Synthesis of New Benzimidazole-1,2,3-triazole Hybrids as Tyrosinase Inhibitors

Article abstract of DOI:10.1002/cbdv.201800120

A novel series of benzimidazole-1,2,3-triazole hybrids containing substituted benzyl moieties were designed, synthesized and evaluated for their inhibitory activity against mushroom tyrosinase. The results indicated that 2-(4-{[1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl]methoxy}phenyl)-1H-benzimidazole (6g) and 2-(4-{[1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl]methoxy}phenyl)-1H-benzimidazole (6h) exhibited effective inhibitory activity with IC₅₀ values of 9.42 and 10.34 μm, respectively, comparable to that of kojic acid as the reference drug (IC₅₀ = 9.28 μm). Kinetic study of compound 6g confirmed mixed-type inhibitory activity towards tyrosinase indicating that it can bind to free enzyme as well as enzyme-substrate complex. Also, molecular docking analysis was performed to determine the binding mode of the most potent compounds (6g and 6h) in the active site of tyrosinase. Consequently, 6g and 6h derivatives might serve as promising candidates in cosmetics, medicine or food industry, and development of such compounds may be of an interest.

Full text of DOI:10.1002/cbdv.201800120

Article Type: Full Paper
Synthesis of new benzimidazole-1,2,3-triazole hybrids as tyrosinase inhibitors
a
b
c,d
c,d
Mohammad Mahdavi , Arsalan Ashtari , Mahsima Khoshneviszadeh , Sara Ranjbar
,
c,d
e,f
a
Ameneh Dehghani , Tahmineh Akbarzadeh , Bagher Larijani , Mehdi Khoshneviszadeh
c,d*
g,f,*
,
Mina Saeedi
Corresponding author mail id:- m-saeedi@tums.ac.ir
a
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
b
School of Chemistry, College of Science, University of Tehran, PO Box 14155-6455,
Tehran, Iran
c
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
d
Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran
e
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
f
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
g
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201800120
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This paper is dedicated to the memory of our unique teacher in Chemistry and
Medicinal Chemistry, Professor Abbas Shafiee.
A novel series of benzimidazole-1,2,3-triazole hybrids containing substituted benzyl moieties
were designed, synthesized and evaluated for their inhibitory activity against mushroom
tyrosinase. The results indicated that 2-(4-((1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)-1H-benzo[d]imidazole (6g) and 2-(4-((1-(4-bromobenzyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6h) exhibited effective inhibitory
activity with IC50 values of 9.42 and 10.34 μM, respectively, comparable to that of kojic acid
as the reference drug (IC50 = 9.28 μM). Kinetic study of compound 6g confirmed mixed-type
inhibitory activity towards tyrosinase indicating that it can bind to free enzyme as well as
enzyme-substrate complex. Also, molecular docking analysis was performed to determine the
binding mode of the most potent compounds (6g and 6h) in the active site of tyrosinase.
Consequently, 6g and 6h derivatives might serve as promising candidates in cosmetics,
medicine or food industry, and development of such compounds may be of an interest.
Key words: Benzimidazole; 1,2,3-Triazole; Tyrosinase inhibitor
Introduction
Benzimidazole and 1,2,3-triazole derivatives are known as the impressive
heterocyclic scaffolds owing to their various valuable biological activities. Benzimidazole
moiety is a fused heterocyclic aromatic system and found in the structure of vitamin B12. It
has also depicted antiviral, anticancer, antibacterial, anticonvulsant, analgesic and anti-
[1]
inflammatory, anti-diabetic, antifungal, antioxidant, and antihypertensive activities.
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Recently,
benzo[d]imidazoles have shown inhibitory activity towards tyrosinase.
,2,3-Triazoles have emerged as the versatile pharmacophore since they are able to
construct hydrogen bonding and dipole-dipole interactions which have made them very stable
some
alkylbenzimidazole
and
2-substitutedbenzyl-4(7)-phenyl-1H-
[2][3]
1
[
4]
to hydrolysis and oxidative/reductive conditions. Moreover, 1,2,3-triazloles have been
introduced as the peptide bond isosteres and 1,2-3-triazole-containing analogues of the
naturally occurring cyclo-[Pro-Val-Pro-Tyr] have shown potent tyrosinase inhibitory
[
5]
activity. 1,2,3-Triazole moiety is not naturally produced; hence, synthetic molecules have
attracted lots of attention and in this regard, copper(I)-catalyzed formation of 1,2,3-triazoles
through azides and terminal alkynes has been a powerful tool in drug discovery
[6-12]
developments.
Tyrosinase (EC 1.14.18.1), a copper containing monooxygenase and also known as
polyphenol oxidase (PPO), is widely distributed in mammals, plants, bacteria and fungi. It
catalyzes two reactions: hydroxylation of L-tyrosine to 3,4-dihydroxy phenylalanine (L-
[13][14]
DOPA) and oxidation of L-DOPA to dopaquinone.
In humans, dopaquinone is
converted to melanin by a series of reactions and tyrosinase plays a crucial role in the
synthesis of melanin which can cause to hyperpigmentation disorders such as melasma,
[
15]
seborrheic, etc. In addition, tyrosinase is responsible for fruits and vegetables enzymatic
browning due to oxidation of phenolic compounds leading to the reduction of nutritional
[
16]
values. Moreover, tyrosinase is associated with some neurodegenerative disorders such as
[
17][18]
Parkinson’s and Huntington’s diseases.
Therefore, safe and effective tyrosinase
inhibitors are essential in the field of clinical medicine, cosmetics, agricultural, and food
[
13][19]
industries.
