Anti-melanogenesis and anti-tyrosinase properties of aryl-substituted acetamides of phenoxy methyl triazole conjugated with thiosemicarbazide: Design, synthesis and biological evaluations

Article abstract of DOI:10.1016/j.bioorg.2021.104979

A series of aryl phenoxy methyl triazole conjugated with thiosemicarbazides were designed, synthesized, and evaluated for their tyrosinase inhibitory activities in the presence of L-dopa and L-tyrosine as substrates. All the compounds showed tyrosinase inhibition in the sub-micromolar concentration. Among the derivatives, compound 9j bearing benzyl displayed exceptionally high potency against tyrosinase with IC₅₀ value of 0.11 μM and 0.17 μM in the presence of L-tyrosine and L-dopa as substrates which is significantly lower than that of kojic acid as the positive control with an IC₅₀ value of 9.28 μM for L-tyrosine and 9.30 μM for L-dopa. According to Lineweaver–Burk plot, 9j demonstrated an uncompetitive type of inhibition in the kinetic assay. Also, in vitro antioxidant activities determined by DPPH assay recorded an IC₅₀ value of 68.43 μM for 9i. The melanin content of 9j was determined on B16F10 melanoma human cells which demonstrated a significant reduction of the melanin content. Moreover, the binding energies corresponding to the same ligand as well as computer-aided drug-likeness and pharmacokinetic studies were also carried out. Compound 9j also possessed metal chelation potential correlated to its high anti-TYR activity.

