–8
Archiv der Pharmazie
Full Paper
Synthesis of Some Novel 2-Substitutedbenzyl-(4)7-phenyl-1H-
benzo[d]imidazoles in Mild Conditions as Potent Anti-Tyrosinase
and Antioxidant Agents
1
2
1
2
1
3
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Inci S. Dogan , Arzu Ozel , Zeynep Birinci , Burak Barut , Hasan E. Sellitepe , and Bahittin Kahveci
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Karadeniz Technical University,
Trabzon, Turkey
2
Faculty of Pharmacy, Department of Biochemistry, Karadeniz Technical University, Trabzon, Turkey
Faculty of Health Sciences, Department of Nutrition and Dietetics, Karadeniz Technical University,
Trabzon, Turkey
3
Novel 2-substitutedbenzyl-4(7)-phenyl-1H-benzo[d]imidazole compounds were synthesized and char-
acterized. Although 2a and 2b were reported previously in the literature, 11 compounds were
synthesized (nine of them were newly synthesized) and the tyrosinase inhibitory effects and
antioxidant activities of these compounds were studied for the first time. All of the synthesized
compounds displayed certain inhibitory effects on tyrosinase, with IC50 values ranging from
37.86 ꢁ 0.24 to 75.81 ꢁ 2.49 mM. Among the compounds, 2j exhibited similar tyrosinase inhibitory
effect (IC50 ¼ 37.86 ꢁ 0.24 mM) to the positive control, kojic acid (IC50 ¼ 21.93 ꢁ 0.11 mM). Kinetic studies
revealed it to act as non-competitive tyrosinase inhibitor with a Ki value of 50.2 mM. The antioxidant
activities of the compounds were investigated by using in vitro antioxidant assays, including 2,2-
diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). All of these results
indicated that the compounds might have potential application as tyrosinase inhibitors.
Keywords: Assay / Inhibitors / Substituent effect / Synthesis
Received: August 2, 2016; Revised: September 11, 2016; Accepted: September 14, 2016
DOI 10.1002/ardp.201600224
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
melanin pigments which can cause to hyperpigmentation
such as melasma freckles, seborrheic, melanoma etc., and
Introduction
Tyrosinase (E.C.1.14.18.1) is a copper-containing multifunc-
tional enzyme and universally distributed in bacteria,
mammals, fungi, and plants [1]. Tyrosinase can catalyze both
the hydroxylation of monophenols to o-diphenols such as
L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and
oxidation of o-diphenols to o-quinone such as L-DOPA to
dopaquinone [2]. This enzyme has crucial role in formation of
decrease the nutritional values of the fruits and vegetables
due to browning problems [3–5]. Also, tyrosinase, associated
with Parkinson’s disease because of responsible for the
neurodegeneration, increases in dopamine level, inducing
high production of melanine and triggers cell death in the
brain [6, 7]. Thus tyrosinase inhibition is a current method to
prevent these problems. Tyrosinase inhibitors are used for
clinical treatments such as kojic acid, arbutin, azeleic acid, and
hydroquinone but they have lower thearapeutic effect,
mutagenicity, and cytotoxicity potential [8, 9]. Therefore, it
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Correspondence: Dr. Inci Selin Dogan, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Karadeniz Technical
University, Trabzon 61080, Turkey.
E-mail: isdogan@ktu.edu.tr
Fax: þ90-462-3256717
ꢀAdditional correspondence: Prof. Bahittin Kahveci,
E-mail: bahittin@ktu.edu.tr
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is important to find a new tyrosinase inhibitor without any
side effects.
.HCl
HN
Diethyl ether
0–5 oC
R
CN
C2H5OH
HCl (g)
OC2H5
Benzimidazoles are among the most important heterocyclic
compounds used as drugs [10]. Since benzimidazole was first
synthesized by Hobrecker, it has been a major area of
interest [11]. The reason for this interest, the compounds
bearing benzimidazole ring were obtained biologically active
molecules. These pharmacological activities are antimicro-
bial [12], antiproliferative [13], antibacterial [14], receptor
antagonists [15], antitumor activity [16], cytotoxic activity [17],
antihelmintic resistance [18], sirtuin inhibitor activity [19],
anti-MRSA and anti-VRE agonists [20], anticancer [21], anti-
tubercular activity [22], hepatitis C virus inhibitor [23],
antidiabetic [24], antihypertensive [25], and lipase inhibition
activity [26].
R
1a–k
R
NH2
N
H2N
1a–k
HN
Methanol
2a–k
Scheme 1. Synthesis of 2-substitutedbenzyl-4(7)-phenyl-1H-
benzo[d]imidazole derivatives (2a–k). Reaction conditions:
a: methyl; b: phenyl; c: benzyl; d: ethyl; e: 4-methylbenzyl;
f: 4-chlorobenzyl; g: 2-chlorobenzyl; h: 3-chlorobenzyl; i:
2-methylbenzyl; j: 3-methylbenzyl; k: 3-chloropropyl.
In this study, we synthesized a novel series of 2-substitu-
tedbenzyl-4(7)-phenyl-1H-benzo[d]imidazole derivative com-
pounds, and tyrosinase inhibitory properties and antioxidant
activities of these synthesized compounds were investigated
first time.
band from 3300 up to 2800 cm. This band suggests that the
–NH group makes strong intermolecular hydrogen bonds. C–H
vibration band often cannot be distinguished from the –NH
band because they are within this same range [35]. In the
synthesized compounds, these bands were observed in the
3166–3025 cmꢂ1. In the 1H NMR spectra of the products,
methylene bridges in the compounds were seen in the range
of 4.15–4.42 ppm, the phenyl ring protons were observed in
the range of 7–8 ppm. The signals of N1-H protons on the
benzimidazole ring appeared as a singlet at about 12.31–
12.41 ppm except products 2c–e, 2g–i. In product 2c–e, 2g–i
(because of resonances of N1-H proton) were seen as a
doublet due to the 1,3-tautomerism of the benzimidazole
ring. The structures of the synthesized compounds were also
proved with 13C NMR spectra. And signals were found to
comply with the literature data [34, 35] as 13C signals in
aromatic field were observed at 110.78–157.84 ppm. And also,
signals of aliphatic methylene chains had disappeared
as expected. The findings obtained from the mass spectra
of compounds confirmed the structures of the compounds.