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In this study, we designed and synthesized a wide range of benzimidazole-1,2,3-triazole
hybrids. All 18 synthesized compounds were evaluated for their tyrosinase inhibitory activity
and the most active derivative was investigated for its inhibition kinetic. Finally, docking
analysis was performed to gain better insight into the ligand-receptor interactions of the most
potent compounds.
Results and Discussion
Design strategy
The structure of target benzimidazole-1,2,3-triazole hybrids was designed by the molecular
hybridization approach based on the structure of some potent tyrosinase inhibitors as reported
in the literature (compounds A-D) (Fig. 1). 2-(3-Methylbenzyl)-4(7)-phenyl-1H-
benzo[d]imidazole (A) demonstrated tyrosinase inhibitory activity with IC50 value of 37.86
[
20]
μM. It has been reported that benzothiazole analogs possessing phenolic substituent (such
as B) were effective tyrosinase inhibitors which showed higher inhibition than kojic acid at
[
21]
5
0 µM.
Moreover, it has been described that 1,2-3-triazole-containing analogues of the
naturally occurring cyclo-[Pro-Val-Pro-Tyr] (such as C) were effective tyrosinase inhibitors
[
3]
(
IC50 = 0.5 mM). It has been also reported that the kojic acid derivative containing 1,2,4-
triazole (D) exhibited better tyrosinase inhibitor activity (IC50 = 4.50 μM) than kojic acid
[22]
(
IC50 = 19.00 μM). Considering these structural features and principles of bioisoterism, we
designed a series of tyroinase inhibitors by employing fragment-based drug design strategy.
Chemistry
The synthetic procedure for the preparation of benzimidazole-1,2,3-triazole hybrids 6 is
shown in Scheme 1. The required substrates 3 were prepared through the reaction of 4-(prop-
2
2 2 5
-yn-1-yloxy)benzaldehyde 1 and o-phenylenediamines 2 in the presence of Na S O in
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dimethylacetamide (DMA) at 120 °C for 12 h. It should be noted that aldehyde 1 was
prepared by the reaction of 4-hydroxybenzaldehyde and propargyl bromide in DMF at 80 °C.
[
7]
[23]
The title compounds 6 were synthesized through click reaction described by Kolb et al.
For this purpose, various organic azides 5 were prepared by the reaction of different benzyl
chlorides/bromides 4 and sodium azide in the presence of triethylamine (NEt ) in the mixture
of H O/t-BuOH at room temperature. Then, compound 3, sodium ascorbate, and catalytic
amount of CuSO .5H O (7 mol%) were added to the freshly prepared azides 5 leading to the
3
2
4
2
formation of different benzimidazole-1,2,3-triazole hybrids 6a-r. The final synthesized
derivatives are listed in Table 1.
Biological activity
All synthesized compounds (6a-r) and kojic acid (as the positive control) were
evaluated using tyrosinase inhibition assay and the IC50 values were reported in Table 1. As it
is stated in Table 1, compounds 6c, 6d, 6g, 6h, 6i, 6j, and 6l showed inhibitory activity
against mushroom tyrosinase (IC50s = 9.42-48.00 µM). Among them, compounds 6g and 6h
exhibited potent inhibitory activity towards tyrosinase with IC50 values of 9.42 and 10.34
µM, respectively, which were comparable to that of kojic acid (IC50 = 9.28 µM). However,
compounds 6a, 6b, 6e, 6f, 6k, 6l-r demonstrated no anti-tyrosinase activity indicating the fact
that the inhibitory activity is dependent on the nature and position of the substitutions on the
benzimidazole and arylidene moieties. The most potent compound 6g contained 3,4-
dichloroarylidene group on 1,2,3-triazole ring and lacked substituents on the benzimidazole
moiety. Changing the number and position of chlorine atom in compounds 6e and 6f deleted
anti-tyrosinase activity. In addition, it was found that compounds 6m-p having methyl group
at 6-position of benzimidazole ring and chlorine (chlorines) on the arylidene moiety, showed
no inhibitory activity.
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The second potent compound 6h possessing 4-bromoarylidene group connected
to1,2,3-triazole ring also lacked substituents on the benzimidazole moiety. Its counterpart,
compound 6q having methyl group at 6-position of benzimidazole ring did not show
inhibition activity on tyrosinase enzyme. Introduction of fluorine into aryliden moiety of
compounds 6c and 6d having 2- and 4-fluorobenzyl group connected to 1,2,3-triazole ring
showed moderate inhibitory activity towards tyrosinase with IC50 values of 45.22 and 46.89
µM, respectively. However, the better inhibitory activity was obtained by compound 6l
containing 4-fluorobenzyl group connected to 1,2,3-triazole ring and methyl group at 6-
position of benzimidazole moiety (IC50 = 29.31 µM).
Generally, it can be stated that compounds bearing methyl group at C6 position of
benzimidazole core (compounds 6j-n) showed less inhibitory activity comparing with their
counterparts lacking methyl group at the same position (6a-i). However, there is an
exception, compound 6l showed anti-tyrosinase activity with IC50 value of 29.31 µM, more
active than compound 6d.
Kinetic studies
The enzyme inhibition mode by the most potent derivative 6g was determined by
Lineweaver–Burk plot analysis as shown in Fig. 2. The Lineweaver–Burk plots (plot of 1/V
versus 1/[S]) for the inhibition of tyrosinase by the selected compound 6g was obtained with
several concentrations of the corresponding compound and L-DOPA as the substrate. The
Lineweaver-Burk double reciprocal plots yielded a group of lines that intercepted in the
m
second quadrant. It was found that K value increased and Vmax value decreased by the
increase of the concentration of 6g confirming that compound 6g demonstrated mixed-type
inhibition towards tyrosinase. It can inhibit both free enzyme and enzyme-substrate complex.