Full text of DOI:10.1016/j.bioorg.2021.104979

Journal Pre-proofs
Anti-melanogenesis and anti-tyrosinase properties of aryl-substituted acet-
amides of phenoxy methyl triazole conjugated with thiosemicarbazide Design,
synthesis and biological evaluations
Hona Hosseinpoor, Aida Iraji, Sadegh Askari, Sara Moghadam Farid, Najmeh
Edraki, Samanesadat Hosseini, Akram Jamshidzadeh, Bagher Larijani,
Somayeh Pirhadi, Mahshid Attarroshan, Mohammad Mahdavi, Mehdi
Khoshneviszadeh
PII:
S0045-2068(21)00356-4
DOI:
https://doi.org/10.1016/j.bioorg.2021.104979
YBIOO 104979
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 January 2021
27 April 2021
5 May 2021
Please cite this article as: H. Hosseinpoor, A. Iraji, S. Askari, S. Moghadam Farid, N. Edraki, S. Hosseini, A.
Jamshidzadeh, B. Larijani, S. Pirhadi, M. Attarroshan, M. Mahdavi, M. Khoshneviszadeh, Anti-melanogenesis
and anti-tyrosinase properties of aryl-substituted acetamides of phenoxy methyl triazole conjugated with
thiosemicarbazide Design, synthesis and biological evaluations, Bioorganic Chemistry (2021), doi: https://
doi.org/10.1016/j.bioorg.2021.104979
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Anti-melanogenesis and anti-tyrosinase properties of aryl-substituted acetamides of phenoxy
methyl triazole conjugated with thiosemicarbazide
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Design, synthesis and biological evaluations
This work is dedicated to the memory of Professor Abbas Shafiee (1937‐2016).
Hona Hosseinpoora, Aida Irajib, Sadegh Askaric, Sara Moghadam Faridf, Najmeh Edrakib, Samanesadat
Hosseinid, Akram Jamshidzadehe, Bagher Larijanif, Somayeh Pirhadib, Mahshid Attarroshanb,
Mohammad Mahdavif*, Mehdi Khoshneviszadeha,b*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences,
71345 Shiraz, Iran
b Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences,
71348 Shiraz, Iran
c School of Chemistry, College of Science, University of Tehran, Tehran, Iran
d Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
e Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran
f Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences
Institute, Tehran University of Medical Sciences, Tehran, Iran
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*Corresponding authors:
Mohammad Mahdavi, PhD
Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences,
Tehran, Iran.
E-mail: mahdavi.chem@gmail.com
Mehdi Khoshneviszadeh, PharmD, PhD
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz,
Iran
Tel: +98 713 230 3872; Fax: +98 713 230 2225
E-mail: M.khoshneviszadeh@gmail.com
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1
Abstract
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A series of aryl phenoxy methyl triazole conjugated with thiosemicarbazides were designed, synthesized,
and evaluated for their tyrosinase inhibitory activities in the presence of L-dopa and L-tyrosine as
substrates. All the compounds showed tyrosinase inhibition in the sub-micromolar concentration. Among
the derivatives, compound 9j bearing benzyl displayed exceptionally high potency against tyrosinase with
IC50 value of 0.11ꢀμM and 0.17 μM in the presence of L-tyrosine and L-dopa as substrates which is
significantly lower than that of kojic acid as the positive control with an IC50 value of 9.28 μM for L-
tyrosine and 9.30 μM for L-dopa. According to Lineweaver–Burk plot, 9j demonstrated an uncompetitive
type of inhibition in the kinetic assay. Also, in vitro antioxidant activities determined by DPPH assay
recorded an IC50 value of 68.43 μM for 9i. The melanin content of 9j was determined on B16F10 melanoma
human cells which demonstrated a significant reduction of the melanin content. Moreover, the binding
energies corresponding to the same ligand as well as computer-aided drug-likeness and pharmacokinetic
studies were also carried out. Compound 9j also possessed metal chelation potential correlated to its high
anti-TYR activity.
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Keywords: Tyrosinase inhibitor; Organic synthesis; Molecular docking; Copper chelation; Melanin
content.
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1. Introduction
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Melanin as the main natural biological polymer pigment has a high determining role in skin and hair
pigmentation [1] which results in photoprotective function in humans [2]. However, the accumulation of an
abnormal amount of melanin causes dermatological disorders such as freckles, melasma, lentigo [3] as well
as increases the risk of cancer and Parkinson's disease [4]. Although melanogenesis is a complicated process,
it is highly accepted that tyrosinase enzyme (TYR) regulates the biosynthetic pathway for melanin formation
[5]. Tyrosinase (EC 1.14.18.1, polyphenol oxidase) is type-3 copper metalloprotein that contains two copper
atoms in its active sites that enable catalysis of two reactions in the presence of oxygen: 1) hydroxylation and
oxidation of monophenols to o-diphenols 2) oxidation of o-diphenols to o-quinones [6]. The TYR takes α-
helical structure with the dinuclear copper, and each atom of the copper is ligated to three histidines
comprised of six histidine residues. It seems that the role of Cu(I) and/or Cu(II) is critical in charge transfer
and melanin synthesis [7]. Mentioned undesirable phenomena related to melanin have persuaded researchers
to search for new potent tyrosinase inhibitors using in skin whitening and anti-browning of foods [8].
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Some substances have been well described as tyrosinase inhibitors (TYRIs) such as kojic acid and arbutin;
however, these compounds possess undesirable side effects, including carcinogenicity and cytotoxicity [9,
10]. Mentioned side effects promote researchers to find novel TYRIs. In this regard, numerous TYRIs from
natural or synthetic sources have been reported such as thiosemicarbazones [11], phenyl benzoic acid [12],
thiazole [13], triazole [14], pyrimidine [15], benzonitriles [16], xanthone [17], carbazole [18] and
apocarotenoids derivatives [19]. It seems that TYRIs have become increasingly important in medical and
cosmetic products. Therefore, in this study hybridization designing approaches were performed to connect
