1,2,3-Triazole-based kojic acid analogs as potent tyrosinase inhibitors: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2018.10.069

A series of kojic acid-derived compounds 6a-p bearing aryloxymethyl-1H-1,2,3-triazol-1-yl moiety were designed by modifying primary alcoholic group of kojic acid as tyrosinase inhibitors. The target compounds 6a-p were synthesized via click reaction. All

Full text of DOI:10.1016/j.bioorg.2018.10.069

Accepted Manuscript
1,2,3-Triazole-based kojic acid analogs as potent tyrosinase inhibitors: Design,
synthesis and biological evaluation
Morteza Ashooriha, Mehdi Khoshneviszadeh, Mahsima Khoshneviszadeh,
Seyed Ershad Moradi, Alireza Rafiei, Mostafa Kardan, Saeed Emami
PII:
S0045-2068(18)30861-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.069
YBIOO 2603
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 August 2018
30 October 2018
30 October 2018
Please cite this article as: M. Ashooriha, M. Khoshneviszadeh, M. Khoshneviszadeh, S. Ershad Moradi, A. Rafiei,
M. Kardan, S. Emami, 1,2,3-Triazole-based kojic acid analogs as potent tyrosinase inhibitors: Design, synthesis
and biological evaluation, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.069
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1,2,3-Triazole-based kojic acid analogs as potent tyrosinase inhibitors: Design, synthesis
and biological evaluation
Morteza Ashooriha a, Mehdi Khoshneviszadeh b,c, Mahsima Khoshneviszadeh c, Seyed Ershad
Moradi d, Alireza Rafiei e, Mostafa Kardan e, Saeed Emami*,f
a
Department of Medicinal Chemistry, Student Research Committee, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences,
Shiraz, Iran
c Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran
d Pharmaceutical Sciences Research Center, Student Research Committee, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
e Department of Immunology and Molecular and Cell Biology Research Center, Hemoglobinopathy
Institute, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
f Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
Abstract: A series of kojic acid-derived compounds 6a-p bearing aryloxymethyl-1H-1,2,3-
triazol-1-yl moiety were designed by modifying primary alcoholic group of kojic acid as
tyrosinase inhibitors. The target compounds 6a-p were synthesized via click reaction. All
compounds showed very potent anti-tyrosinase activity (IC50s = 0.06-6.80 µM, being superior
to reference drug, kojic acid. In particular, the naphthyloxy analogs 6o and 6p were found to
be 31-155 times more potent than kojic acid. The metal-binding study of selected compound
6o revealed that the prototype compound possesses metal-chelating ability, particularly with
Cu2+ ions. The promising compounds 6o and 6p had acceptable safety profile as
demonstrated by cytotoxicity assay against melanoma (B16) cell line and Human Foreskin
Fibroblast (HFF) cells.
Keywords: Click reaction; hyperpigmentation; kojic acid; 1,2,3-triazole; tyrosinase inhibitor
*Correspondence: Saeed Emami, Department of Medicinal Chemistry, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
E-mail: sdemami12@gmail.com; semami@mazums.ac.ir
1

1. Introduction
Melanin is a general name for wide range of natural pigments found in many species of living
organisms and microorganisms such as animals, plants, fungi and bacteria. In mammals,
melanin is synthesized in various part of their body like skin, eyes, hair and brain [1]. Various
roles are attributed to melanin, but its significant function is protection of cells. For example,
this molecule is pivotal for skin and eyes as a protective molecule against UV light [2].
Melanin is made and secreted by melanocytes in the basal layer of dermis. Despite the key
role of melanin, its overproduction results in several hyperpigmentation disorders, such as
freckles, senile lentigines and melasma [3]. Also, there are some evidences about correlation
between neuromelanin and the pathogenesis of Parkinson’s disease [4]. In agriculture
industry, huge amount of vegetables and fruits are throwing away due to browning. This
color change is also because of melanin overproduction [5]. Skin-whitening and
depigmenting agents, which inhibit melanin formation, are used extensively in cosmetic
products, hence they have great economic value in the cosmetic industry [6].
The process of melanin formation contains several steps including the hydroxylation of L-
tyrosine to L-3-(3,4-dihydroxyphenyl)-alanine (L-DOPA) and also the subsequent oxidation
of DOPA to dopaquinone. Dopaquinone is a reactive substance that can spontaneously
polymerize to form melanin [7]. Two elementary oxidation reactions are rate-limiting steps of
melanin production which catalyzed by tyrosinase enzyme [8]. Thus, this enzyme can be a
suitable target to inhibit melanin formation. Tyrosinase is a metalloenzyme with coupled
binuclear copper active site in which each of copper ion chelate with three histidine residue.
This multifunctional enzyme catalyzes the oxidation of phenol and catechol by the coppers
in presence of oxygen molecule [9, 10].
Many natural and synthetic compounds such as, arbutin, hydroquinone, azelaic acid and kojic
acid (Fig. 1) have been reported as tyrosinase inhibitors [11,12]. However, few of them
possess enough safety and potency in order to use in industry and medications. For example,
there are some overwhelming evidences which indicate mutagenic and cytotoxic effects of
hydroquinone against melanocytes. Furthermore, hydroquinone can cause local skin dryness
or irritation, thereby leading to post-inflammatory hyperpigmentation [11]. Kojic acid is the
most widely used compound among the skin-whitening agents in cosmetic products, but this
compound has poor efficacy, insufficient stability and skin penetration. To overcome these
drawbacks, higher concentration of kojic acid should be used, which results in incidence of
2