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Molecular docking analysis
The binding models of the compounds 6g and 6h with tyrosinase (PDB code: 2Y9X)
were represented in Fig. 3 and Fig. 4, respectively. The results revealed that both compounds
were well accumulated in the binding pocket of tyrosinase by hydrogen bond, Pi-H and Pi-Pi
interactions. The following interactions could be seen in the case of compound 6g (Fig. 3).
The benzimidazole core exhibited a hydrogen bond and a Pi-H interaction with Asn81
(distance: 2.08 Å) and His85, respectively. Oxygen of compound 6g formed another
hydrogen bond with His244 (distance: 1.99 Å) and additionally, 3,4-dichlorophenyl ring
involved in a Pi-Pi interaction with Val283. It seems that the binding orientation of
compound 6h (Fig. 4) is different from that of 6g. Consequently, 1,2,3-triazole ring in
compound 6h involved in two interactions including a hydrogen bond with His244 (distance:
1
.72 Å) and a Pi-Pi interaction with His85. Furthermore, benzimidazole core formed a
hydrogen bonding interaction with Val283 (distance: 2.40 Å).
Conclusion
In conclusion, hybridization strategy was utilized to prepare and evaluate
benzimidazole-1,2,3-triazole hybrids as tyrosinase inhibitors. According to the obtained
results, compounds 6g and 6h lacking substituents on the imidazole moiety and possessing
3
,4-dichlorobenzyl and 4-bromobenzyl on the 1,2,3-triazole ring were the most potent
tyrosinase inhibitors respectively with IC50 values of 9.42 and 10.34 μM. The inhibitory
potential was dependent on the substituents on the arylidene and benzimidazole moieties.
Consequently, derivatives bearing hydrogen at C6 position of benzimidazole ring had
superior activity over their counterparts possessing methyl substituent at the same position.
Also, compounds containing halogen atoms at the para position of phenyl ring connected to
1
,2,3-triazole core showed good inhibitory activity. The binding patterns of compounds 6g
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and 6h within the active site of tyrosinase were predicted through docking analysis. Docking
results showed that benzimidazole, 1,2,3-triazole and arylidene moieties provided some
favorite interactions with the enzyme active site. The key residues forming interactions were
Asn81, His244, His85 and Val283. Therefore, compounds 6g and 6h could be introduced as
the potent tyrosinase inhibitors and might be promising compounds in the field of drug
discovery.
Experimental Section
General chemistry
1
Melting points were measured on a Kofler hot stage apparatus and are uncorrected. H
1
3
and C NMR spectra were recorded on a Bruker FT-500, using TMS as an internal standard.
IR spectra were obtained on a Nicolet Magna FTIR 550 spectrophotometer (KBr disks).
Elemental analysis was performed on an Elementar Analysensystem GmbH VarioEL CHNS
mode. All chemicals were obtained from Merck and Aldrich and used without further
purification.
General procedure for the synthesis of compounds 6
A mixture of 4-(prop-2-yn-1-yloxy)benzaldehyde 1 (1 mmol), o-phenylenediamine
derivative 2 (1 mmol), and sodium metabisulfite (1.1 mmol) in DMA (12 mL) was heated at
1
20 °C for 12 h. After completion of reaction, the mixture was poured into crushed ice and
the precipitate was filtered off, washed with water, and dried. It was used for next step with
no further purification. Then, a solution of benzyl chloride/bromide derivative 4 (1.1 mmol),
sodium azide (0.9 mmol), and Et
mL) was stirred at room temperature for 1 h. Subsequently, mixture of compound 3 (1
mmol), sodium ascorbate (0.1 mmol), and CuSO .5H O (7 mol %) was added to the freshly
3
N (1.3 mmol) in the mixture of water (4 mL)/t-BuOH (4
4
2
prepared azide derivative 5 and the reaction mixture was stirred at room temperature for 24 h.
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Upon completion of the reaction (checked by TLC), the reaction mixture was diluted with
water, poured into crushed ice; the precipitated product was filtered off, and washed with
cold water. All products were recrystallized from petroleum ether/ethyl acetate to give pure
products 6.
2
-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6a)
Yield: 75%. Mp: 249-250 °C. IR (KBr): 3436, 3135, 3057, 2958, 2876, 1610, 1589 cm . H
NMR (500 MHz, DMSO-d ): 5.24 (s, 2H, CH ), 5.62 (s, 2H, CH ), 7.17-7.39 (m, 9H, H5,
H6, H3′, H5′, H2′′, H3′′, H4′′, H5′′, H6′′), 7.51 (d, J = 7.5 Hz, 1H, H7), 7.63 (d, J = 7.5 Hz,
H, H4), 8.12 (d, J = 7.0 Hz, 2H, H2′, H6′), 8.31 (s, 1H, triazole), 12.74 (s, 1H, NH).
-1 1
6
2
2
1
1
3
6
CNMR (125 MHz, DMSO-d ): 52.8, 61.2, 111.0, 115.1, 118.5, 121.4, 122.0, 123.0, 124.7,
1
5
2
27.9, 128.1, 128.7, 135.9, 142.7, 143.9, 159.3. Anal. Calcd for C23
.02; N, 18.36. Found: C, 72.21; H, 4.88; N, 18.19.