potent pharmacophores in a single molecule.
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2. Results and discussion
3
2.1. Design strategy
4
Thiosemicarbazide is now widely recognized as an important biological and pharmacological molecule. In
the past few years, studies have revealed that thiosemicarbazide plays a critical role in targeting TYR. It
was found that thiosemicarbazide-containing compounds A-B (Fig. 1) displayed potent tyrosinase
inhibition in comparison to kojic acid [20]. Soares et al reported thiosemicarbazone derivatives (compound
C, Fig. 1.) as a strong inhibitor of the mushroom tyrosinase with IC50= 0.35 μM [21]. Recently, we have
reported aryl-thiosemicarbazide as highly potent tyrosinase inhibitors. Specifically, compound D was the
most potent compound with a 128 fold increase in potency compared to kojic acid (IC50= 0.05 μM). The
results indicated that thiosemicarbazones significantly inhibited the tyrosinase activity with an IC50 value
lower than positive control in most cases. This potency may be due to the presence of highly active groups
with the capability of H-bound interactions with TYR active site [22]. However, limited data are available
related to cytotoxicity, cell-based melanin content, and copper chelation capacity of mentioned compounds.
Triazole moiety is an important heterocyclic ring that possesses a high dipole moment, rigidity, and
solubility with the potency of forming hydrogen bond interactions with the biological receptors [23-25].
However, there are a few 1,2,3-triazole-containing hybrids as TYRIs that include triazole-benzoimidazole
(E, IC50 = 9.42 μM) [26], triazole-isoindoline (F, IC50 = 26.55 μM) [27], triazole-5-hydroxy-pyranone (G,
IC50 = 0.52 μM) [28]. Based on the biological activities of triazole compounds, recently a series of new
triazole derivatives (H) have been reported. According to the SARs analysis of the previous studies, it can
be understood that in potent TYR inhibitors, there were moieties with the ability to form hydrogen bonds
with His85 and His244 [29].
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Based on the above findings and our previous studies, we designed and synthesized novel aryl-substituted
acetamides of phenoxy methyl triazole conjugated with thiosemicarbazide with hope to be enhanced
activity with optimum drug-likeness and calculated-pharmacokinetic criteria (Table 5 and 6). The triazole
ring synthesized by one pot multicomponent click reaction [30] gives a wide range of groups at R increasing
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the opportunity to synthesis a series of new compounds. Meanwhile, phenoxy linker and
thiosemicarbazones act as hydrogen bond donors and /or receptors and may improve TYR inhibition.
N
Cl
Cl
N
N
S
N
O
N
O
N
NH2
N
H
H
N
E
, IC50= 9.42 µM
A
, IC50=0.508 µM
O
H
N
Br
N
S
N
O
O
N
N
NH2
N
H
F
, IC50= 26.55 µM
N
H
B
, IC50=0.372 µM
O
H
HO
N
O
N
NH2
N
HO
O
N
N
S
O
C
, IC50= 0.35 µM
G
, IC50= 0.52 µM
H
O
N
N
N
N
O
O
S
O
N+
NH2
NH
-O
N
Cl
N
H
O
D
, IC50=0.05 µM
H
, IC50= 29.9 µM
S
NH
n
N
N
NH2
O
N
N
N
H
O
(Phenoxy)triazole moiety
Thiosemicarbazide Fragment
Tyrosinase inhibitory activity
Anti-melanogenesis assay
In silico anti-tyrosinase evaluation
Antioxidant assay
Cu+ chelatory activity
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Fig.1. Design of novel triazole-acetamide derivatives bearing phenoxy thiosemicarbazide
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2.2. Chemistry
A series of aryl phenoxy methyl triazole conjugated with thiosemicarbazides derivatives was synthesized
according to the general synthetic route depicted in Scheme 1. First, commercially available 4-hydroxy
benzaldehyde (1) was reacted with propargyl bromide (2) in DMF in the presence of potassium carbonate
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under reflux condition for 24 hours to give the corresponding compound (3). The reaction of different
aniline derivatives (4a-n) and chloro-acethylchloride (5) in DMF led to the formation of intermediates (6a-
n).
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The target compounds 7a-n were synthesized via click reaction of compound 3, sodium azide and different
aryl acetamide derivative (6a-n) in the presence of the catalytic amount of CuSO4,5H2O and sodium
ascorbate in DMF/H2O [31]. In the end, the reaction of compound 7a-n with thiosemicarbazide in refluxing
acetic acid gave the final product (9a-n). After cooling, the precipitate was filtered and washed with cold
water, and recrystallization from methanol. The chemical structure and some physical properties of
synthesized derivatives are presented in Table S1 (Supplementary file). The structures of purified products
were confirmed by IR, Mass, 1HNMR, 13CNMR, and CHNSO spectroscopy.
5
6
7
8
9
10
S
CHO
CHO
CHO
HN
N
NH2
Br
a
+
O
O
N
OH
1
H
d
C
N
NH2
3
2
H2N
O
NaN3
S
N
N
R
N
N
O
8
N
R
NH
H
NH2
N
b
Cl
Cl
R
O
Cl
R
+
NH
O
O
5
4a-n
6a-n
9a-n
7a-n
9j, R: Benzyl
9e, R: 4-Chloro phenyl
9f, R: 4-Flouro phenyl
9g, R: 3-Methoxy phenyl
9h, R: 4-Methoxy phenyl
9i, R: 4-Nitro phenyl
9a, R: Phenyl
9k, R: 4-Flouro benzyl
9l, R: 2,3-Flouro phenyl
9m, R: 3-Chloro 2-methyl phenyl
9n, R: 2-Methyl 3-nitro phenyl
9b, R: 3-Bromo phenyl
9c, R: 4-Bromo phenyl
9d, R: 3-Chlorophenyl
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Scheme 1. Synthesis pathway for the preparation 1,2,3 triazole hybrids & phenoxy thiosemicarbazone
derivatives, reagents and conditions: (a) DMF, K2CO3, reflux, 3 h; (b) DMF, K2CO3, 0 ºc, 20 min,
quenched with NaHCO3, (c) DMF, TEA, sodium ascorbate, CuSO4.5H2O, reflux, 24h; (d) CH3COOH,
reflux, 2 h
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2.3. Tyrosinase inhibitory activity in the presence of L-tyrosine as a substrate
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3
4
In vitro anti-tyrosinase activity of synthesized compounds, 9a-n were performed based on the calorimetric
method against mushroom tyrosinase comparing with kojic acid as the reference drug. The results were
summarized in Table 1 and the SAR of synthetic derivatives was investigated. All tests were undertaken at
least three times in triplicate and the IC50 was calculated at five different concentrations.
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6