side effects [13,14]. Furthermore, kojic acid has been reported to have a high-sensitizing
potential which can cause irritant contact dermatitis [15]. Thus, modification of kojic acid
structure may be a new way to design of potent and safe tyrosinase inhibitors. Kojic acid-
derived tyrosinase inhibitors have been reviewed comprehensively by us [14]. The
esterification of alcoholic group and conversion of the 4-pyron structure into 4-pyridinone
were the main modifications on kojic acid framework. For example, 3,4-methylenedioxy
cinnamic acid ester of kojic acid has been reported as a potent tyrosinase inhibitor, being 14-
fold more potent than parent compound, kojic acid [16]. Also, Ahn et al. described a kojic–
trolox conjugate with more potent tyrosinase inhibitory activity compared to kojic acid [17].
It has been proven that alpha-hydroxy ketone structure of kojic acid has key role in tyrosinase
inhibitory activity. By protecting of the enolic hydroxyl group, the tyrosinase inhibitory
activity of kojic acid is also eliminated. Due to the structural similarity to tyrosine (native
ligand), kojic acid can enter in the active site of tyrosinase and form a chelate complex with
the copper ion [14]. In order to design new kojic acid analogs, we decide to reserve the main
skeleton of kojic acid and substitute alcoholic hydroxyl group with a suitable substituent. For
this purpose, we use click chemistry reaction and formation of 1,2,3-triazole ring. The click
reaction has some outstanding features, including convenience in doing, stereo selectivity,
and stability of the triazole in biologic conditions [18]. Thus we report here, the synthesis and
biological evaluation of kojic acid analogs 6a-p bearing aryloxymethyl-1H-1,2,3-triazol-1-yl
moiety as potent tyrosinase inhibitors (Fig. 1).
2. Results and discussion
2.1. Chemistry
The kojic acid-derived compounds 6a-p were synthesized in several steps as illustrated in
Scheme 1. The commercially available starting material 1 was converted to chlorokojic 2
using thionyl chloride at room temperature [19]. The treatment of chlorokojic 2 with sodium
azide in DMF afforded azidokojic derivative 3 under mild condition [20,21]. On the other
hand, the phenolic compounds 4a-p were reacted with propargyl bromide in the presence of
K2CO3 in refluxing acetone to give the corresponding alkyne ethers 5a-p [22]. Eventually,
two key intermediates 3 and 5 were coupled by click reaction (azide–alkyne 1,3-dipolar
cycloaddition) in a mixture of tert-BuOH: H2O: THF (1:3:10) and catalytic amount of copper
3

sulphate pentahydrate and sodium ascorbate at room temperature to afford target compounds
6a-p.
2.2. Tyrosinase inhibition
The effect of kojic acid derivatives 6a-p on diphenolase activity was determined using
mushroom tyrosinase enzyme in accordance to the reported protocol [23]. For this purpose,
L-DOPA was used as the tyrosinase substrate in spectrophotometric assay. The obtained IC50
values of compounds 6a-p against tyrosinase were reported in Table 1, in comparison with
kojic acid as reference drug.
Generally, all compounds had significant inhibitory activity against tyrosinase, displaying
IC50 values in the range of 0.06-6.80 µM. Their activities were higher than that of kojic acid
(IC50 = 9.28 µM). The naphthyloxy derivatives 6o and 6p with IC50 values of 0.06 and 0.3
µM were found to be the most potent compounds. In particular, 1-naphthyloxy analog 6o was
155 times more active than kojic acid.
From structure-activity relationship (SAR) point of view, the obtained results revealed that
there is a strong relation between tyrosinase inhibition and the type of substituent on the
aryloxy moiety of synthesized compounds. The comparison of unsubstituted phenoxy
compound 6a with halo-substituted derivatives 6b-f indicated that the introduction of fluoro
or chloro group on para or meta position increased the potency. In contract, substitution of
bromo group on para position diminished the inhibitory activity. The differences between
para- and meta-halo compounds were not significant. The presence of second chlorine atom
in compound 6g cannot improve the activity against tyrosinase.
The activity of 4-nitrophenoxy derivative 6i was 2-fold superior to that of parent phenoxy
compound 6a. Thus, the presence of 4-nitro group has positive effect on activity.
Furthermore, cyano and acetamido analogs (6h and 6j) were less potent than 6a. Also,
hydroxymethyl and methoxy derivatives (6k and 6l) were as potent as parent compound 4a,
indicating the tolerance of framework to HOCH2 and CH3O substituents for remaining
intrinsic potency. Interestingly, the presence of 3,4,5-trimethoxy or 3,5-dimethyl substituents
in compounds 6m and 6n resulted in greater potency.
4

Finally, annulation of benzene ring with phenoxy moiety of parent congener 6a resulted in 1-
naphthyloxy and 2-naphthyloxy analogs (6o and 6p, respectively) with highest inhibitory
activity against tyrosinase. However, the 1-naphthyloxy regioisomer 6o was 5-fold more
potent than 2-naphthyloxy derivative 6p.
The mode of inhibition of the enzyme by the most potent derivative, 6o, was determined by
Lineweaver–Burk plot analysis. As shown in Fig. 2, the plots of 1/V versus 1/[S] gave a
series of lines with slightly different slopes, intersecting in the lower left quadrant. The
analysis showed that Vmax and Km values decreased in the presence of increasing
concentrations of compound 6o. With these results, it can presume that the type of inhibition
for compound 6o is a mixed, predominantly uncompetitive inhibition.
2.3. Antioxidant activity evaluation
As mentioned above (Introduction section), tyrosinase enzyme has monophenolase and
diphenolase activities, catalyzing hydroxylation of monophenols to catechols and further
oxidation to o-quinones using oxygen [24]. Probably, compounds with redox capability and
antioxidant property may inhibit the oxidation process of substrates without interacting with
the tyrosinase enzyme [25]. Thus, the potent inhibitors of tyrosinase reported in this
communication were assessed for their possible free radical scavenging activity by using
DPPH test.
The obtained results demonstrated that kojic acid-derived compounds 6a-p possess high anti-
tyrosinase activity with no DPPH free radical scavenging property. Thus, there is no evidence
for indirect action of compounds on oxidization process of the tyrosinase enzyme and the
observed inhibitions are related to the interaction with the enzyme.
2.4. Studies of metal-chelating properties
The metal-chelating properties of the promising compound 6o toward biometals such as Cu2+,
Zn2+, Fe2+ and Al3+ was studied by ultraviolet–visible (UV–Vis) spectrometry. The spectrum
of 6o was shown in Fig. 3A. When CuSO4 was added, the absorption bands at 240 to 300 nm
were enhanced. This enhancement for Cu2+ ion was greater than that of other ions.
Additionally, the absorption in the range of 400 to 600 nm exhibited an enhancement, which
5