19 5
H N O: C, 72.42; H,
-(4-((1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6b)
-1
1
Yield: 78%. Mp>250 °C. IR (KBr): 3340, 3138, 3055, 2920, 2880, 1611, 1584 cm . H
NMR (500 MHz, DMSO-d ): 2.27 (s, 3H, CH ), 5.23 (s, 2H, CH ), 5.56 (s, 2H, CH ), 7.17-
.23 (m, 8H, H5, H6, H3′, H5′, H2′′, H3′′, H5′′, H6′′), 7.50 (d, J = 7.5 Hz, 1H, H7), 7.61 (d, J
6
3
2
2
7
=
7.5 Hz, 1H, H4), 8.10 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.25 (s, 1H, triazole), 12.70 (s, 1H,
1
3
NH). CNMR (125 MHz, DMSO-d
6
): 20.6, 52.6, 61.2, 110.9, 115.1, 118.4, 121.4, 122.0,
23.0, 124.5, 127.9, 129.2, 132.9, 137.4, 142.7, 143.8, 159.3. Anal. Calcd for C24 O: C,
2.89; H, 5.35; N, 17.71. Found: C, 72.62; H, 5.70; N, 17.90.
-(4-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6c)
1
7
21 5
H N
2
1
H NMR (500 MHz, DMSO-d
6
): Yield: 80%. Mp: 234-235°C. IR (KBr): 3418, 3132, 3056,
-1
2
963, 2877, 1611, 1495 cm . 5.23 (s, 2H, CH ), 5.69 (s, 2H, CH ), 7.19-7.27 (m, 6H, H5,
2
2
H6, H3′, H5′, H5′′, H6′′), 7.35-7.45 (m, 2H, H3′′, H4′′), 7.50 (d, J = 7.5 Hz, 1H, H7), 7.61 (d,
J = 7.5 Hz, 1H, H4), 8.10 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.29 (s, 1H, triazole), 12.70 (s, 1H,
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1
3
NH). CNMR (125 MHz, DMSO-d
6
): 46.9, 61.1, 110.5, 115.0, 115.5 (d, JC-F = 21.2 Hz),
18.5, 121.4, 121.8, 123.0, 124.8 (d, JC-F = 7.5 Hz), 126.3, 127.9, 130.7, 138.2, 142.6, 143.2,
59.3, 161.2 (d, JC-F = 245.0 Hz). Anal. Calcd for C23 O: C, 69.16; H, 4.54; N, 17.53.
1
1
H18FN
5
Found: C, 69.35; H, 4.35; N, 17.67.
-(4-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6d)
2
-1 1
Yield: 84%. Mp: 236-238 °C. IR (KBr): 3420, 3139, 3067, 2924, 2882, 1609, 1507 cm . H
NMR (500 MHz, DMSO-d ): 5.27 (s, 2H, CH ), 5.82 (s, 2H, CH ), 7.17-7.23 (m, 6H, H5,
6
2
2
H6, H3′, H5′, H3′′, H5′′), 7.41 (dd, J = 8.2, 5.5 Hz, 2H, H2′′, H6′′), 7.49 (d, J = 7.0 Hz, 1H,
H7), 7.62 (d, J = 7.0 Hz, 1H, H4), 8.11 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.30 (s, 1H, triazole),
1
3
1
2
1
2.70 (s, 1H, NH). CNMR (125 MHz, DMSO-d
1.2 Hz), 118.5, 122.0, 123.0, 124.6, 127.9, 130.2 (d, JC-F = 8.7 Hz), 132.1, 142.8, 143.8,
59.3, 161.5 (d, JC-F = 241.0 Hz). Anal. Calcd for C23 O: C, 69.16; H, 4.54; N, 17.53.
6
): 52.0, 61.2, 111.0, 115.1, 115.5 (d, JC-F =
H18FN
5
Found: C, 69.31; H, 4.63; N, 17.38.
-(4-((1-(2-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6e)
2
-1
1
Yield: 80%. Mp>250 °C. IR (KBr): 3335, 3189, 2984, 2805, 1617, 1589, 1497 cm . H
NMR (500 MHz, DMSO-d ): 5.25 (s, 2H, CH ), 5.37 (s, 2H, CH ), 7.14-7.26 (m, 4H, H5,
H6, H3′, H5′), 7.36-7.42 (m, 2H, H4′′, H5′′), 7.49 (m, 3H, H4, H7, H6′′), 7.62 (d, J = 7.5 Hz,
H, H3′′), 8.11 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.29 (s, 1H, triazole), 12.72 (s, 1H, NH).
6
2
2
1
1
3
CNMR (125 MHz, DMSO-d
6
): 50.6, 61.1, 111.0, 115.1, 118.5, 121.4, 122.0, 123.0, 125.1,
O:
1
27.7, 127.9, 129.6, 130.2, 130.5, 133.1, 142.6, 143.8, 159.3. Anal. Calcd for C23H18ClN
5
C, 66.43; H, 4.36; N, 16.84. Found: C, 66.61; H, 4.51; N, 16.62.
-(4-((1-(2,3-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole
6f)
Yield: 79%. Mp: 248-250 °C. IR (KBr): 3350, 3190, 3160, 1615, 1589, 1498 cm . H NMR
500 MHz, DMSO-d ): 5.26 (s, 2H, CH ), 5.79 (s, 2H, CH ), 7.17-7.19 (m, 3H, H5, H6,
2
(
-1 1
(
6
2
2
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H6′′), 7.21 (d, J = 9.0 Hz, 2H, H3′, H5′), 7.40 (t, J = 8.0 Hz, 1H, H5′′), 7.50 (d, J = 7.5 Hz,
H, H7), 7.62 (d, J = 7.5 Hz, 1H, H4), 7.66 (dd, J = 8.0, 2.0 Hz, 1H, H4′′), 8.11 (d, J = 9.0
1
1
3
Hz, 2H, H2′, H6′), 8.32 (s, 1H, triazole), 12.72 (s, 1H, NH). CNMR (125 MHz, DMSO-d
6
):
1.1, 61.1, 110.9, 115.1, 118.5, 121.4, 122.0, 123.0, 125.2, 127.9, 128.5, 128.9, 130.5, 130.7,
35.7, 141.8, 142.6, 159.3. Anal. Calcd for C23 O: C, 61.34; H, 3.81; N, 15.75.