LC-mass spectra of compounds 2a–k showed the molecular ion
[M]þ peaks of different intensity which confirmed their
molecular weights. For phenyl ring bearing the chlorine atom
on the compound (2f–h, 2k), [Mþ2]þ peaks were observed.
The intensity of the peak [Mþ2]þ was 1/3 of [Mþ] peak.
Results and discussion
Chemistry
In this study, reaction of [1,10-biphenyl]-2,3-diamine com-
pound with iminoester hydrochloride derivatives (1a–k) was
investigated. As a result of this reaction, 2-substitutedbenzyl-
4(7)-phenyl-1H-benzo[d]imidazole compounds (2a–k) were
obtained. The two compounds that carry a methyl and a
phenyl group (2a and 2b) were reported previously in the
literature [27, 28]; herein, these two compounds were
synthesized via
a different method and the tyrosinase
inhibition properties and antioxidant activities of all com-
pounds were investigated.
Several methods for the synthesis of benzimidazole are
used in the literatures, among the most preferred is the
cyclization reaction between 1,2-phenylenediamine and
carboxyl derivatives [29–31]. We tried a conventional method,
using the [1,10-biphenyl]-2,3-diamine with iminoester hydro-
chloride in methanol in mild conditions.
The iminoester hydrochloride derivatives (1a–k) were
synthesized by the Pinner method. [1,10-Biphenyl]-2,3-di-
amine as the starting compound was purchased. These
obtained iminoester hydrochloride derivatives (1a–k) and
[1,10-biphenyl]-2,3-diamine compounds reacted in methanol
under suitable conditions and our target compounds (2a–k)
were synthesized (Scheme 1).
Pharmacology
Inhibition of tyrosinase
The structures of the novel compounds were confirmed by
using IR, 1H NMR, 13C NMR spectra, and mass analysis. Spectral
(IR, 1H NMR, 13C NMR) data of the synthesized compounds
(2a–k) were consistent with the literature [32–34]. The IR
spectra of the products are characterized by the presence of
absorption bands at 2936–2730 and 1588–1441 cmꢂ1 corre-
In this study, we investigated the tyrosinase inhibitory effects
of compounds 2a–k. The tyrosinase inhibitor effectiveness of
compounds 2a–k is shown in Table 1. The lower IC50 value
showed the higher inhibition of tyrosinase. Our results
showed that all the synthesized compounds exhibited certain
inhibition effects on mushroom tyrosinase with IC50 values
ranging from 37.86 ꢁ 0.24 to 75.81 ꢁ 2.49 mM. Especially,
compounds 2f, 2g, 2j showed more potent inhibitory effects
–
–
sponding to the aliphatic C–H, C C, and C N functions in the
–
–
structure. IR spectra of benzimidazole give a broad and strong
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Table 1. Inhibitory effect and kinetic analysis of kojic acid and compounds (2a–k) on mushroom tyrosinase activity.
Inhibitors
IC50 (mM)
Type of inhibition
Ki (mM)
2a
2b
2c
2d
2e
2f
2g
2h
55.44 ꢁ 0.82
55.61 ꢁ 0.52
51.49 ꢁ 0.47
52.92 ꢁ 1.37
62.11 ꢁ 1.42
50.72 ꢁ 0.68
59.13 ꢁ 1.51
44.82 ꢁ 0.52
75.81 ꢁ 2.49
37.86 ꢁ 0.24
58.54 ꢁ 1.28
21.93 ꢁ 0.11
Non-competitive
54.0
55.8
60.3
34.0
23.2
22.7
64.0
67.2
49.8
50.2
26.0
10.2
Non-competitive
Non-competitive
Non-competitive
Mixed-type
Non-competitive
Non-competitive
Non-competitive
Mixed-type
2i
2j
2k
Non-competitive
Mixed-type
Mixed-type
Kojic acid
than most of the other compounds. In addition, compound 2j
demonstrated similar inhibitory effect to the reference
standard inhibitor kojic acid and exhibited the most potent
tyrosinase inhibition with IC50 value of 37.86 ꢁ 0.24 mM. These
results show that substituted benzyl groups at position of
benzimidazole skeleton decreased tyrosinase activity. Tyrosi-
nase inhibition is complex and it is the interaction of several
substituents and the structural framework that leads to
effective binding at the enzyme causing inhibition of enzyme
function [36]. According to these results, the inhibitory
activities of substituted benzyl derivatives compounds 2e–j
showed the most effective inhibition at third position and the
least inhibition at second position, the inhibitory activities
also increased gradually. Second position might cause steric
hindrances and decrease the binding affinity. Also, among
compounds 2e–j, chloride substituted benzyl derivatives
were more effective inhibitors than methyl substituted benzyl
derivatives. According to the inhibitory activities of com-
pounds 2e–j, electron acceptor groups were more effective
than electron donor groups. Karatas et al. [37] have reported
the inhibitory effect of some benzimidazole derivatives
on tyrosinase activity and among the compounds studied,
1,4-di(1H-benzo[d]imidazol-1-yl)butane was found to be the
most active tyrosinase inhibitor (IC50 ¼ 310 mM).
(Table 1). The inhibition of mushroom tyrosinase by com-
pounds 2f, 2h, and 2k are illustrated in Fig. 1, representatively.