was same for all metal ions. These results indicated the formation of complexes between 6o
and tested metal ions.
To evaluate the stoichiometry of the 6o–Cu2+ complex, titration experiment was performed
using UV-vis spectrophotometry. The final concentration of 6o was maintained at 50 µM,
and the absorption spectra were recorded after addition of different concentrations of Cu2+.
The stoichiometry of the 6o–Cu2+ complex was evaluated by determining the changes in
absorbance at 249 nm, where the absorption band was enhanced (Fig. 3B). As shown in Fig.
4, the absorption increased with an increase in Cu2+ concentration and reached a plateau at
Cu2+ molar ratio of 0.5, which indicated that the stoichiometry of 6o–Cu2+ complex was 2:1.
2.5. Cytotoxicity evaluation
In order to verify the safety profile of kojic-derived compounds with potential tyrosinase
inhibitory activity, the most potent compounds 6o and 6p were selected to test their
cytotoxicity against B16 melanoma cell line and human foreskin fibroblast cells in
comparison to reference drug kojic acid. As presented in Table 2, the IC50 values of
compounds 6o and 6p against B16 melanoma cells were 65.0-67.5 µM, being less than that
of kojic acid (IC50= 144.4 µM). Furthermore, the test compounds exhibited cytotoxic activity
against normal HFF cells at very high concentrations (IC50 values ≥138.7 µM). Since, the
promising compounds 6o and 6p showed anti-tyrosinase activity at sub-micromolar
concentrations; we could conclude that these compounds have good safety for potential
application in the treatment of tyrosinase-related disorders.
2.6. Molecular docking study
Molecular docking was carried out to understand the mode of action and type of interactions
between the most potent inhibitor 6o and target enzyme, tyrosinase. As shown in Figure 5,
the inhibitor adopts a folded U-shape conformation which is stabilized by coordination,
hydrogen bonds and hydrophobic interactions. Importantly, the tyrosinase inhibition of
compound 6o apparently depended on the kojic acid residue of the molecule. Indeed, the 3-
hydroxy-4-pyrone moiety of compound 6o can locate into the active site pocket near the
copper center. The 4-carbonyl group coordinates with copper ions, and also forms hydrogen
6

bond with His85. Furthermore, hydrogen bonds are generated between nitrogens of 1,2,3-
triazole ring and His244. It was found the naphthyl moiety is involved in the hydrophobic
interactions with Asn81, Cys83, and Thr324 (Figure 6). These findings confirm the potent
inhibition of tyrosinase by compound 6o, and agreed well with our hypothesis for design of
prototype compounds 6 by inserting the 1,2,3-triazole ring and aryloxy moiety onto the kojic
acid structure.
3. Conclusion
In order to find new potent tyrosinase inhibitors, we have designed and synthesized kojic
acid-derived analogs 6a-p bearing aryloxymethyl-1H-1,2,3-triazol-1-yl moiety via click
reaction. The aryloxy part of the designed molecule was varied for molecular optimization
and structure-activity relationships study. All compounds showed very potent anti-tyrosinase
activity with IC50 values in the range of 0.06-6.80 µM, being superior to reference drug kojic
acid. Certainly, the naphthyloxy analogs 6o and 6p were found to be 31-155 times more
potent than kojic acid. The DPPH free radical scavenging assay showed that the synthesized
compound had no significant antioxidant activity. Thus, there is no evidence for indirect
action of compounds on oxidization process of the tyrosinase enzyme and the observed
inhibitions are related to the interaction with the enzyme. The metal-binding study of selected
compound 6o revealed that the prototype compound possesses metal-chelating ability,
particularly with Cu2+ ions. The promising compounds displayed acceptable safety profile as
revealed by cytotoxicity assay against B16 melanoma cell line and Human Foreskin
Fibroblast (HFF) cells. These properties make the prototype kojic acid-derived compound 6o
as a good candidate for development of potential agents for treatment of tyrosinase-related
disorders in agriculture, food industry, and cosmetics.
4. Experimental
4.1. General
All starting materials, reagents and solvents were purchased from Sigma-Aldrich or Merck
companies and were used as received without further purification. Chlorokojic acid (2) was
synthesized from kojic acid in accordance with the literature method [19].
7

The completion of reaction was checked by TLC using precoated silica gel 60 F254
aluminum sheets. The UV lamp (254 nm) was used for TLC visualization and detection of
spots. Melting points were determined in open capillary tubes using Bibby Stuart Scientific
SMP3 apparatus (Stuart Scientific, Stone, UK) and are uncorrected. The IR spectra were
recorded on a PerkinElmer FT-IR spectrophotometer using KBr disks. The NMR spectra
were recorded using Bruker 400 or 500 spectrometers and chemical shifts are expressed as δ
(ppm). The mass spectra of compounds were obtained using a HP 5937 Mass Selective
Detector (Agilent Technologies, CA, USA). Elemental analyses for C, H and N were
performed by using a CHN-O-rapid elemental analyzer (Heraeus GmbH, Hanau, Germany),
and the results are within ± 0.4% of the theoretical values.
4.2. Preparation of 2-(azidomethyl)-5-hydroxy-4H-pyran-4-one (3)
The procedure of Atkinson et al. [26] was used with some modifications. A mixture of
sodium azide (2.0 g, 30.77 mmol) in DMF (3 mL) was heated at 85 °C for 20 min. After
cooling down to room temperature, chlorokojic (2, 1.5 g, 9.34 mmol) was added to the
mixture and stirred overnight at ambient temperature. After completion of the reaction as
monitored by TLC, the reaction was quenched by addition of water (5 mL). The precipitated
crude product was separated by filtration, washed with water and dried in vacuum oven. The
obtained brown solid was dissolved in tetrahydrofuran and filtered to obtain a clear solution.
Then, the solvent was evaporated under reduced pressure to give pure compound 3. Light
1
brown solid; yield: 80%; m.p. 129-131 °C; H NMR (500 MHz, DMSO-d6) δ: 9.20 (s, 1H,
OH), 8.07 (s, 1H, H-6 pyran), 6.42 (s, 1H, H-3 pyran), 4.32 (s, 2H, methylene).
4.3. General procedure for the preparation of propargyl ethers 5a-p
Propargyl bromide (1.2 mmol) was added to a mixture of phenolic compounds 4a-p (1.0
mmol) and K2CO3 (1.2 mmol) in acetone (5 mL) and the mixture was stirred under reflux for
10 h. After completion of the reaction confirmed by TLC, the solid salts were separated by
filtration and the filtrate was concentrated under reduced pressure to give corresponding pure
propargyl ethers 5a-p.
8