5
1
2 5
H17Cl N
Found: C, 61.54; H, 3.66; N, 15.51.
-(4-((1-(3,4-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole
6g)
Yield: 84%. Mp: 230-231 °C. IR (KBr): 3400, 3189, 3150, 2942, 1614, 1581, 1496 cm . H
NMR (500 MHz, DMSO-d ): 5.25 (s, 2H, CH ), 5.65 (s, 2H, CH ), 7.17-7.18 (m, 4H, H5,
H6, H3′, H5′), 7.31 (dd, J = 8.2, 2.0 Hz, 1H, H6′′), 7.50 (d, J = 7.5 Hz, 1H, H7), 7.61-7.67
2
(
-1 1
6
2
2
(m, 3H, H4, H2′′, H5′′), 8.11 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.36 (s, 1H, triazole), 12.72 (s,
1
3
1
1
H, NH). CNMR (125 MHz, DMSO-d
27.9, 128.3, 129.2, 129.3, 130.1, 130.9, 131.2, 136.9, 142.9, 143.8, 159.3. Anal. Calcd for
O: C, 61.34; H, 3.81; N, 15.55. Found: C, 61.11; H, 3.70; N, 15.39.
-(4-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6h)
6
): 51.4, 61.2, 115.1, 121.7, 121.9, 123.0, 124.9,
23 2 5
C H17Cl N
2
-1 1
Yield: 82%. Mp: 239-241 °C. IR (KBr): 3400, 3139, 3061, 2956, 1610, 1496 cm . H NMR
500 MHz, DMSO-d ): 5.24 (s, 2H, CH ), 5.62 (s, 2H, CH ), 7.18-7.22 (m, 4H, H5, H6, H3′,
H5′), 7.29 (d, J = 8.0 Hz, 2H, H2′′, H6′′), 7.53-7.62 (m, 4H, H4, H7, H3′′, H5′′), 8.14 (d, J =
(
6
2
2
1
3
8
d
1
5
2
.5 Hz, 2H, H2′, H6′), 8.33 (s, 1H, triazole), 12.82 (s, 1H, NH). CNMR (125 MHz, DMSO-
6
): 52.0, 61.2, 115.1, 121.3, 122.0, 123.8, 123.9, 124.8, 127.8, 130.1, 131.6, 135.3, 140.8,
42.8, 146.8, 159.3. Anal. Calcd for C23
9.87; H, 4.18; N, 15.40.
5
H18BrN O: C, 60.01; H, 3.94; N, 15.21. Found: C,
-(4-((1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-benzo[d]imidazole (6i)
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-
Yield: 78%. Mp: 120-122 °C. IR (KBr): 3425, 3142, 3077, 2945, 1608, 1520, 1497, 1346 cm
1
1
.
H NMR (500 MHz, DMSO-d
6
): 5.27 (s, 2H, CH
2
), 5.82 (s, 2H, CH
2
), 7.17-7.18 (m, 2H,
H5, H6), 7.22 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.54-7.56 (m, 4H, H4, H7, H2′′, H6′′), 8.12 (d, J
8.5 Hz, 2H, H2′, H6′), 8.24 (d, J = 8.5 Hz, 2H, H3′′, H5′′), 8.39 (s, 1H, triazole), 12.73 (s,
=
1
3
1
1
6
2
6
H, NH). CNMR (125 MHz, DMSO-d ): 51.9, 61.2, 115.1, 121.7, 121.8, 123.0, 123.8,
25.1, 127.9, 128.0, 129.0, 142.9, 143.3, 147.2, 151.2, 159.3. Anal. Calcd for C23
4.78; H, 4.25; N, 19.71. Found: C, 64.61; H, 4.41; N, 19.56.
18 6 3
H N O : C,
-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-benzo[d]imidazole (6j)
-1 1
Yield: 77%. Mp: 190-191 °C. IR (KBr): 3235, 3033, 2923, 1611, 1584, 1495 cm . H NMR
500 MHz, DMSO-d ): 2.42 (s, 3H, Me), 5.23 (s, 2H, CH ), 5.62 (s, 2H, CH ), 6.99 (d, J =
.5 Hz, 1H, H4), 7.19 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.32-7.43 (m, 7H, H5, H7, H2′′, H3′′,
H4′′, H5′′, H6′′), 8.08 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.31 (s, 1H, triazole), 12.58 (s, 1H, NH).
(
6
2
2
8
1
3
CNMR (125 MHz, DMSO-d
25.2, 126.8, 127.8, 127.9, 128.1, 128.7, 130.5, 135.9, 142.7, 150.5, 159.2. Anal. Calcd for
O: C, 72.89; H, 5.35; N, 17.71. Found: C, 72.68; H, 5.18; N, 17.56.