The inhibition kinetics was illustrated by Dixon plots, which
were obtained by plotting 1/V versus [I] with varying
concentrations of L-DOPA. Dixon plots gave families of lines
passing through the same point at the second quadrant,
giving the inhibition constant (Ki) (Fig. 2). The Ki values
estimated from these Dixon plots were 22.7, 67.2, and 26.0 mM
for the compounds 2f, 2h, and 2k, respectively. A comparison
of the Ki values of the compounds showed that especially
compounds 2f and 2k had the highest tyrosinase binding
affinity among the other compounds but these compounds
were found to have twofold lower binding affinity than kojic
acid (10.2 mM) as the reference drug.
Antioxidant activities
It is well-known that tyrosinase inhibitors possess antioxidant
activity. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) accepts hydro-
gen atoms or an electron to be a diamagnetic molecule [40].
DPPH assay is the most simple, rapid, and low costly method
among the antioxidant assay, that is why it is a well-known
scavenger method [41, 42]. DPPH gives a strong absorption
band at 517nm because of an odd electron and this method is
based on reduction of absorbance. The SC50 values for DPPH
radical scavenging activities of the compounds are summarized
in Table 2. The radical scavenging activities of synthesized
compounds are displayed, with SC50 values ranging from
0.504ꢁ 0.009 to 0.549ꢁ 0.014mM. In this study, compound 2j
showed the lowest SC50 values with 0.504ꢁ 0.009mM followed
by compound 2h (0.508ꢁ 0.009 mM) and compound 2g
(0.519 ꢁ 0.016mM), respectively. These results claimed that,
these compounds showed low scavenging activities compared
to the standard gallic acid.
The ferric reducing antioxidant power (FRAP) is one of the
antioxidant mechanisms which are used to examine reducing
power. In this method, synthesized compounds ability to
reduce Fe3þ to Fe2þ is investigated using butylated hydrox-
yanisole (BHA) as standard compound which is measured at
700 nm [43]. The results are presented in Table 2. Ferric
reducing ability of compounds has the following order (mM
Kinetic studies
Kojic acid has been reported to exhibit a mixed type of
inhibition on the diphenolase activity of the mushroom
tyrosinase enzyme [38, 39] as confirmed in the current study.
So, a mixed inhibitor also binds at a site distinct from the
substrate active site, but it binds to either enzyme or enzyme–
substrate complex. To determine the type of enzyme
inhibition of compounds 2a–k, tyrosinase activity was ana-
lyzed by Lineweaver–Burk plots using data derived from
enzyme assay containing different concentrations of L-DOPA
in the absence or presence of each inhibitor. The Lineweaver–
Burk plots analysis indicated that the compounds 2a–d, 2f–h,
and 2j were non-competitive inhibitors which affect the Vmax
but not the Km, and 2e, 2i, 2k, and kojic acid (as a standard)
were mixed inhibitors which usually affect both Vmax and Km
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Figure 2. Dixon plot for the inhibitory effect of compounds 2f,
2h, and 2k on L-DOPA oxidation catalyzed by mushroom
tyrosinase.
Figure 1. Lineweaver–Burk plot for inhibition of compounds 2f,
2h, and 2k on mushroom tyrosinase.
using IR, 1H NMR, 13C NMR spectra, and mass analysis.
Tyrosinase inhibitory effects and antioxidant activities
of the synthesized benzimidazole derivative compounds
were evaluated. Compounds 2j, 2h, and 2f showed more
potent inhibitory effects than most of the other com-
pounds. The kinetic analysis of compounds 2j, 2h, and 2f
indicated that they are non-competitive inhibitors. The
radical scavenging assay claimed that these compounds
showed low-scavenging activities compared to gallic acid.
The results of the ferric reducing power assay demonstrated
that all of the synthesized compounds have moderate
antioxidant capacity when compared to standard com-
pound. All of these results claimed that synthesized
compounds might have potential usage as tyrosinase
inhibitors.
BHA/mM compounds); 2j (5.84 ꢁ 0.17) > 2h (5.62 ꢁ 0.15) > 2g
(4.88 ꢁ 0.05) > 2a
(3.98 ꢁ 0.09) > 2b
(4.56 ꢁ 0.07) > 2c
(3.88 ꢁ 0.12) > 2d
(4.46 ꢁ 0.03) > 2e
(3.72 ꢁ 0.07) > 2k
(3.40 ꢁ 0.08) > 2i (2.98 ꢁ 0.13) > 2f (2.62 ꢁ 0.01). These data
showed that all of the synthesized compounds have moderate
ferric reducing antioxidant power when compared to the
standard compound.
Conclusion
In conclusion, we synthesized 2-substitutedbenzyl-4(7)-phenyl-
1H-benzo[d]imidazole derivatives (2a–k) and characterized by
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Table 2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ferric
reducing antioxidant power (FRAP) of the compounds
(2a–k).
Generalprocedureforthesynthesisof2-substituted-benzyl-
4(7)-phenyl-1H-benzo[d]imidazole derivatives 2a–k
[1,10-Biphenyl]-2,3-diamine (0.002 mol) and an equivalent
amount of appropriate iminoester hydrochloride compounds
(1a–k) were stirred in 5 mL methanol for 8 h at room
temperature. The reaction progress was monitored by thin
layer chromatography (TLC). Water was added into the
resulting solution. The crude product was filtered and
recrystallized from the mixture of ethanol/water (1:3).
DPPH (SC50
values of
compounds
(mM))
FRAP mM BHAE/
mM compounds
Compounds
2a
2b
2c
2d
2e
2f
2g
2h
0.531 ꢁ 0.009
0.533 ꢁ 0.012
0.549 ꢁ 0.014
0.537 ꢁ 0.022
0.546 ꢁ 0.018
0.548 ꢁ 0.031
0.519 ꢁ 0.016
0.508 ꢁ 0.009
0.549 ꢁ 0.014
0.504 ꢁ 0.009
0.539 ꢁ 0.013
0.064 ꢁ 0.003
4.56 ꢁ 0.07
3.88 ꢁ 0.12
4.46 ꢁ 0.03
3.72 ꢁ 0.07
3.98 ꢁ 0.09
2.62 ꢁ 0.01
4.88 ꢁ 0.05
5.62 ꢁ 0.15
2.98 ꢁ 0.13
5.84 ꢁ 0.17
3.40 ꢁ 0.08
–
2-Methyl-4(7)-phenyl-1H-benzo[d]imidazole (2a)
Yield (82.26%); m.p. 180–182°C, lit. [27].