4.4. General procedure for the preparation of 5-hydroxy-2-{(4-(aryloxymethyl)-1H-1,2,3-
triazol-1-yl)methyl}-4H-pyran-4-ones (6a-p)
Copper sulfate pentahydrate (30 mg, 10 mol%) and sodium ascorbate (95.1 mg, 40 mol%)
were suspended in deoxygenated H2O (3 mL) and stirred until the reaction mixture turned
yellow. Then, azidokojic 3 (1.0 mmol) and alkyne 5 (1.2 mmol) were dissolved in THF–tert-
butanol (10:1), and the solution was added to aqueous mixture containing in situ generated
Cu(I). This reaction mixture was stirred for 3 h at ambient temperature. After completion of
the reaction (checked by TLC), the solvent was removed and the residue was dissolved in hot
THF. Then, this reaction mixture was filtered and evaporated to remove solvent under
reduced pressure. The residue was washed with small amount of acetone or methanol and
dried in vacuo to give desired compounds 6a-p.
4.4.1. 5-Hydroxy-2-((4-(phenoxymethyl)-1H-1,2,3-triazol-1-yl)methyl)-4H-pyran-4-one (6a)
White solid; yield 83%; m.p. 161-164 °C; IR (KBr, cm-1): 3246, 3112, 1626, 1500, 1458,
1
1391, 1219, 1033, 857, 756, 689, 482. H NMR (500 MHz, DMSO-d6) δ: 9.27 (br s, 1H,
OH), 8.32 (s, 1H, H-6 pyran), 8.04 (s, 1H, triazole), 7.29 (dd, 2H, J = 8.6 and 7.5 Hz, H-3
and H-5 Ar), 7.02 (d, 2H, J = 8.6 Hz, H-2 and H-6 Ar), 6.94 (t, 1H, J = 7.3 Hz, H-4 Ar), 6.40
(s, 1H, H-3 pyran), 5.61 (s, 2H, OCH2), 5.15 (s, 2H, NCH2). 13C NMR (125 MHz, DMSO-d6)
δ: 174.08, 161.04, 158.44, 146.51, 143.69, 140.50, 129.98, 125.84, 121.35, 115.14, 113.54,
61.31, 50.43. MS (m/z, %): 299 (M+, 20), 178 (100), 151 (12), 125 (75), 112 (16), 94 (54), 77
(16), 67 (33), 51 (12). Anal. Calcd for C15H13N3O4: C, 60.20; H, 4.38; N, 14.04. Found: C,
60.22; H, 4.16; N, 14.00.
4.4.2. 2-((4-((4-Fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-pyran-4-
one (6b)
White solid; yield 85%; m.p. 128-131 °C; IR (KBr, cm-1): 3265, 3112, 3068, 1638, 1506,
1452, 1381, 1276, 1030, 898, 838, 663, 465. 1H NMR (500 MHz, DMSO-d6) δ: 9.28 (br s,
1H, OH), 8.32 (s, 1H, H-6 pyran), 8.05 (s, 1H, triazole), 7.12 (t, 2H, J = 8.7 Hz, H-3 and H-5
Ar), 6.95-7.07 (m, 2H, H-2 and H-6 Ar), 6.39 (s, 1H, H-3 pyran), 5.60 (s, 2H, OCH2), 5.13 (s,
13
2H, NCH2). C NMR (125 MHz, DMSO-d6) δ: 174.08, 161.03, 157.15 (d, JC,F= 234.7 Hz),
154.76, 146.52, 143.55, 140.49, 125.88, 116.55 (d, 2C, JC,F= 8.0 Hz), 116.31 (d, 2C, JC,F=
22.7 Hz), 113.52, 61.99, 50.44. MS (m/z, %): 317 (M+, 20), 207 (25), 178 (100), 151 (16),
9

125 (87), 112 (83), 97 (25), 83 (41), 67 (40), 57 (43). Anal. Calcd for C15H12FN3O4: C, 56.78;
H, 3.81; N, 13.24. Found: C, 56.96; H, 3.89; N, 13.11.
4.4.3. 2-((4-((4-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-pyran-
4-one (6c)
White solid; yield 73%; m.p. 158-160 °C; IR (KBr, cm-1): 3260, 3095, 1615, 1500, 1386,
1
1224, 1024, 789, 690, 466. H NMR (500 MHz, DMSO-d6) δ: 9.32 (s, 1H, OH), 8.34 (s, 1H,
H-6 pyran), 8.06 (s, 1H, triazole), 7.34 (d, 2H, J = 9.0 Hz, H-3 and H-5 Ar), 7.07 (d, 2H, J =
9.0 Hz, H-2 and H-6 Ar), 6.41 (s, 1H, H-3 pyran), 5.62 (s, 2H, OCH2), 5.16 (s, 2H, NCH2).
13C NMR (125 MHz, DMSO-d6) δ: 174.08, 160.99, 157.74, 146.52, 143.32, 140.49, 132.61,
125.96, 117.53, 113.55, 112.82, 61.67, 50.46. MS (m/z, %): 333 (M+, 1), 207 (15), 149 (12),
125 (15), 111 (23), 97 (38), 85 (53), 71 (75), 57 (100). Anal. Calcd for C15H12ClN3O4: C,
53.99; H, 3.62; N, 12.59. Found: C, 54.17; H, 3.54; N, 12.80.
4.4.4. 2-((4-((4-Bromophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-pyran-4-
one (6d)
White solid; yield 78%; m.p. 144-146 °C, IR (KBr, cm-1): 3255, 3097, 1616, 1579, 1490,
1
1458, 1387, 1244, 1027, 914, 792, 756, 685, 596, 502. H NMR (500 MHz, DMSO-d6) δ:
9.32 (s, 1H, OH), 8.34 (s, 1H, H-6 pyran), 8.06 (s, 1H, triazole), 7.46 (d, 2H, J = 9.0 Hz, H-3
and H-5 Ar), 7.02 (d, 2H, J = 8.5 Hz, H-2 and H-6 Ar), 6.41 (s, 1H, H-3 pyran), 5.62 (s, 2H,
OCH2), 5.16 (s, 2H, NCH2). MS (m/z, %): 352 (8), 280 (12), 207 (83), 170 (25), 144 (50),
131 (100), 112 (40), 103 (67), 71 (40), 55 (67). Anal. Calcd for C15H12BrN3O4: C, 47.64; H,
3.20; N, 11.11. Found: C, 47.53; H, 3.25; N, 10.97.
4.4.5. 2-((4-((3-Fluorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-pyran-4-
one (6e)
White solid; yield 81%; m.p. 177-178 °C; IR (KBr, cm-1): 3166, 3110, 2936, 1633, 1501,
1
1381, 1425, 1276, 1032, 897, 676, 466. H NMR (500 MHz, DMSO-d6) δ: 9.28 (s, 1H, OH),
8.34 (s, 1H, H-6 pyran), 8.05 (s, 1H, triazole), 7.31 (dd, 1H, J = 15.9 and 7.3 Hz, H-4 Ar),
6.94 (dt, 1H, J = 11.4 and 2.4 Hz, H-2 Ar), 6.87 (dd, J = 8.4 and 2.3 Hz, H-6 Ar), 6.77 (dt, J
= 8.8 and 2.3 Hz, H-5 Ar), 6.40 (s, 1H, H-3 pyran), 5.61 (s, 2H, OCH2), 5.17 (s, 2H, NCH2).
MS (m/z, %): 317 (M+, 21), 206 (20), 178 (100), 151 (15), 125 (85), 111 (40), 97 (30), 83
(35), 67 (45). Anal. Calcd for C15H12FN3O4: C, 56.78; H, 3.81; N, 13.24. Found: C, 56.76; H,
3.77; N, 13.31.
10