-Methyl-2-(4-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-
benzo[d]imidazole (6k)
Yield: 80%. Mp: 171-173 °C. IR (KBr): 3235, 3030, 2921, 2858, 1612, 1584, 1477 cm . H
NMR (500 MHz, DMSO-d ): 2.27 (s, 3H, Me), 2.42 (s, 3H, Me), 5.22 (s, 2H, CH ), 5.56 (s,
), 7.00 (d, J = 7.5 Hz, 1H, H4), 7.17-7.23 (m, 6H, H3′, H5′, H2′′, H3′′, H5′′, H6′′),
.41-7.55 (m, 2H, H5, H7), 8.08 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.27 (s, 1H, triazole), 12.57
6
): 21.2, 52.8, 61.2, 115.0, 118.0, 121.8, 123.1, 123.2, 124.7,
1
24 21 5
C H N
6
-1 1
6
2
2
7
H, CH
2
1
3
(
bs, 1H, NH). CNMR (125 MHz, DMSO-d
6
): 20.6, 21.2, 52.6, 61.2, 110.8, 115.1, 118.0,
1
1
1
23.1, 124.5, 127.8, 127.9, 128.0, 129.2, 131.3, 132.5, 132.9, 137.5, 142.7, 144.0, 151.0,
59.2. Anal. Calcd for C25
6.88.
23 5
H N O: C, 73.33, H, 5.66; N, 17.10. Found: C, 73.58; H, 5.80; N,
This article is protected by copyright. All rights reserved.

2
-(4-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-
benzo[d]imidazole (6l)
Yield: 83%. Mp: 183-185 °C. IR (KBr): 3201, 2924, 2871, 1613, 1492 cm . H NMR (500
MHz, DMSO-d ): 2.42 (s, 3H, Me), 5.23 (s, 2H, CH ), 5.69 (s, 2H, CH ), 6.99 (d, J = 7.5 Hz,
H, H4), 7.19 (d, J = 8.0 Hz, 2H, H3′, H5′), 7.23-7.48 (m, 6H, H5, H4, H3′′, H4′′, H5′′, H6′′),
-1 1
6
2
2
1
8
1
3
.08 (d, J = 8.0 Hz, 2H, H2′, H6′), 8.30 (s, 1H, triazole), 12.56 (s, 1H, NH). CNMR (125
): 21.2, 46.9, 61.1, 110.7, 115.0, 115.5 (d, JC-F = 21.2 Hz), 118.1, 122.1,
22.3, 122.4, 122.6, 123.2, 123.9, 124.0, 124.8, 127.2, 127.8, 130.7, 142.6, 144.2, 159.1,
61.0 (d, JC-F = 245.2 Hz). Anal. Calcd for C24 O: C, 69.72, H, 4.88; N, 16.94. Found:
MHz, DMSO-d
6
1
1
H20FN
5
C, 69.58; H, 4.67; N, 17.14.
-(4-((1-(2-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-
benzo[d]imidazole (6m)
Yield: 76%. Mp: 227-228 °C. IR (KBr): 3202, 2916, 2855, 1611, 1473, 1447 cm . H NMR
500 MHz, DMSO-d ): 2.42 (s, 3H, Me), 5.27 (s, 2H, CH ), 5.82 (s, 2H, CH ), 6.99 (d, J =
.5 Hz, 1H, H4), 7.19 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.24 (d, J = 7.5 Hz, 1H, H5), 7.35-7.42
m, 4H, H7, H4′′, H5′′, H6′′), 7.53 (dd, J = 7.0, 1.0 Hz, 1H, H3′′), 8.08 (d, J = 8.5 Hz, 2H,
2
-1 1
(
6
2
2
7
(
1
3
H2′, H6′), 8.28 (s, 1H, triazole), 12.55 (s, 1H, NH). CNMR (125 MHz, DMSO-d
6
): 21.2,
5
1
4
0.6, 61.1, 115.1, 120.9, 121.5, 123.2, 123.7, 125.1, 126.6, 127.7, 127.8, 127.9, 129.6, 130.2,
30.5, 130.6, 132.6, 133.1, 142.6, 144.8, 159.1. Anal. Calcd for C24
.69; N, 16.29. Found: C, 66.87; H, 4.80; N, 16.45.
5
H20ClN O: C, 67.05, H,
2
-(4-((1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-
benzo[d]imidazole (6n)
Yield: 80%. Mp: 237-238 °C. IR (KBr): 3225, 2919, 2850, 1612, 1495 cm . H NMR (500
MHz, DMSO-d ): 2.43 (s, 3H, Me), 5.24 (s, 2H, CH ), 5.65 (s, 2H, CH ), 6.97-7.01 (m, 2H,
-1 1
6
2
2
H4, H6''), 7.19 (d, J = 8.0 Hz, 2H, H3′, H5′), 7.27-7.49 (m, 5H, H5, H7, H2'', H4'', H5''), 8.08
This article is protected by copyright. All rights reserved.

1
3
(
d, J = 8.0 Hz, 2H, H2′, H6′), 8.35 (s, 1H, triazole), 12.57 (s, 1H, NH). CNMR (125 MHz,
DMSO-d ): 21.2, 52.0, 61.1, 110.7, 115.0, 118.0, 118.2, 120.8, 122.8, 123.2, 123.4, 124.8,
26.6, 127.8, 128.1, 130.6, 131.2, 131.5, 138.3, 141.7, 142.8, 159.3. Anal. Calcd for
O: C, 67.05, H, 4.69; N, 16.29. Found: C, 67.28; H, 4.84; N, 16.10.