The unsubstituted derivatives (9a, R= phenyl) showed strong TYR inhibitory potency with an IC50
value of 0.79 μM with around 12 times improvement in potency compares to that of kojic acid as
a positive control.
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8

Comparison of the mono halogenated hybrids on phenyl ring (9b-f) revealed that the presence of
halogen atom on different positions of phenyl moiety led to relative improvement in the activity
compared to 9a although the difference was not significant in some derivatives. In this halogenated
series, compound 9f bearing para-fluorophenyl moiety showed the best inhibitory activity with
IC50 value of 0.14 μM. It would be interesting to note that no statistical differences were seen
between bromine (9b and 9c) and chlorine (9d and 9e) analog even at different positions of the
phenyl ring.
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
Also, the evaluation of the multi-substituted compounds demonstrating that 9l possessing 3,4-
dichloro-phenyl and 9m with 2CH3-3Cl-phenyl pendant did not demonstrate significant changes
compare to mono-substituted counterpart although potency improvement was seen compared to
unsubstituted one (9a). The exception in this series as 9n bearing 2CH3-3NO2- phenyl with IC50
value of 0.25 μM.


The elongation of the linker between amide moiety and substituted derivatives resulted in
compounds 9j with the best TYR inhibitory potency (R= benzyl with IC50 = 0.11 ± 0.01 µM)
compared to the phenyl counterpart 9a. However, this trend was not seen between 9k (R= para
fluoro benzyl with IC50 = 0.83 ± 0.15 µM) and 9f (R= para fluorophenyl; IC50 = 0.14 ± 0.02 µM).
From the screening data, the presence of methoxy as a bulk-electron donating group (9g and 9h)
led to an improvement of TYR inhibitory activity comparing with the unsubstituted one. In this
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4
regard, the other top potent TYR inhibitor belonged to 9h (R= Para-methoxyphenyl) with an IC50
value of 0.12 µM while changing para to meta getting compound 9g (IC50= 0.22 μM) in which the
TYR inhibitory potency slightly decreased.
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Table 1. The anti-TYR activity of 9a-na in the presence of L-tyrosine
R
S
NH
N
N
NH2
O
N
N
N
O
H
6
Comp
ounds
R
%
%
%
%
%
IC50 (µM)
Inhibition
at 50 µM
89.94±0.8
Inhibition
at 10 µM
82.1±1.1
Inhibition Inhibition
Inhibition
at 0.1 µM
36.1±4.9
at 1 µM
at 0.5 µM
9a
9b
56.02±5.5
40.4±5.1
0.79±0.14
0.51±0.19
Br
90.2±1.1
86.3±1.6
65.4±1.8
49.7±2.7
46±2.5
9c
92.4±1.4
90.1±0.8
80.1±1.1
87.5±1.1
68.98±0.8
70.12±0.6
55.5±1.08
65.27±3.2
40±0.54
41.6±0.6
0.35±0.15
0.51±0.13
Br
9d
Cl
9e
9f
90.3±1.1
96.2±1.2
88.1±1.8
85.4±2.6
66.3±0.5
73.2±3.5
50±1.6
47.8±0.2
46±2.9
0.50±0.21
0.14±0.02
Cl
58.9±6.2
F
8

OCH3
9g
94.3±0.6
88.8±1.2
69.7±4.3
55.09±1.4
44.9±2.5
0.22±0.02
9h
9i
98.2±0.6
94.5±1.2
98.6±1.1
91.2±1.1
78.4±1.6
84.4±1.6
74.1±2.4
68.1±1.9
61.5±1.08
52.8±1.3
68.9±1.6
43.07±0.1
42.3±1.9
48.7±0.81
0.12±0.01
0.42±0.13
0.11±0.01
OCH3
NO2
9j
76.05±1.35
C
H2
9k
9l
94.2±1.4
93.8±1.8
80.2±1.6
76.8±1.2
53.1±1.3
66.1±2.3
49.7±0.5
52.8±2.6
23.2±2.1
33±1.08
0.83±0.15
0.42±0.16
F
C
H2
Cl
Cl
9m
9n
95.2±1.4
95.9±1.1
78.4±1.4
81.1±2.1
66.6±6.1
69.3±3.5
51±6.2
32.6±2.9
42.1±0.2
0.46±0.20
H3C
Cl
H3C
NO2
56.9±1.6
0.25±0.020
Kojic
-
9.28±1.15
Acid[b]
[22]
1
2
3
4
[a] Data represented in terms of mean±SD
[b] Positive control
2.4 Tyrosinase inhibitory activity in the presence of L-dopa as a substrate
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Next, the effect of the 9a-n on TYR inhibitory activity in the presence of L-dopa was investigated (Table
2). Considering the attributes of the substitutions on the triazole pendant, it was concluded that
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


Compound 9j possessing benzyl at R induced significant TYR inhibitory activity (IC50= 0.17± 0.04
μM); however, the elimination of CH2 linker (9a, R=phenyl with IC50= 2.18 µM) changed the
activity in such a manner that decline in potency was seen. A similar trend was seen between 9f
(R= 4-fluorophenyl) and 9k (R= 4-fluorobenzyl).
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4
5
In the case of compounds containing an electron-donating group, it can be seen that the substitution
of MeO on phenyl ring led to a significant improvement in the potency (9g, R= 3-methoxyphenyl,
IC50= 0.84 µM, and 9h, R= 4-methoxyphenyl, IC50= 0.52 µM) against TYR compared to the
unsubstituted derivative 9a.
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7
8
9
The investigation of electron-withdrawing substitution on phenyl pendant showed enhancement in
the activity compared to 9a. Also, it seems that the para-substituted derivatives (9c: R= 4-Br and
9e: R= 4-Cl) afford approximately better TYR inhibitory activity compared to the meta-substituted
derivatives (9b: R= 3-Br and 9d: R= 3-Cl) although the differences were not significant.
Considering the type and position of halogen the most potent compound was 9e (R= 4-Cl ) with an
IC50 value of 0.14 µM demonstrating 66% improvement in the inhibitory potency compared to that
of positive control.
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11
12
13
14
15
16
17
18
19