4.4.6. 2-((4-((3-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-pyran-
4-one (6f)
White solid; yield 79%; m.p. 135-136 °C; IR (KBr, cm-1): 3258, 3126, 1636, 1460, 1388,
1229, 1004, 900, 860, 766, 680. 1H NMR (500 MHz, DMSO-d6) δ: 9.28 (s, 1H, OH), 8.33 (s,
1H, H-6 pyran), 8.04 (s, 1H, triazole), 7.31 (t, 1H, J = 8.2 Hz, H-5 Ar), 7.14 (dd, 1H, J = 2.0
and 2.2 Hz, H-2 Ar), 6.85-7.05 (m, 2H, H-4 and H-6 Ar), 6.40 (s, 1H, H-3 pyran), 5.61 (s,
2H, OCH2), 5.19 (s, 2H, NCH2). 13C NMR (125 MHz, DMSO-d6) δ: 174.08, 160.99, 159.39,
146.52, 143.23, 140.49, 134.21, 131.34, 126.02, 121.35, 115.26, 114.34, 113.56, 61.73,
50.46. MS (m/z, %): 333 (M+, 13), 307 (8), 203 (66), 178 (70), 125 (68), 84 (50), 56 (100).
Anal. Calcd for C15H12ClN3O4: C, 53.99; H, 3.62; N, 12.59. Found: C, 53.90; H, 3.73; N,
12.60.
4.4.7. 2-((4-((2,4-Dichlorophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-
pyran-4-one (6g)
White solid; yield 85%; m.p. 150-151 °C; IR (KBr, cm-1): 3300, 1640, 1476, 1381, 1262,
1
1217, 1112, 1059, 875, 804, 712. H NMR (400 MHz, DMSO-d6) δ: 9.32 (s, 1H, OH), 8.37
(s, 1H, H-6 pyran), 8.06 (s, 1H, triazole), 7.58 (br s, 1H, Ar), 7.40 (br s, 2H, Ar), 6.41 (s, 1H,
13
H-3 pyran), 5.63 (s, 2H, OCH2), 5.28 (s, 2H, NCH2). C NMR (125 MHz, DMSO-d6) δ:
174.07, 160.98, 152.93, 146.52, 142.92, 140.47, 129.83, 128.51, 126.167, 125.40, 123.08,
116.22, 113.55, 62.78, 50.48. MS (m/z, %): 365 (2), 342 (7), 279 (14), 207 (72), 167 (31),
149 (100), 111 (17), 97 (65), 85 (35), 71 (55), 57 (86). Anal. Calcd for C15H11Cl2N3O4: C,
48.94; H, 3.01; N, 11.41. Found: C, 49.13; H, 2.64; N, 11.66.
4.4.8. 4-((1-((5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methoxy)benzonitrile (6h)
White solid; yield 93%; m.p. 204-206 °C; IR (KBr, cm-1): 3742, 3251, 3091, 2925, 2464,
1
1618, 1501, 1458, 1401, 1350, 1216, 1133, 1020, 947, 866, 763, 676, 508. H NMR (500
MHz, DMSO-d6) δ: 9.32 (s, 1H, OH), 8.38 (s, 1H, H-6 pyran), 8.06 (s, 1H, triazole), 7.79 (d,
2H, J = 8.5 Hz, H-3 and H-5 Ar), 7.22 (d, 2H, J = 8.5 Hz, H-2 and H-6 Ar), 6.41 (s, 1H, H-3
pyran), 5.62 (s, 2H, OCH2), 5.28 (s, 2H, NCH2). 13C NMR (125 MHz, DMSO-d6) δ: 174.08,
161.88, 160.96, 146.52, 142.86, 140.50, 134.67, 126.19, 119.53, 116.29, 113.57, 103.69,
61.80, 50.48. MS (m/z, %): 324 (M+, 15), 206 (38), 178 (80), 125 (100), 97 (26), 67 (38).
Anal. Calcd for C16H12N4O4: C, 59.26; H, 3.73; N, 17.28. Found: C, 59.42; H, 3.71; N, 17.14.
11