2-Phenyl-4(7)-phenyl-1H-benzo[d]imidazole (2b)
Yield (84.96%); m.p. 231.5–232°C, lit. [28] IR (Vmax, cmꢂ1): 3035,
3030 (N-H and aromatic C-H), 2785 (aliphatic C-H), 1588, 1546,
2i
2j
2k
1474, 1457 (C C and C N); 1H NMR (DMSO-d6, 400MHz) d
–
–
–
–
(ppm):7.27–7.31(1H,t,Ar-H),7.34–7.37(1H,t,Ar-H),7.41(2H,d,
Ar-H), 7.50–7.56 (5H, m, Ar-H), 7.64–7.69 (2H, t, Ar-H), 8.15–8.19
(1H, t, Ar-H), 8.27 (1H, d, Ar-H), 12.52 (1H; s; N1-H); 13C NMR
(DMSO-d6, 400MHz) d ppm: 120.95, 123.36, 126.97, 127.54,
128.72, 129.28, 129.40, 130.34, 130.54, 131.06, 136.31, 138.79
Gallic acid
–
(Ar–C) and 151.76 (C N); ESI-MS m/z calculated for C19H14N2
Experimental
–
[M]þ 270.12. Found: 270.83 (100%) and 271.95 [Mþ1]þ (20%).
Material and methods
Melting points were determined in open capillaries on
“Thermo Scientific Capillary Melting Point Apparatus” and
were uncorrected. IR spectra were recorded on a PerkinElmer
100 FT-IR spectrophotometer taken with ATR technique and
were evaluated in terms of wavenumbers (cmꢂ1). 1H and
13C NMR spectra were measured on a Varian 400 spectrometer
using DMSO-d6 as solvent and TMS as internal standard.
Chemical shifts were given in parts per million (ppm), was
evaluated d (ppm) in scale. LC-mass spectra (LC-MS) were
taken in H-ESI mode on an Agilent 1260 Infinity Series LC-MS.
Studies of tyrosinase inhibitory and antioxidant activity were
performed by MultikanTM Go Microplate spectrophotometer.
Gallic acid (GA), kojic acid, L-DOPA, methanol, tyrosinase
from mushroom, Trisma-base, sodium phosphate buffer,
potassium ferricyanide, trichloroacetic acid, and iron(III)
chloride were purchased from Sigma-Aldrich (St. Louis, MO).
The InChI codes of the investigated compound are provided
as Supporting Information.
2-Benzyl-4(7)-phenyl-1H-benzo[d]imidazole (2c)
Yield (86.27%); m.p. 220–221°C; IR (Vmax, cmꢂ1): 3158, 3063,
3026 (N-H and aromatic C-H), 2730 (aliphatic C-H), 1526, 1498,
1453, 1399 (C C and C N); 1H NMR (DMSO-d6, 400 MHz) d
–
–
–
–
(ppm): 4.2 (2H; s; –CH2–), 7.18–7.22 (1H, t, Ar-H), 7.30 (1H, d,
Ar-H), 7.39 (2H, d, Ar-H), 7.43–7.46 (5H, m, Ar-H), 7.52–7.53
(2H, t, Ar-H), 7.61–7.63 (1H, t, Ar-H), 8.06 (1H, d, Ar-H), 12.38
(1H; s; N1-H); 13C NMR (DMSO-d6, 400 MHz) d ppm: 35.44
(–CH2–), 110.79, 120.39, 122.05, 122.48, 126.80, 126.98, 127.38,
128.63, 128.80, 128.96, 129.10, 129.24, 129.39, 130.62, 135.76,
–
–
138.15, 138.95, 141.37 (Ar–C) and 154.00 (C N); ESI-MS m/z
calculated for C20H16N2 [M]þ 284.35. Found: 284 (100%) and
285 [Mþ1]þ (10%).
2-Ethyl-4(7)-phenyl-1H-benzo[d]imidazole (2d)
Yield (88.27%); m.p. 182.5–183°C; IR (Vmax, cmꢂ1): 3154, 3059,
3027 (N-H and aromatic C-H), 2936 (aliphatic C-H), 1529, 1505,
1415, 1327 (C C and C N); 1H NMR (DMSO-d6, 400 MHz) d
–
–
–
–
(ppm): 1.30 (3H; t; –CH3), 2.8 (2H; q; –CH2–), 7.17–7.20 (1H, t,
Ar-H), 7.31 (2H, d, Ar-H), 7.38–7.50 (2H, t, Ar-H), 7.61 (2H, d,
Ar-H), 8.05 (1H, d, Ar-H), 12.29 (1H; s; N1-H); 13C NMR (DMSO-
d6, 400 MHz) d ppm: 12.93 (–CH3), 22.55 (–CH2–), 110.53,
117.85, 120.17, 121.77, 122.17, 127.76, 128.57, 129.21, 130.37,
Chemistry
Synthesis of compounds 1a–k
Our initial compounds, iminoester hydrochloride derivatives
were synthesized by the Pinner method [44]. In this method,
absolute ethanol (0.103 mol) was added to nitrile derivatives
(0.101 mol) in anhydrous ether. Dry HCl gas was passed
through the reaction mixture until saturation. The mixture
was kept in the freezer for 12 h, and precipitated by addition
of anhydrous ether. The precipitated crude product was
filtered, washed with anhydrous ether, and dried over
anhydrous CaCl2. And the corresponding iminoester hydro-
chloride compounds 1a–k were obtained [45].
–
135.58, 139.04, 141.31 (Ar–C) and 156.75 (C N); ESI-MS m/z
–
calculated for C15H14N2 [M]þ 222.19. Found: 222.8 (100%) and
224 [Mþ1]þ (10%).