Slight enhancement in the potency of multi-substitution on the phenyl rings was seen compared to
a single substituted one. This is obvious in the IC50 values for compounds 9l (R= 2,3-
dichlorophenyl) with IC50= 0.42 µM and 9m (R=3-chloro-2-methylphenyl) with an IC50 value of
0.26 µM compare to 9d (R= 3-chlorophenyl, IC50= 0.50µM).
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Table 2. The anti-TYR activity of 9a-na in the presence of L-dopa
R
S
NH
N
N
NH2
O
N
N
N
O
H
22
10

%
%
%
%
%
Compo
unds
9a
R
Inhibition
at 50 µMa
80.5±1.6
Inhibition
at 10 µMa
75.8±3.2
Inhibition
at 1 µMa
54.57±5.5
Inhibition
at 0.5 µMa
36.1±5.1
Inhibition
at 0.1 µMa
8.82±4.9
IC50 (µM) a
2.18±0.13
9b
92.9±1.8
85.7±2.6
74.9±1.8
52.5±2.7
18.11±2.5
0.31±0.15
Br
9c
94.1±1.2
88.6±1.3
82.9±2.1
81.3±1.9
76.7±0.8
68.7±0.7
65±1.8
53±0.54
26.2±3.9
0.21±0.02
0.50±0.21
Br
9d
46.9±5.05
Cl
9e
9f
98.9±1.1
87.1±1.3
85.6±2.5
89±2.3
79.5±2.6
81.3±3.2
77.7±0.5
61.3±3.5
54.9±4.3
70.4±1.6
50.9±6.2
33.6±1.4
45±0.2
22.1±2.9
13.5±2.5
0.14±0.02
0.60±0.09
0.84±0.17
Cl
F
9g
OCH3
9h
9i
88.7±3.2
87.5±2.3
98.1±1.8
96.5±1.1
96.7±3.2
81.9±1.4
72.6±1.4
80.4±2.6
83.9±2.3
86.7±2.6
68.1±2.4
61.1±1.9
76.9±1.35
70.1±1.3
73.4±1.4
61.5±1.08
53.6±1.3
70±1.6
43.07±0.1
46.5±1.9
49±0.81
38.8±2.1
24±1.08
0.52±0.15
0.68±0.11
0.17±0.04
0.43±0.10
0.42±0.15
OCH3
NO2
9j
9k
9l
C
H2
61.15±0.5
54±2.6
F
C
H2
Cl
Cl
11

9m
9n
98.9±2.1
88.6±2.1
81.9±1.6
78.5±1.8
75.6±6
50.9±6.2
56.9±1.6
22.1±2.9
42.1±0.2
0.26±0.01
0.56±0.16
H3C
Cl
62.5±3.5
H3C
NO2
Kojic
Kojic Acid
9.30±1.27
[22]
Acid[b]
1
2
3
[a] Data represented in terms of mean±SD
[b] Positive control
2.5 SAR assessments regarding the potential of both substrate
4
5
6
7
8
Taking the in vitro data in presence of L-tyrosine and L-dopa together, all of the compounds exhibited
remarkable anti-tyrosinase activity displaying IC50 values in the range of 0.11–0.83ꢀµM in the case of L-
tyrosine as the substrate and 0.14-2.18 µM in the case of L-dopa. Although the first look at IC50 ranges did
not demonstrate significant changes in the activity; however, several important considerations were
purposed.
9
10
11
12
13
14
15
16
17
18

9j (R= benzyl; IC50= 0.11 µM) followed by 9h (R= methoxyphenyl; IC50= 0.12 µM) regarded as
two important TYR inhibitors in presence of L-tyrosine. This trend is completely different in the
presence of L-dopa in such a way that 9h (R= methoxyphenyl) is known as one of the least active
compounds in this series. Compound 9e (R= 4-chlorophenyl) was proposed as the top potent TYR
inhibitor in the presence of L-dopa with IC50 of 0.14 µM.