4.4.9. 5-Hydroxy-2-((4-((4-nitrophenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4H-pyran-4-
one (6i)
White solid; yield 71%; m.p. 212-214 °C; IR (KBr, cm-1): 3271, 3123, 3073, 2936, 1643,
1512, 1386, 1276, 1134, 1024, 909, 887, 673. 1H NMR (500 MHz, DMSO-d6) δ: 9.33 (s, 1H,
OH), 8.41 (s, 1H, H-6 pyran), 8.23 (d, 2H, J = 9.0 Hz, H-3 and H-5 Ar), 8.07 (s, 1H,
triazole), 7.28 (d, 2H, J = 9.5 Hz, H-2 and H-6 Ar), 6.42 (s, 1H, H-3 pyran), 5.64 (s, 2H,
OCH2), 5.35 (s, 2H, NCH2). MS (m/z, %): 344 (M+, 4), 206 (42), 178 (80), 125 (100), 97
(27), 67 (38). Anal. Calcd for C15H12N4O6: C, 52.33; H, 3.51; N, 16.27. Found: C, 52.19; H,
3.43; N, 16.02.
4.4.10. N-(4-((1-((5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methoxy)phenyl)acetamide (6j)
White solid; yield 69%; m.p. 216-218 °C, IR (KBr, cm-1): 3333, 3140, 3099, 1657, 1543,
1
1507, 1447, 1401, 1236, 1126, 1042, 957, 823, 672, 595, 520. H NMR (500 MHz, DMSO-
d6) δ: 9.81 (s, 1H, NH), 9.32 (s, 1H, OH), 8.33 (s, 1H, H-6 pyran), 8.07 (s, 1H, triazole), 7.48
(d, 2H, J = 9.0 Hz, H-3 and H-5 Ar), 6.97 (d, 2H, J = 9.0 Hz, H-2 and H-6 Ar), 6.42 (s, 1H,
H-3 pyran), 5.62 (s, 2H, OCH2), 5.11 (s, 2H, NCH2), 2.01 (s, 3H, CH3). MS (m/z, %): 356
(M+, 8), 232 (15), 178 (8), 151 (70), 125 (15), 109 (100), 80 (20), 53 (8). Anal. Calcd for
C17H16N4O5: C, 57.30; H, 4.53; N, 15.72. Found: C, 57.11; H, 4.65; N, 15.71.
4.4.11. 5-Hydroxy-2-((4-((4-(hydroxymethyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-
4H-pyran-4-one (6k)
White solid; yield 80%; m.p. 200-201 °C; IR (KBr, cm-1): 3255, 3172, 2910, 1629, 1510,
1
1458, 1395, 1218, 1012, 758, 689. H NMR (500 MHz, DMSO-d6) δ: 9.33 (s, 1H, OH), 8.34
(s, 1H, H-6 pyran), 8.07 (s, 1H, triazole), 7.24 (d, 2H, J = 8.5 Hz, H-3 and H-5 Ar), 6.99 (d,
2H, J = 8.5 Hz, H-2 and H-6 Ar), 6.42 (s, 1H, H-3 pyran), 5.62 (s, 2H, OCH2), 5.14 (s, 2H,
NCH2), 5.07 (br s, 1H, OH), 4.42 (s, 2H, CH2OH). MS (m/z, %): 329 (M+, 4), 311 (20), 207
(11), 178 (100), 151 (15), 125 (92), 107 (30), 67 (42). Anal. Calcd for C16H15N3O5: C, 58.36;
H, 4.59; N, 12.76. Found: C, 58.40; H, 4.67; N, 12.53.
4.4.12. 5-Hydroxy-2-((4-((4-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4H-
pyran-4-one (6l)
12

White solid; yield 93%; m.p. 132-133 °C; IR (KBr, cm-1): 3124, 1642, 1589, 1506, 1260,
1
1117, 1008, 789. H NMR (500 MHz, DMSO-d6) δ: 9.33 (br s, 1H, OH), 8.32 (s, 1H, H-6
pyran), 8.07 (s, 1H, triazole), 6.97 (d, 2H, J = 8.5 Hz, Ar), 6.87 (d, 2H, J = 9.0 Hz, Ar), 6.40
(s, 1H, H-3 pyran), 5.62 (s, 2H, OCH2), 5.09 (s, 2H, NCH2), 3.70 (s, 3H, OCH3). MS (m/z,
%): 329 (M+, 61), 178 (38), 124 (100), 109 (70), 95 (30), 67 (23). Anal. Calcd for
C16H15N3O5: C, 58.36; H, 4.59; N, 12.76. Found: C, 58.62; H, 4.62; N, 12.49.
4.4.13. 5-Hydroxy-2-((4-((3,4,5-trimethoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-
4H-pyran-4-one (6m)
White solid; yield 78%; m.p. 146-147°C; IR (KBr, cm-1): 3234, 1679, 1613, 1506, 1405,
1
1267, 1182, 1090, 838, 580, 482. H NMR (500 MHz, DMSO-d6) δ: 9.33 (br s, 1H, OH),
8.34 (s, 1H, H-6 pyran), 8.07 (s, 1H, triazole), 6.40 (s, H, H-3 pyran), 6.35 (s, 2H, H-2 and H-
6 Ar), 5.62 (s, 2H, OCH2), 5.14 (s, 2H, NCH2), 3.75 (s, 6H, 2×CH3O), 3.57 (s, 3H, CH3O).
13C NMR (125 MHz, DMSO-d6) δ: 174.07, 161.10, 154.96, 153.79, 146.53, 143.71, 140.48,
125.79, 113.49, 93.32, 93.23, 61.77, 60.57, 56.33, 50.44. MS (m/z, %): 389 (M+, 45), 374 (8),
184 (88), 169 (100), 144 (18), 125 (45), 97 (12), 69 (25). Anal. Calcd for C18H19N3O7: C,
55.53; H, 4.92; N, 10.79. Found: C, 55.31; H, 5.14; N, 11.02.
4.4.14. 2-((4-((3,5-Dimethylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-5-hydroxy-4H-
pyran-4-one (6n)
White solid; yield 88%; m.p. 174-175°C; IR (KBr, cm-1): 3250, 3091, 2927, 1618, 1501,
1
1401, 1350, 1269, 1219, 1133, 1020, 947, 857, 764, 675, 504. H NMR (500 MHz, DMSO-
d6) δ: 9.32 (br s, 1H, OH), 8.31 (s, 1H, H-6 pyran), 8.06 (s, 1H, triazole), 6.64 (s, 2H, H-2 and
H-6 Ar), 6.59 (s, 1H, H-4 Ar), 6.40 (s, 1H, H-3 pyran), 5.61 (s, 2H, OCH2), 5.10 (s, 2H,
13
NCH2), 2.22 (s, 6H, 2×CH3). C NMR (125 MHz, DMSO-d6) δ: 174.08, 161.08, 158.48,
146.52, 143.85, 140.48, 139.11, 125.72, 122.97, 113.50, 112.87, 113.50, 112.87, 61.23,
50.42, 21.51. MS (m/z, %): 327 (M+, 3), 281 (8), 266 (8), 207 (30), 160 (16), 145 (100), 112
(60), 91 (45), 71 (67), 55 (90). Anal. Calcd for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84.
Found: C, 62.42; H, 5.19; N, 13.01.
4.4.15. 5-Hydroxy-2-((4-((naphthalen-1-yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4H-
pyran-4-one (6o)
White solid; yield 70%; m.p. 130-131 °C; IR (KBr, cm-1): 3742, 3059, 1640, 1586, 1505,
1
1452, 1390, 1269, 1212, 1151, 1097, 971, 773, 578, 469. H NMR (500 MHz, DMSO-d6) δ:
13