2-(4-Methylbenzyl)-4(7)-phenyl-1H-benzo[d]imidazole
(2e)
Yield (63.76%); m.p. 215–216°C; IR (Vmax, cmꢂ1): 3154, 3056,
3025 (N-H and aromatic C-H), 2918, 2765 (aliphatic C-H), 1540,
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1512, 1480, 1451, 1399 (C C and C N); 1H NMR (DMSO-d6,
1504, 1492 (C C and C N); 1H NMR (DMSO-d6, 400 MHz) d
–
–
–
–
–
–
–
–
400 MHz) d (ppm): 2.23 (3H; s; –CH3), 4.15 (2H; s; –CH2–), 7.11
(2H, d, Ar-H), 7.21 (2H, d, Ar-H), 7.31–7.33 (1H, t, Ar-H), 7.38
(2H, d, Ar-H), 7.41–7.46 (2H, t, Ar-H), 7.50–7.52 (1H, t, Ar-H),
7.60–7.62 (1H, t, Ar-H), 8.06 (1H, d, Ar-H), 12.33 (1H; s; N1-H);
13C NMR (DMSO-d6, 400 MHz) d ppm: 21.06 (CH3), 35.06 (CH2),
110.78, 118.03, 120.35, 121.99, 122.43, 127.37, 127.88,
128.602, 129.01, 129.37, 130.59, 135.20, 135.88, 138.670,
(ppm): 1.70 (3H; s; –CH3), 4.42 (2H; s; –CH2–), 7.18–7.21 (4H,
t, Ar-H), 7.29–7.38 (1H, t, Ar-H), 7.44–7.47 (3H, t, Ar-H), 7.56
(3H, d, Ar-H), 8.10 (1H, d, Ar-H), 12.31 (1H; s; N1-H); 13C NMR
(DMSO-d6, 400 MHz) d ppm: 20.87 (CH3), 39.15 (CH2),
110.78, 120.33, 122.4, 126.99, 127.3, 127.71, 128.65,
128.95, 129.24, 130.65, 135.69, 138.98, 141.02, 144.10
–
(Ar–C), and 157.84 (C N); ESI-MS m/z calculated for
–
138.97, 141.38, 154.22 (Ar–C) and 155.22 (C N); ESI-MS m/z
C
21H18N2 [M]þ 298.38 (20%). Found: 298.90 (100%),
–
–
calculated for C21H18N2 [M]þ 298.38. Found: 300 (Mþ2)
299.88 [Mþ1]þ (10%).
(100%), 299 [Mþ1]þ (20%).
2-(3-Methylbenzyl)-4(7)-phenyl-1H-benzo[d]imidazole (2j)
Yield (83.89%); m.p. 184–5°C; IR (Vmax, cmꢂ1): 3154, 3057,
2-(4-Chlorobenzyl)-4(7)-phenyl-1H-benzo[d]imidazole (2f)
Yield (97%); m.p. 228–230°C; IR (Vmax, cmꢂ1): 3154, 3062, 3027
(N-H and aromatic C-H), 2739 (aliphatic C-H), 1526, 1504, 1488
3028 (N-H and aromatic C-H), 2898, 2861, 2812 (aliphatic C-H),
1
–
–
1528, 1490, 1414 (C C and C N); H NMR (DMSO-d , 400 MHz)
–
–
6
(C C and C N); 1H NMR (DMSO-d6, 400 MHz) d (ppm): 4.2
d (ppm): 2.24 (3H; s; –CH3), 4.16 (2H; s; –CH2–), 7.01 (3H, d, Ar-
H), 7.09 (1H, s, Ar-H), 7.11–7.315 (1H, t, Ar-H), 7.17–7.20 (1H, t,
Ar-H), 7.22–7.33 (3H, t, Ar-H), 7.47 (3H, d, Ar-H), 8.07 (1H, d,
Ar-H), 12.36 (1H; s; N1-H); 13C NMR (DMSO-d6, 400 MHz) d ppm:
21.44 (CH3), 35.40 (CH2), 122.42, 126.22, 127.58, 128.83,
129.24, 129.74, 138.00, 138.91 (Ar–C); ESI-MS m/z calculated
for C21H18N2 [M]þ 298.38. Found: 300 [Mþ2]þ (10%), 299.18
[Mþ1]þ (100%), 284, 258, 230.
–
–
–
–
(2H; s; –CH2–), 7.19 (2H, d, Ar-H), 7.21 (2H, d, Ar-H), 7.22 (1H, t,
Ar-H), 7.34 (3H, d, Ar-H), 7.45 (3H, t, Ar-H), 8.05 (1H, d, Ar-H),
12.39 (1H; s; N1-H); 13C NMR (DMSO-d6, 400 MHz) d ppm: 34.65
(CH2), 120.48, 122.52, 127.44, 128.86, 129.24, 131.02, 131.65,
137.16, 138.85 (Ar–C); ESI-MS m/z calculated for C20H15ClN2
[M]þ 318.09. Found: 320.9 [Mþ2] (%30), 319 [M]þ (100%).