Mostly, structural modification via introducing methoxy on phenyl pendant had a positive effect
on anti-TYR activity in presence of L-tyrosine and destructive effects on TYR inhibitory activity
in the presence of L-dopa compare to electron-withdrawing groups.
Taking the in vitro data together, compound 9j as the most potent compound was selected to determine the
uncompetitive type of inhibition and evaluated via molecular dynamic studies.
19
12

1
2
3
4
5
6
2.6 Kinetic studies of enzyme inhibition
Kinetic studies were conducted to examine the mechanism and type of inhibition by compound 9j towards
TYR in the presence of L-dopa. Graphical analysis of the reciprocal Lineweaver–Burk plot related to
compound 9j presented in Fig. 2. Lineweaver–Burk is a useful plot to determine the inhibition type as
mixed-type, competitive, uncompetitive. As presented in Table 3, 9j revealed an uncompetitive type of
inhibition.
80
No inhibitor
100 nM
70
173 nM
60
500 nM
50
40
30
20
10
0
-4
-2
0
2
4
6
8
10
12
-10
1/[S] mM-1
7
8
Fig. 2. Lineweaver–Burk plot for the inhibition of mushroom tyrosinase-catalyzed L-dopa oxidation by 9j
Table 3. Kinetic parameters for the compounds 9j against mushroom tyrosinase inhibition assay
9
Compound
Control (without 9j)
9j (100 nM)
Km (mM)
2.05
Vm (OD/Min)
0.55
1.06
0.21
9j (173 nM)
0.96
0.18
9j (500 nM)
0.63
0.11
10
11
13

1
2.7 Cell viability
2
3
Cell-based MTT assay was performed to examine toxicity on invasive melanoma B16F10 cell line. Our
results show that compounds 9c, 9e, and 9j, had no significant toxicity at tested concentrations.
100
80
60
40
20
0
Conrtol kojic
acid
kojic
9c
9c
9e
9e 9j (4µM)9j (8µM)
acid (4µM) (8µM) (4µM) (8µM)
(4µM) (8µM)
4
5
Fig 3. Toxicity of 9c, 9e, and 9j compounds against B16F10 cells examined by MTT assay
2.8 Melanin content assay
6
7
8
B16/F10 cell lines were incubated with concentrations 4 and 8 µM of compounds 9c, 9e, and 9j for 48 h.
Data were expressed as mean ± SD of at least three independent experiments. As can be seen in Fig. 4, 9e
and 9j significantly reduced the melanin content in skin melanoma cells to 69.6% and 39.8% at 8 µM
respectively compared to kojic acid with 94.3% reduction at 8 µM.
9
10
14

*
*
*
*
*
100
80
60
40
20
0
**
**
Control Kojic
Kojic 9c-4µM 9c-8µM 9e-4µM 9e-8µM 9j-4µM 9j-8µM
acid4µMacid8µM
1
2
3
Fig. 4. Effect of compounds 9c, 9e, and 9j on melanin content in B16/F10 murine melanoma cells.
*p<0.05 and **p<0.001 statistically significant from control.
4
5
2.9 Docking analyses
6
7
The molecular docking analysis was performed to resolve interactions and binding modes of all synthesized
compounds inside the binding site of the tyrosinase enzyme. Trying all available scoring functions in the
GOLD, the RMSD value of the redocking process for ChemScore was 2.00 Å and for other scoring,
functions were more than 2.00 Å. Hence, the score value and binding modes of tyrosinase inhibitors with
the enzyme with PDB code 2Y9X was carried out in terms of ChemScore fitness function. Fig. 5 shows the
docking interactions of potent compound 9j. It showed three hydrogen bonds, one pi-pi stacked, and one
metal acceptor interaction inside the binding site of tyrosinase. NH2 in thiourea made two hydrogen bonds
with Glu256 and Asn260 residues. Methoxybenzene group made a pi-pi stacked interaction with His263
and a pi-alkyl interaction with Ala286. Residue Val283 formed a hydrogen bond interaction with the
triazole ring and two pi-sigma interactions with triazole and methoxybenzene rings. The terminal benzyl
group showed a pi-alkyl interaction with Pro277. The critical metal acceptor interaction was made between
15
8
9
10
11
12
13
14
15
16

1
2
one Cu2+ ion andnitrogen in methylidenehydrazine. Making three hydrogen bonds beside having critical pi-
pi and metal acceptor interaction chose this structure to be the most active compound in the set.
3
4
5
Compound 9j was aligned on Kojic acid in the active site of tyrosinase and was shown in Fig. 5b. They
shared a common binding mode and Kojic acid made metal acceptor, hydrogen bond, and pi-pi interactions
with Cu2+ ion, Met280, and His263 respectively.
6
7
8
9
10
Fig.5 Compounds 9j in the active site of the TYR enzyme. 3D interaction pattern of compound 9j (a) and
alignment of 9j (green) on Kojic acid (yellow) (b). Hydrogen bonds and pi-pi interactions are green and
pink dotted lines respectively
11
12
2.10 Copper chelating
13
14
15
16
17
18
19
Since the active site of TYR contains two critical Cu2+ ions which play an important role during the mono
and diphenolase process, trapping mentioned bio-metals confirming the desired inhibitory potential of the
designed scaffold. In this regard, compound 9j was tested for its metal chelating ability towards Cu2+ (Fig.
6) The UV spectrum of methanolic solution of 9j (20 µM) showed a characteristic absorption peak at 320
nm (depicted with an orange arrow in Fig.6). After around 30 min incubation of compound 9j with the
Cu2+, hypochromic shift (reducing in absorption intensity) was observed in the spectra confirming biometal-
ligand interactions.
16