9.33 (br s, 1H, OH), 8.47 (s, 1H, H-6 pyran), 8.12 (d, 1H, J = 8.0 Hz, H-8 naphthalene), 8.07
(s, 1H, triazole), 7.87 (d, 1H, J = 8.0 Hz, H-5 naphthalene), 7.45-7.54 (m, 3H, H-4, H-6 and
H-7 naphthalene), 7.44 (t, 1H, J = 8.0 Hz, H-3 naphthalene), 7.18 (d, J = 7.5 Hz, H-2
13
naphthalene), 6.44 (s, 1H, H-3 pyran), 5.65 (s, 2H, OCH2), 5.37 (s, 2H, NCH2). C NMR
(500 MHz, DMSO-d6) δ: 174.10, 161.06, 153.90, 146.53, 143.77, 140.52, 134.51, 127.94,
126.95, 126.59, 125.83, 125.36, 121.98, 120.86, 113.56, 106.33, 99.48, 62.08, 50.48. MS
(m/z, %): 349 (M+, 25), 178 (33), 144 (100), 115 (78), 97 (7), 67 (13). Anal. Calcd for
C19H15N3O4: C, 65.32; H, 4.33; N, 12.03. Found: C, 65.18; H, 4.54; N, 11.87.
4.4.16. 5-Hydroxy-2-((4-((naphthalen-2-yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4H-
pyran-4-one (6p)
White solid; yield; 65%; m.p. 200-201 °C; IR (KBr, cm-1): 3066, 1643, 1508, 1393, 1219,
1
1127, 1013, 830, 758, 475. H NMR (500 MHz, DMSO-d6) δ: 9.33 (s, 1H, OH), 8.40 (s, 1H,
H-6 pyran), 8.07 (s, 1H, triazole), 7.84 (d, 2H, J = 9.0 Hz, H-5 and H-8 naphthalene), 7.82 (d,
1H, J = 8.0 Hz, H-4 naphthalene), 7.51 (d, 1H, J = 2.5 Hz, H-1 naphthalene), 7.47 (dt, 1H, J
= 8.0 and 1.2 Hz, H-6 naphthalene), 7.36 (dt, 1H, J = 8.0 and 1.2 Hz, H-7 naphthalene), 7.20
(dd, 1H, J = 9.0 and 2.5 Hz, H-3 naphthalene), 6.44 (s, 1H, H-3 pyran), 5.63 (s, 2H, OCH2),
13
5.29 (s, 2H, NCH2). C NMR (125 MHz, DMSO-d6) δ: 174.10, 161.03, 156.34, 146.54,
143.57, 140.50, 134.64, 129.86, 129.09, 127.99, 127.22, 126.92, 125.97, 124.21, 119.11,
113.58, 107.71, 61.52, 50.48. MS (m/z, %): 349 (M+, 27), 178 (15), 144 (100), 115 (61), 67
(11). Anal. Calcd for C19H15N3O4: C, 65.32; H, 4.33; N, 12.03. Found: C, 65.34; H, 4.26; N,
12.09.
4.5. Anti-tyrosinase assay
Mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.) was subjected to enzymatic
inhibition assay using L-DOPA as substrate [23]. The stock solutions of test compounds 6a-
p and kojic acid were prepared in DMSO (40 mM) and then diluted with phosphate buffer
(pH= 6.8) to the required concentrations. Firstly, 10 L of mushroom tyrosinase (0.5 mg/mL)
was mixed with 160 µL of phosphate buffer (50 mM, pH= 6.8) in 96-well microplates, and
then 10 µL of the test compound was added. After the mixture was pre-incubated at 28 °C for
20 min, 20 L of L-DOPA solution (0.5 mM) was added to each well and dopachrome
formation was monitored at 475 nm for 10 min. Each assay was conducted as triplicate and
14

DMSO without test compounds was used as control. The final concentration of DMSO in the
test solution was less than 2.0%. The percent of inhibition was calculated according to the
following equation:
Inhibition (%) =100 × (Abscontrol − Abscompound)/Abscontrol
The inhibitory effect of each compound was expressed as the required concentration for 50%
inhibition of the enzyme activity (IC50).
4.6. Kinetic analysis of the tyrosinase inhibition
A series of experiments were performed to determine the inhibition kinetics of compound 6o.
The inhibitor concentrations were: 0.25, 0.5, 1 µM. Substrate L-DOPA concentrations were
0, 0.1, 0.25, 0.75, and 1.5 mM in all kinetic studies. Pre-incubation and measurement time
were the same as reported in the mushroom tyrosinase inhibition assay protocol. Maximum
initial velocity was determined from initial linear portion of absorbance up to ten minutes
after addition of L-DOPA with 1 min interval.
4.7. DPPH radical scavenging assay
The antioxidant activity of all synthesized compounds 6a-p was determined by the
diphenylpicrylhydrazine (DPPH) radical scavenging assay [27]. Various concentrations (1-
100 µM) of the compounds 6a-p in methanol were prepared in a 96-well microtiter plate.
Then, the DPPH solution )5 µL, 1 mg/mL dissolved in methanol) was added to each well and
the plate was shaken gently and left in the dark for 30 min. The absorbance of the solution in
each well was measured at 517 nm by an ELISA reader (BioTek). The percentage of total
radical scavenging activity was calculated based on the formula: (%) = 100× [(Abs(control)
–
Abs(sample) )/Abs(control)], where Abs(control) = Abs(DPPH) + Abs(solvent)
.
4.8. Metal-chelating studies
The metal-chelating studies were performed with a JASCO V-630 spectrophotometer
(Tokyo, Japan). The UV absorption spectra of compound 6o, in the absence or presence of
sulfate salts of Cu2+, Al3+, Fe2+, and Zn2+ were recorded with wavelength ranging from 200 to
15