2-(2-Chlorobenzyl)-4(7)-phenyl-1H-benzo[d]imidazole
(2g)
2-(3-Chloropropyl)-4(7)-phenyl-1H-benzo[d]imidazole
(2k)
Yield (92.62%); m.p. 229–230°C; IR (Vmax, cmꢂ1): 3146, 3061,
3027 (N-H and aromatic C-H), 2900, 2736 (aliphatic C-H), 1521,
Yield (82.47%); m.p. 185–186°C; IR (Vmax, cmꢂ1): 3166, 3028
(N-H and aromatic C-H), 2957 (aliphatic C-H), 1592, 1544,
1476, 1434 (C C and C N); 1H NMR (DMSO-d6, 400 MHz) d
–
–
–
–
1428, 1405 (C C and C N); 1H NMR (DMSO-d6, 400 MHz) d
–
–
–
–
(ppm): 4.33 (2H; s; –CH2–), 7.20 (1H, d, Ar-H), 7.26 (2H, t, Ar-H),
7.33 (1H, d, Ar-H), 7.39–7.44 (1H, t, Ar-H), 7.51–7.54 (3H, t, Ar-
H), 7.63–7.65 (3H, d, Ar-H), 8.04 (1H, d, Ar-H), 12.33 (1H; s; N1-
H); 13C NMR (DMSO-d6, 400 MHz) d ppm: 33.24 (CH2), 110.91,
118.13, 120.45, 122.04, 122.01, 122.55, 125.68, 127.65, 128.67,
129.43, 130.59, 131.54, 133.59, 135.74, 138.74, 141.39, 144.59,
(ppm): 2.24 (2H; m; –CH2–), 2.97 (2H; t; –CH2–), 3.75 (2H; t;
–CH2–), 7.18–7.22 (1H, t, Ar-H), 7.27–7.35 (1H, t, Ar-H), 7.36–
7.45 (2H, t, Ar-H), 7.47 (1H, d, Ar-H), 7.49 (2H, d, Ar-H), 7.91
(1H, d, Ar-H), 12.34 (1H; s; N1-H); 13C NMR (DMSO-d6,
400 MHz) d ppm: 30.84, 39.54, 45.26 (–CH2–), 122.48, 122.52,
122.54, 122.59, 127.63, 127.65, 128.92, 128.99, 129.78,
–
152.53 (Ar–C), and 153.68 (C N); ESI-MS m/z calculated
–
for C20H15ClN2 [M]þ 318.09. Found: 321 [Mþ2] (%30), 318.5
138.62, 138.64, 138.65, 138.66 (Ar–C), and 154.75 (C N);
–
–
[M]þ (100%).
ESI-MS m/z calculated for C16H15ClN2 [M]þ 270.09. Found:
270.9 (100%) and 272.8 [Mþ2]þ (30%).
2-(3-Chlorobenzyl)-4(7)-phenyl-1H-benzo[d]imidazole
(2h)
Pharmacology
Tyrosinase inhibition assay
Yield (86.34%); m.p. 216–218°C; IR (Vmax, cmꢂ1): 3154, 3058 (N-
H and aromatic C-H), 2990, 2897, 2803 (aliphatic C-H), 1504,
The tyrosinase inhibition of compounds 2a–k was examined
using the method described by Masuda et al. [46]. All of the
compounds were prepared as stock solutions in 20% DMSO.
Then different concentrations were prepared in the buffer
(100 mM phosphate buffer, pH 6.8) from stock solution for
experiment. The final concentration of DMSO solution was
2%. Twenty microliters of the compounds at different
concentrations (8–125 mM), 20 mL of 250 U/mL tyrosinase,
and 100 mL of 100 mM pH 6.8 phosphate buffer solutions
were added in a 96-well microplate. After pre-incubation for
10 min at room temperature, 20 mL of 3 mM L-DOPA was
added and the microplate was further incubated at room
temperature for 20 min. Subsequently, the absorbance of
1475, 1441 (C C and C N); 1H NMR (DMSO-d6, 400 MHz) d
–
–
–
–
(ppm): 4.23 (2H; s; –CH2–), 7.20 (1H, d, Ar-H), 7.26 (2H, t, Ar-H),
7.33 (1H, d, Ar-H), 7.39–7.46 (1H, t, Ar-H), 7.53 (3H, t, Ar-H),
7.61–7.63 (3H, d, Ar-H), 8.05 (1H, d, Ar-H), 12.41 (1H; s; N1-H);
13C NMR (DMSO-d6, 400 MHz) d ppm: 34.66 (CH2), 110.88,
118.14, 120.49, 122.13, 122.63, 126.94, 127.43, 127.91, 128.00,
128.60, 129.19, 130.84, 133.40, 135.71, 138.87, 140.67, 141.31,
–
144.61 (Ar–C), and 153.34 (C N); ESI-MS m/z calculated
–
for C20H15ClN2 [M]þ 318.09. Found: 321 [Mþ2]þ (30%),
318.9 [M]þ (100%).
2-(2-Methylbenzyl)-4(7)-phenyl-1H-benzo[d]imidazole (2i)
Yield (84%); m.p. 179–181°C; IR (Vmax, cmꢂ1): 3058, 3028 (N-
H and aromatic C-H), 2979, 2933, 2726 (aliphatic C-H), 1523,
dopachrome was measured at 475 nm using
a 96-well
microplate reader. a-Kojic acid was used as the reference
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RCHH HARM
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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–8
Potent Anti-Tyrosinase and Antioxidant Agents
Archiv der Pharmazie
drug and the tyrosinase inhibition percentage was calculated
as follows:
mean ꢁ standard deviation (SD).
À
Á
ꢁ
ꢀ
Acontrol ꢂ Acompound
À
Á
ꢀ
ꢁ
Scavenging Effectsð%Þ ¼
ꢃ 100
Acontrol ꢂ Acompound
Acontrol
Tyrosinase inhibitionð%Þ ¼
ꢃ 100
Acontrol
Ferric-reducing antioxidant power (FRAP) assay
where Acontrol is the activity of enzyme without compound/
reference (containing 2% DMSO in the buffer) and Acompound
is the activity of enzyme with compound/reference at
different concentrations. The concentrations of the inhibitors
required for inhibiting 50% of the enzyme activity under the
assay conditions was defined as the IC50. The experiments
were carried out in triplicate and results were expressed as the
mean ꢁ standard deviation (SD).
The ferric-reducing power (FRAP) was examined according to
the Oyaizu method [50] using butylated hydroxyanisole
(BHA) as the standard compound. Briefly, 250 mL of each
compound and various concentrations of BHA as standard
compound were mixed with 250 mL of sodium phosphate
buffer (pH 6.6) and 250 mL potassium ferricyanide solution.