1.4
1.2
1
9j (20 µM)
CuSO4 (20 µM)
9j (20µM)CuSO4 (10µM)
9j (20µM)CuSO4 (20µM)
0.8
0.6
0.4
0.2
0
230
250
270
290
310
330
350
370
390
wavelength (nm)
1
2
Fig. 6 The absorbance change of compound 9j alone and in the presence of Cu2+ ions
3
4
5
6
7
The stoichiometry of complex 9j-Cu2+ was also studied at 320 nm by the molar ratio method. The
concentration of the test compound 9j was set to 20 μM and the final concentration of Cu2+ ranged from
0-50 μM. The plot was obtained by the corresponding absorption against the mole fraction of Cu2+ to ligand
9j. According to Fig. 7, the two straight lines crossed at a mole fraction of around 0.3, indicating a 1:3
stoichiometry for the complex.
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0
0.5
1
1.5
Mole fraction Cu2+/9j
8
9
Fig.7. Determination of the stoichiometry of Cu+2-9j using the molar ratio method.
17

1
2.11 Antioxidant activity
2
3
4
5
6
7
8
9
The potential role of antioxidants to alleviate symptoms of hyperpigmentation was reported in various
studies [32, 33]. To this end, the synthesized compounds were also evaluated for their antioxidant ability,
using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and quercetin as the reference
standard. Compounds 9i bearing 4-nitrophenyl and 9n containing 2-methyl-3-nitrophenyl demonstrated
IC50 values of 68.43 and 58.24ꢀμM, respectively (Table 4). The antioxidant activity of nitro derivatives can
be explained by the electron-withdrawing effect and thus the inactivation of free radicals. However,
replacement of the NO2 substitute with halogen atoms and even OCH3 resulted in a drastic decrease in
antioxidant activity.
10
Table 4. Antioxidant properties synthesized compounds using DPPH assay
Compounds
% Inhibition at 100
µM [a]
% Inhibition at 30
µM[a]
IC50 µM[a]
9a
9b
9c
9d
9e
9f
12.83±4.09
17.07±8.44
10.82±2.88
17.83±2.76
14.28±3.51
34.82±3.81
16.82±0.44
14.72±3.50
89.56±0.87
11.09±4.36
5.29±3.16
7.64±5.59
5.14±3.06
-
-
-
-
-
-
-
6.88±3.21
7.43±1.59
5.87±2.76
16.47±3.22
5.67±3.25
-
9g
9h
9i
-
-
68.43±4.30
-
9j
18

9k
21.87±5.9
30.24±2.35
10.26±4.07
83.87±2.47
-
14.39±3.56
-
9l
9m
5.50±3.18
-
-
-
9n
23.19±3.65
-
58.24±2.84
4.62 ±2.26
Quercetin [b]
1
2
3
[a] Data presented here are the mean ± S.Eof three to five independent experiments
[b] Positive control
4
2.12 Pharmacokinetic and ADME-Tox studies
5
6
The pkCSM online server was applied to predict the ADME-Tox properties and drug-likeness of designed
compounds. As summarized in Table 5, the Human Intestinal Absorption (HIA) amount of compounds is
between 60.798-70.763 which is in the range of [30 ∼ 79%] percent showings of middle HIA [34]. The
volume of distribution (VD) is the computed volume that the total amount of medicine will be spread at an
equal concentration in the blood plasma. The high value of log VDss is for amounts greater than 0.45 log
L/kg, whereas log VDss < -0.15 log L/kg is considered low [35] and our compounds are categorized as low.
For metabolism, all compounds were predicted not to be substrates or inhibitors of CYP450 2D6, and
substrates of 1A2 isoform. Regarding excretion, none of the compounds were expected to be the substrates
of renal Organic Cation Transporter 2 (OCT2). Finally, all compounds were predicted nontoxic in terms of
skin sensitization and inhibition of hERG I channel.
7
8
9
10
11
12
13
14
15
16
Table 6 shows the drug-likeness properties of compounds. All compounds passed Lipinski’s rule of 5 and
their number of rotatable bonds did not exceed 10.
Table 5. ADME-Tox prediction of the designed compounds.
Absorption
Distribution
Metabolism
Excretion
Toxicity
Human Intestinal
Absorption (%
absorbed)
VDss (logL/Kg)
2D6
3A4
1A2
2C19
2C9
2D6
3A4
Renal OCT2
substrate
hERG I
Skin
Sensitization
inhibitor
Substrate
Inhibitor
19