600 nm after incubating for 30 min at room temperature. The final volume of reaction
mixture was 5 mL, and the final concentrations of tested compound and metals were 50 µM.
The stoichiometry of the 6o–Cu2+ complex was determined from molar ratio method [28] by
titrating the methanol solution of tested compound with ascending concentrations of CuSO4.
The final concentration of tested compound was 50 µM, and the final concentration of Cu2+
ranged from 5 to 50 µM. The UV spectra were recorded and treated by numerical subtraction
of CuSO4 and 6o at corresponding concentrations. The absorbance of the absorption peak at
249 nm was plotted versus the mole fraction of Cu2+. The breakpoint indicated the
stoichiometry of the compound–Cu2+ complex.
4.9. Cytotoxicity determination (MTT assay)
B16 melanoma cell line and HFF (Human Foreskin Fibroblast) cells were obtained from
Pasture Institute, Iran. The B16 cells (8000 cells/well) and HFF cells (10000 cells/well) were
seeded in RPMI-1640 and DMEM mediums, respectively. The cultured medium was
supplemented with 10% fetal bovine serum (FBS), 100 unit/ml of penicillin-G, 100 μg/ml of
streptomycin in each well of a 96-well plate and incubated in a humidified incubator
containing 5% CO2 at 37 °C. The selected compounds 6o and 6p, and reference drug kojic
acid were dissolved in dimethyl sulfoxide (DMSO) and then diluted with the desired medium
to the different concentrations. The final concentration of DMSO was less than 1% in
medium culture. The cultured cells were treated with various concentrations of compounds
and incubated at 37 °C for 48 h. The culture medium was replaced with fresh medium
containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) with a final
concentration of 0.5 mg/mL, and incubated for further 4 h. Then, the medium containing
MTT was removed and the remaining formazan crystals were dissolved in DMSO. The
absorbance of each well was recorded in an ELISA reader (BioTek) at 570 nm [23]. Two
independent experiments in triplicate were carried out for determination of cell viability for
each compound. The IC50 values were calculated from concentration–viability rate curves
using Prism 6, GraphPad Software and expressed as means ± SD.
16

4.10. Docking study
The X-ray structure of mushroom tyrosinase with the cognate ligand tropolone was extracted
from the Protein Data Bank (PDB code 2Y9X). Before docking, some corrections should be
made, such as, the removal of water molecules, the addition of Gasteiger charges and polar
hydrogens in enzyme molecule by using the AutoDock tools (ADT) 1.5.4 package. The
ligand was constructed and energy minimized using MOPAC Chem3D Ultra 8.0.3. Finally,
ADT, Discovery Studio, ViewerLite, and LIGPLOT plus were used to evaluate the docking
of ligand in the active site of tyrosinase and 2D and 3D visualization.
Acknowledgments
This work was supported by a grant (No. 2781) from the Research Council of Mazandaran
University of Medical Sciences, Sari, Iran. This work was related to the Ph.D. thesis of MA
(Faculty of Pharmacy, Mazandaran University of Medical Sciences).
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20

Captions:
Figure 1. Structures of well-known tyrosinase inhibitors and designed compounds.
Figure 2. Lineweaver-Burk plots for inhibition of selected compound 6o on mushroom
tyrosinase for the oxidation of L-DOPA.
Figure 3. A) The UV spectrum of compound 6o (50 µM, in methanol) alone or in the
presence of sulfate salt of Cu2+, Fe2+, Zn2+ and Al3+ (50 µM, in methanol). B) UV spectrum of
compound 6o (final concentration was 50 µM) alone or in the presence of Cu(II) (the final
concentration of Cu2+ ranged from 0 µM to 50 µM).
Figure 4. Determination of the stoichiometry of 6o-Cu2+ complex by using molar ratio
method through titration the methanol solution of compound 6o with ascending amounts of
CuSO4. The final concentration of tested compound was 50 µM, and the final concentration
of Cu2+ ranged from 5 to 50 µM.
Figure 5. 3D presentations for binding mode of compound 6o into the binding site of
tyrosinase.
Figure 6. 2D representation for the interactions of compound 6o with the residues in the
active site of tyrosinase, visualized by LIGPLOT.
Scheme 1. Synthesis of compounds 6a-p.
21

Table 1. Tyrosinase inhibitory activity of compounds 6a-p.
Compound
Ar
IC50 (µM)
1.33 ± 0.18
0.88 ± 0.03
6a
6b
0.69 ± 0.091
6.80 ± 0.42
6c
6d
6e
6f
1.07 ± 0.16
0.99 ± 0.15
1.12 ± 0.18
6g
6.29 ± 1.28
0.52 ± 0.13
2.64 ± 0.13
6h
6i
6j
1.32 ± 0.15
1.24 ± 0.25
6k
6l
0.87 ± 0.17
0.74 ± 0.08
0.06 ± 0.02
6m
6n
6o
0.30 ± 0.14
9.28 ± 1.12
6p
Kojic acid
-
22

Table 2. Cytotoxic activity (IC50, µM) of selected compounds 6o, 6p and kojic acid against
melanoma (B16) and Human Foreskin Fibroblast (HFF) cell lines.
Compound
6o
Structure
B16
HFF
65.0 ± 1.1
138.7 ± 1.5
67.5 ± 0.8
149.5 ± 0.9
>150
6p
144.4 ± 2.3
Kojic acid
23

Figure 1. Structures of well-known tyrosinase inhibitors and designed compounds.
24

Figure 2. Lineweaver-Burk plots for inhibition of selected compound 6o on mushroom
tyrosinase for the oxidation of L-DOPA.
25

Figure 3. A) The UV spectrum of compound 6o (50 µM, in methanol) alone or in the
presence of sulfate salt of Cu2+, Fe2+, Zn2+ and Al3+ (50 µM, in methanol). B) UV spectrum of
compound 6o (final concentration was 50 µM) alone or in the presence of Cu(II) (the final
concentration of Cu2+ ranged from 0 µM to 50 µM).
26

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.2
0.4
0.6
0.8
1
1.2
mol Cu2+/mol Ligand
Figure 4. Determination of the stoichiometry of 6o-Cu2+ complex by using molar ratio
method through titration the methanol solution of compound 6o with ascending amount of
CuSO4. The final concentration of tested compound was 50 µM, and the final concentration
of Cu2+ ranged from 5 to 50 µM.
27

Figure 5. 3D presentations for binding mode of compound 6o into the binding site of
tyrosinase.
28

Figure 6. 2D representation for the interactions of compound 6o with the residues in the
active site of tyrosinase, visualized by LIGPLOT.
29