The mixture was incubated at 50 °C for 20 min and then
250 mL trichloroacetic acid was added and centrifuged at
3000ꢃg for 10 min. Then 200 mL of supernatant was mixed
with 200 mL distilled water, after which 200 mL of this mixture
was added 20 mL iron(III) chloride and incubated 30 min at
room temperature in the dark. After incubated, the
absorbance was measured 700 nm. The ferric-reducing
power of extracts was expressed as butylated hydroxyanisole
equivalents (BHAE) per mM of compounds. The experiments
were carried out in triplicate and results were expressed as
the mean ꢁ standard deviation (SD).
Kinetic analysis of tyrosinase inhibition
Enzyme activity was determined according to the method
Jeong et al. [47] using L-DOPA as a substrate in phosphate
buffer (100 mM, pH 6.8). Increasing concentrations of L-
DOPA (1.25–10 mM), 20 mL of 250 U/mL tyrosinase, and
100 mL of 100 mM pH 6.8 phosphate buffer solutions were
added in
a 96-well microplate and the mixture was
incubated at 25°C for 20 min. The absorbance was mea-
sured at 475 nm using a 96-well microplate reader. The data
obtained were plotted as 1/activity (1/V) against 1/substrate
concentration (1/[S]), according to Lineweaver–Burk
graph [48]. Enzyme activity was calculated from the linear
portion of the curve. Compounds 2a–k were further studied
to determine the type of tyrosinase inhibition. Tyrosinase
was incubated with of various concentrations of L-DOPA
(1.25–10 mM) and phosphate buffer, and then different
concentrations of tested compounds (0, 15.625, 31.25, and
62.5 mM) were simultaneously added to the reaction
mixtures. The Lineweaver–Burk graphs were obtained
and inhibition patterns were evaluated. Furthermore,
inhibition constant (Ki) was deduced from the Dixon plot
which is a graphical method (plot of 1/enzyme velocity (1/v)
versus inhibitor concentration [I] with varying concentra-
tions of the substrate (0, 2.5, 5.0, and 10.0 mM) for each
inhibitor.
This work was supported by Scientific and Technological
Research Council of Turkey (TUBITAK) (grant number
1919B011500695).
The authors have declared no conflict of interest.
References
[1] T. Oyama, S. Takahashi, A. Yoshimori, T. Yamomoto,
A. Sato, T. Kamiya, H. Abe, T. Abe, S. Tanuma, Bioorg.
Med. Chem. 2016, 24, 4509–4515.
[2] Z. Ashraf, M. Rafiq, S. Seo, M. Babar, N. Zaidi, J. Enzyme
Inhib. Med. Chem. 2015, 30, 874–883.
[3] D. S. Robinson, N. A. M. Eskin (Eds.), Oxidative Enzymes
in Foods, Elsevier, London 1991, p. 217.
[4] S. Y. Lee, N. Baek, T. Nam, J. Enzyme Inhib. Med. Chem.
2016, 31, 1–13.
[5] W. Yi, R. Cao, W. Peng, H. Wen, Q. Yan, B. Zhou, L. Ma,
H. Song, Eur. J. Med. Chem. 2010, 45, 639–646.
[6] Z.Aumeeruddy-Elalfi, A. Gurib-Fakim, M. F. Mahomoodally,
S. Afr. J. Bot. 2016, 103, 89–94.
[7] Y. Xu, A. Stokes, W. Freeman, S. C. Kumer, B. A. Vogt,
K. E. Vrana, Mol. Brain Res. 1997, 45, 159–162.
[8] S. Suk Kim, C. Hyun, Y. Choi, N. Lee, J. Enzyme Inhib.
Med. Chem. 2013, 28, 685–689.
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activities
The DPPH radical scavenging activities were determined
using method of Blois et al. [49] compared to gallic acid
as the reference compound. Hundred microliters of com-
pounds at various concentration and 900 mL of methanolic
DPPH solution (0.4 mM) were mixed. The mixtures were
kept for 30 min at room temperature in the dark. After
incubation, the absorbance of the compounds (Acompound
)
was measured at 517 nm. Assay mixtures without com-
pounds were used as a control (Acontrol). DPPH scavenging
was calculated by using the formula 2 and SC50 values
obtained plotting the inhibition percentage against
the compounds or reference. The experiments were
carried out in triplicate and results were expressed as the
[9] R. Uchida, S. Ishikowa, H. Tamoda, Acta Pharmacol. Sin.
2014, 4, 141–145.
[10] S. C. Shilcrat, M. K. Mokhallalati, J. M. Fortunak,
L. N. Pridgen, J. Org. Chem. 1997, 28, 8449–8454.
[11] F. Hobrecker, Berichte der Deutschen Chemischen
Gesellschaft 1872, 5, 920–924.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
7

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–8
I. S. Dogan et al.
_
ꢀ
Archiv der Pharmazie
[12] X.-J. Fang, P. Jeyakkumar, S. Rao Avula, Q. Zhou,
C.-H. Zhou, Bioorg. Med. Chem. Lett. 2016, 26, 2584–2588.
[13] V. Onnis, M. Demurtas, A. Deplano, G. Balboni,
A. Baldisserotto, S. Manfredini, S. Pacifico, S. Liekens,
J. Balzarini, Molecules 2016, 21, 579.
[14] D. Song, S. Ma, Chem. Med. Chem. 2016, 11, 646–659.
[15] M. Mochizuki, M. Kori, K. Kobayashi, T. Yano, Y. Sako,
M. Tanaka, N. Kanzaki, A. Gyorkos, C. P. Corrette,
S. Y. Cho, S. A. Pratt, K. Aso, J. Med. Chem. 2016, 59,
2551–2566.
[29] C. Hofmann, Chemistry of Heterocyclic Compounds:
Imidazole and Its Derivatives, Part I, Volume 6, Inter-
science Publ. Inc., New York 1953, p. 258.
[30] M. R. Grimmett, A. R. Katritzky, C. W. Rees; Potts, K. T.