-(4-((1-(2,3-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-
benzo[d]imidazole (6o)
Yield: 80%. Mp: 248-250 °C. IR (KBr): 3198, 2918, 2855, 1612, 1583, 1448 cm . H NMR
500 MHz, DMSO-d ): 2.42 (s, 3H, Me), 5.25 (s, 2H, CH ), 5.79 (s, 2H, CH ), 6.99 (d, J =
.5 Hz, 1H, H4), 7.18-7.23 (m, 3H, H3′, H5′, H6′′), 7.38-7.44 (m, 3H, H5, H7, H5''), 7.67 (d,
J = 8.0 Hz, 1H, H4′′), 8.09 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.33 (s, 1H, triazole), 12.59 (s, 1H,
6
1
24 5
C H20ClN
2
-1 1
(
6
2
2
7
1
3
NH). CNMR (125 MHz, DMSO-d
6
): 21.2, 51.1, 61.1, 115.1, 118.5, 123.2, 123.3, 125.2,
25.3, 126.2, 127.8, 128.5, 128.9, 130.5, 132.2, 132.3, 133.3, 134.1, 135.8, 142.6, 145.2,
59.1. Anal. Calcd for C24 O: C, 62.08, H, 4.12; N, 15.08. Found: C, 62.29; H, 4.40;
1
1
2 5
H19Cl N
N, 15.32.
-(4-((1-(3,4-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-
benzo[d]imidazole (6p)
Yield: 83%. Mp: 225-226 °C. IR (KBr): 3201, 3157, 3031, 2919, 1612, 1585 cm . H NMR
500 MHz, DMSO-d ): 2.42 (s, 3H, Me), 5.24 (s, 2H, CH ), 5.65 (s, 2H, CH ), 6.99 (d, J =
.0 Hz, 1H, H4), 7.18 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.32 (dd, J = 7.2, 1.5 Hz, 1H, H6′′), 7.40-
.66 (m, 4H, H5, H7, H2′′, H5′′), 8.08 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.36 (s, 1H, triazole),
2
-1 1
(
6
2
2
8
7
1
1
1
6
1
3
2.57 (s, 1H, NH). CNMR (125 MHz, DMSO-d
6
): 21.2, 51.4, 61.2, 115.0, 118.1, 123.2,
24.2, 124.9, 125.2, 125.8, 126.1, 126.3, 127.8, 128.3, 130.1, 130.9, 131.2, 136.8, 138.8,
42.9, 148.5, 159.1. Anal. Calcd for C24
1.87; H, 3.89; N, 14.85.
2 5
H19Cl N O: C, 62.08, H, 4.12; N, 15.08. Found: C,
This article is protected by copyright. All rights reserved.

2
-(4-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-methyl-1H-
benzo[d]imidazole (6q)
Yield: 81%. Mp: 208-209 °C. IR (KBr): 3421, 3194, 2983, 2930 cm . H NMR (500 MHz,
DMSO-d ): 2.42 (s, 3H, Me), 5.23 (s, 2H, CH ), 5.61 (s, 2H, CH ), 6.99 (d, J = 7.5 Hz, 1H,
H4), 7.18 (d, J = 8.5 Hz, 2H, H3′, H5′), 7.28 (d, J = 8.5 Hz, 2H, H2′′, H6′′), 7.39-7.47 (m,
-1 1
6
2
2
2
1
1
1
6
H, H5, H7), 7.58 (d, J = 8.5 Hz, 2H, H3′′, H5′′), 8.08 (d, J = 8.5 Hz, 2H, H2′, H6′), 8.30 (s,
1
3
H, triazole), 12.52 (s, 1H, NH). CNMR (125 MHz, DMSO-d
6
): 21.2, 52.0, 61.2, 110.8,
15.0, 118.1, 121.4, 122.7, 122.8, 123.2, 123.5, 124.7, 127.8, 130.1, 131.6, 133.9, 135.3,
42.8, 143.5, 159.1. Anal. Calcd for C24
0.91; H, 4.42; N, 14.53.
5
H20BrN O: C, 60.77, H, 4.25; N, 14.76. Found: C,
6
-Methyl-2-(4-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-1H-
benzo[d]imidazole (6r)
Yield: 85%. Mp: 114-116 °C. IR (KBr): 3201, 3157, 3031, 2919, 1612, 1585 cm . H NMR
500 MHz, DMSO-d ): 2.42 (s, 3H, Me), 5.26 (s, 2H, CH ), 5.81 (s, 2H, CH ), 6.99 (d, J =
.0 Hz, 1H, H4), 7.20 (d, J = 8.0 Hz, 2H, H3', H5'), 7.35 (s, 1H, H7), 7.44 (d, J = 8.0 Hz, 1H,
H5), 7.55 (d, J = 8.5 Hz, 2H, H2′′, H6′′), 8.10 (d, J = 8.0 Hz, 2H, H2′, H6′), 8.24 (d, J = 8.5
-1 1
(
6
2
2
8
1
3
Hz, 2H, H3′′, H5′′), 8.38 (s, 1H, triazole), 12.60 (s, 1H, NH). CNMR (125 MHz, DMSO-
d
1
4
6
): 21.2, 51.9, 61.2, 115.1, 115.2, 118.2, 123.0, 123.2, 123.8, 124.0, 124.8, 125.1, 125.5,
27.8, 129.0, 134.8, 141.0, 142.9, 143.3, 159.2. Anal. Calcd for C24
.58; N, 19.08. Found: C, 65.30; H, 4.37; N, 18.84.
20 6 3
H N O : C, 65.45, H,
Tyrosinase inhibition assay
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) inhibitory activity was
evaluated using L-DOPA as substrate and formation of dopachrome was observed at 475 nm.
All test samples were first dissolved in DMSO at 40 mM and diluted to the required
This article is protected by copyright. All rights reserved.