(eds.): Comprehensive Heterocyclic Chemistry, Vol. 5,
Pergamon Press, Oxford 1984, p. 345.
[31] L. M. Dudd, E. Venardou, E. Garcia Verdugo, P. Licence,
A. J. Blake, C. Wilson, M. Poliakoff, Green Chem. 2003, 5,
187–192.
[32] R. M. Silverstein, F. X. Webster, (6th edn.), Spectrometric
Identification of Organic Compounds, John Wiley & Sons
Inc., New York 1998.
[16] I. Castillo, M. Suwalsky, M. J. Gallardo, V. Troncoso,
ꢁ
ꢁ
~
B. N. Sanchez-Eguıa, E. Santiago-Osorio, I. Aguiniga,
ꢁ
A. K. Gonzalez-Ugarte, J. Inorg. Biochem. 2016, 156,
98–104.
[33] E. Pretsch, T. Clerk, J. Seibl, W. Simon, Tables of Spectral
Data for Structure Determination of Organic Com-
pounds, Springer-Verlag, Berlin 1983.
[34] D. J. Rabiger, M. M. Ioullie, J. Org. Chem. 1964, 29,
476–482.
[17] H. Bui, Q. Ha, W. Oh, D. Vo, Y. Chau, C. Tu, E. Pham,
P. Tran, L. Tran, H. Mai, Tetrahedron Lett. 2016, 57,
887–891.
[18] D. Traversa, G. Von Samson-Himmelstjerna, Small Rumin.
Res. 2016, 135, 75–80.
[19] Y. K. Yoon, T. S. Choon, Archiv. Der. Pharmazie 2016,
349, 1–8.
[35] R. W. Layer, Chem. Rev. 1963, 63, 489–510.
[36] S. Radhakrishan, R. Shimman, C. Conn, A. Baker, Bioorg.
Med. Chem. Lett. 2015, 25, 4085–4091.
[37] M. O. Karatas, B. Alici, E. Cetinkaya, CS Bilen, N. Gencer,
O. Arslan, Russ. J. Bioorg. Chem. 2014, 40, 461–466.
[38] J. Cabanes, S. Chazarra, F. Garcia-Carmona, J. Pharm.
Pharmacol. 1994, 46, 982–985.
€
[20] H. Goker, C. Karaaslan, M. O. Puskullu, S. Yildiz,
Y. Duydu, A. Ustundag, C. O. Yalcin, Chem. Biol. Drug.
Des. 2016, 87, 57–68.
[21] M. Chhabra, S. Sinha, S. Banerjee, P. Paira, Bioorg. Med.
Chem. Lett. 2016, 26, 213–217.
[39] Z. Ashraf, M. Rafiq, S. Y. Seo, K. S. Kwon, M. M. Babar,
N. U. Zaidi, Eur. J. Med. Chem. 2015, 98, 203–211.
[40] A. Ozel, B. Barut, U. DemirbasS, Z. Biyiklioglu, J. Photo-
chem. Photobiol. B 2016, 157, 32–38.
€
€
[22] N. S. Chandrasekera, T. Alling, M. A. Bailey, M. Files,
J. V. Early, J. Ollinger, Y. Ovechkina, T. Masquelin,
P. V. Desai, J. W. Cramer, P. A. Hipskind, J. O. Odingo,
T. Parish, J. Med. Chem. 2015, 58, 7273–7285.
[23] A. V. Ivachtchenko, P. M. Yamanushkin, O. D. Mit’kin,
E. V. Ezhova, O. Korzinov, E. A. Bulanova, V. V. Bichko,
A. A. Ivashchenko, Pharmaceut. Chem. J. 2015, 49,
352–361.
[41] Md. Nur Alam, N. Jahan Bristi, Md. Rafiquazzaman,
Saudi Pharm. J. 2013, 21, 143–152.
€
€
[42] A. Ozel, B. Barut, U. DemirbasS, Z. Biyiklioglu, J. Photo-
chem. Photobiol. B. 2016, 157, 32–38.
[43] S. Shukla, J. Park, D. Kim, S. Hong, J. S. Lee, M. Kim, Food
Control 2016, 59, 854–861.
[24] S. Hemalatha, T. Ayyappan, S. Shanmugam, D. Nagavalli,
T. Shrivijaya Kirubha, Indian J. Tradit. Know. 2006, 5,
468–470.
[25] K. Kubo, Y. Kohara, E. Imamiya, Y. Sugiura, Y. Inada,
Y. Furukawa, K. Nishikawa, T. Nakat, J. Med. Chem. 1993,
36, 2182–2195.
[26] E. Mentese, H. Bektas, S. U. Ilker, O. Bekircan, B. Kahveci,
J. Enzyme Inhib. Med. Chem. 2014, 29, 64–68.
[27] C. M. Atkinson, C. J. Sharpe, J. Chem. Soc. 1959,
2858–2864
[44] A. Pinner, Dieimidoether und ihre derivate, 1. Auflage,
Oppenheim, Berlin 1892.
[45] B. Kahveci, Molecules 2005, 10, 376–382.
[46] T. Masuda, D. Yamashita, Y. Takeda, S. Yonemori,
Bioschi. Biotechnol. Biochem. 2005, 69, 197–201.
[47] S. H. Jeong, Y. B. Ryu, M. J. Curtis-Long, H. W. Ryu,
Y. S. Baek, J. E. Kang, W. S. Lee, K. H. Park, J. Agric. Food
Chem. 2009, 57, 1195–1203.
[48] H. Lineweaver, D. Burk, J. Am. Chem. Soc. 1934, 56,
658–661.
[28] M. Devulapally, S. Pradeep, P. Tharmalingam, J. Org.
Chem. 2016, 81, 3227–3234.
[49] M. S. Blois, Nature 1958, 181, 1199–1200.
[50] M. Oyaizu, Jpn. J. Nutr. 1986, 44, 307–315.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
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