-1
concentrations. First, 10 L of tyrosinase (0.5 mg.mL ) was mixed with 160 µL of 50 mM
phosphate buffer (pH 6.8) and then 10 µL of the test sample was added to 96-well
°
microplates. After pre-incubation at 28 C for 20 min, 20 L of L-DOPA solution (0.5 mM)
was added to the each well. DMSO without test compound was used as the control and kojic
acid was used as the positive control. Each assay was conducted as three separate replicates.
The inhibitory effects of the tested compounds were expressed as the concentrations that
inhibited 50% of the enzyme activity (IC50).
Kinetic study
A series of experiments were performed to determine the inhibition kinetics of
compound 6g. The inhibitor concentrations were 10 and 50 µM. Substrate L-DOPA
concentrations were 0.000, 0.125, 0.250, 1.000 and 2.000 mM in all kinetic studies. Pre-
incubation and measurement time was as the same as mushroom tyrosinase inhibition assay.
The tyrosinase inhibition rate was then calculated using Lineweaver-Burk plot analysis. The
Michaelis constant (K
m
) and maximal velocity (Vmax) were also calculated by Lineweaver-
Burk plots with different concentrations of L-DOPA up to ten minutes after adding L-DOPA
[24]
during a one minute interval.
Molecular docking study
Molecular docking study was achieved using AutoDock 4.2 and AutoDock Tools
.5.4 (ADT). The X-ray crystal structure of tyrosinase carrying tropolone as innate ligand in
1
binding site (PDB code: 2Y9X) were obtained from protein data bank (http://www.rcsb.org).
Water molecules and tropolone were removed and PDB file of protein was regenerated by
adding polar hydrogen and computing Gasteiger charges. Structures of ligands were sketched
and optimized (by molecular mechanics, MM+, and semi-empirical, AM1, methods) using
This article is protected by copyright. All rights reserved.

Hyperchem software. The PDBQT files were generated by adding charges and defining the
degree of torsions. In order to create the grid parameter file, a grid of 40, 40, and 40 points in
x, y, and z directions with 0.375 Å grid spacing was built using AutoGrid. Rigid
macromolecule and Lamarckian Genetic Algorithm (LGA), with 100 GA runs was selected
to generate the docking parameter file.
Acknowledgment
This work was supported by Research Council of Tehran University of Medical
Sciences with project No. 95-03-96-33047 and Shiraz University of Medical Sciences.
Author Contribution Statement
Mohammad Mahdavi, Arsalan Ashtari, Mahsima Khoshneviszadeh, Sara Ranjbar, and
Ameneh Dehghani synthesized compounds, performed biological tests, docking studies, and
wrote the article. Tahmineh Akbarzadeh and Bagher Larijani analysed data. Mehdi
Khoshneviszadeh and Mina Saeedi designed the experiments and wrote the article.
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Firuzi, M. Khoshneviszadeh, ‘6-Methoxy-3,4-dihydronaphthalenone Chalcone-like
Derivatives as Potent Tyrosinase Inhibitors and Radical Scavengers’, Lett. Drug Des.
Dis. In press
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Fig. 1. Design of tyrosinase inhibitors using molecular hybridization approach.
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6
5
4
3
2
1
0
0
0
0
0
0
0
0
1
5
µM
0 µM
0 µM
-
4
-3 -2 -1
0
1
2
3
4
5
6
7
8
9
-
10
1
/[S] mM-1
Concentration (µM)
Vmax (mM/min)
K (mM)
m
No inhibitor
0.16
0.13
0.11
0.24
0.45
0.62
6
6
g-10 µM
g-50 µM
Fig. 2. Lineweaver−Burk plot of mushroom tyrosinase enzyme inhibition by different
concentrations of 6g in the presence of L-DOPA as a substrate. The reciprocal tyrosinase
inhibitory activity was plotted against the reciprocal substrate concentration (double
reciprocal plot, n = 3). K
reaction velocity.
m
is the Michaelis-Menten constant and Vmax is the maximum
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Fig. 3. Interactions and binding mode of compound 6g into tyrosinase (2Y9X).
Fig. 4. Interactions and binding mode of compound 6h into tyrosinase (2Y9X).
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Scheme 1. Synthesis of benzimidazole-1,2,3-triazole hybrids 6.
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Table 1
Structure of synthesized compounds 6a-r and their inhibitory effects on mushroom tyrosinase.
a
Entry
Compound 6
Ar
IC50 (μM)
X
H
1
2
3
4
5
6
7
8
9
6a
6b
6c
6d
6e
6f
Phenyl
>50
H
4-Methylphenyl
2-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
2,3-Dichorophenyl
3,4-Dichlorophenyl
4-Bromophenyl
4-Nitrophenyl
>50
H
45.22 ± 1.15
46.89 ± 0.52
>50
H
H
H
>50
6g
6h
6i
H
9.42 ± 0.60
10.34 ± 1.06
48.00 ± 1.05
43.30 ± 0.75
>50
H
H
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
6j
CH
CH
CH
CH
CH
CH
CH
CH
3
3
3
3
3
3
3
3
Phenyl
6k
6l
4-Methylphenyl
4-Fluorophenyl
2-Chlorophenyl
3-Chlorophenyl
2,3-Dichorophenyl
3,4-Dichorophenyl
4-Bromophenyl
29.31 ± 0.89
>50
6m
6n
6o
6p
6q
>50
>50
>50
>50
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1
2
8
0
6r
CH
3
4-Nitrophenyl
-
>50
Kojic acid
-
9.28±0.81
a
5
0 % inhibitory concentration (IC50). Values represent means ± SE of four independent experiments
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