68.41
69.322
69.322
69.586
69.586
66.523
64.61
-0.435
-0.438
-0.438
-0.453
-0.453
-0.581
-0.538
-0.538
-0.311
-0.474
-0.627
-0.467
-0.409
-0.205
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
9a
9b
9c
9d
9e
9f
No Yes
No Yes
No No No No
No Yes Yes No
Yes
Yes
No No
No Yes
No Yes
No No No No
No No Yes No
No No Yes No
No
Yes
Yes
No Yes
No Yes
No No Yes No
No No No No
Yes
Yes
9g
9h
9i
64.61
No Yes
No No No No
Yes
60.798
65.918
64.03
No Yes
No Yes
No No Yes No
No Yes No No
Yes
Yes
9j
9k
9l
No Yes
No Yes
No Yes No No
No Yes Yes No
Yes
Yes
70.763
70.055
61.368
No Yes
No Yes
No Yes Yes No
No No Yes No
Yes
Yes
9m
9n
1
2
3
Table 6. Drug-likeness properties of synthesized compounds
Compound
MW
Num. rotatable Num.
H-bond Num. H-bond Log P o/w
bonds
acceptors
donors
409.475
488.371
488.371
443.92
8
8
8
8
8
8
9
9
9
9
9
8
7
7
7
7
7
7
8
8
9
7
7
7
3
3
3
3
3
3
3
3
3
3
3
3
1.66
2.42
2.42
2.32
2.32
1.80
1.67
1.67
1.57
1.34
1.48
2.97
9a
9b
9c
9d
9e
9f
443.920
427.465
439.501
439.501
454.472
423.502
441.492
478.365
9g
9h
9i
9j
9k
9l
20

457.947
468.499
8
9
7
9
3
3
2.62
1.88
9m
9n
1
2
3. Conclusion
3
4
Continuing our research to develop possible anti-melanogenesis agents, aryl-thiosemicarbazide moiety was
selected and modified in view of its interesting known properties. In this regard, 14 derivatives were
designed and synthesized. The SAR data obtained by the anti-TYR screening of the aryl-substituted
acetamides of phenoxy methyl triazole conjugated with thiosemicarbazides have shown promising
inhibitory effects for all compounds with IC50 value 0.14–2.18 μM and 0.11-0.83 µM while using L-dopa
and L-tyrosine as substrate, respectively. 9j containing benzyl group was 55 times more potent in presence
of L-dopa and 84 times in presence of L-tyrosine compared to kojic acid as the positive control. To confirm
the anti-melanogenesis activity, a cell-based assay on the B16F10 cell line was performed on 9c, 9e, and 9j
derivatives, the obtained data showed that 9j had high anti-melanogenesis capacities. Moreover, molecular
docking suggested that all of the investigated compounds may have an inhibitory effect on TYR via traping
Cu2+ cofactors which might justify high potency and a potential mechanism of the anti-TYR activity of
compounds. The proposed mechanism was also confirmed in metal chelatory assay which demonstrating
Cu2+ chelation capacity. Additionally, the possible antioxidant activities of all compounds were also
performed demonstrating that compounds 9i and 9n possessing moderate antioxidant potential. In silico
pharmacokinetic and drug-likeness prediction represented an important starting point for hit/lead discovery
of different phenoxy methyl triazole conjugated with thiosemicarbazide. However, further experimental
studies to verify our proposed mechanism are needed to understand the relationship between the synthetic
derivatives, and the ability to inhibit the melanogenesis pathway.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4. Material and method
4.1. Chemistry
21

13
1
2
3
4
1H and C NMR spectra were determined by a Bruker FT-300 MHz spectrometer in DMSO-d6. All the
chemical shifts were reported as (δ) values (ppm). Mass spectra were obtained on the Agilent 7890A
spectrometer at 70 eV. The infrared (IR) spectra were run as KBr disk on Perkin-Elmer Spectrum FTIR.
Melting points of synthesized compounds were determined on a Kofler hot stage apparatus.
5
4.1.1. Procedure for the synthesis of 4-(prop-2-ynyloxy) benzaldehyde (3)
6
7
The 4-hydroxy benzaldehyde (compound 1, 10 mmol) and propargyl bromide (compound 2, 12 mmol) were
dissolved in 15 ml DMF as a solvent in the presence of potassium carbonate (1.5 mmol). The mixture was
refluxed for 24h. The crude product was filtered and purified by column chromatography using a mixture
of hexanes: ethyl acetate (9:1) to give the pale yellow solid product (yield: 80%) [36]. Pale yellow solid;
isolated yield: 80%; 1H NMR (DMSO-d6, 400 MHz) δH (ppm):9.90 (s, 1H), 7.89-7.82 (m, 2H), 7.12-7.06
(m, 2H), 4.78 (d, J= 2.4 Hz, 2H), 2.57 (t, J= 2.40 Hz, 1H).
8
9
10
11
12
4.1.2. Procedure for the synthesis of 2-chloro-N-phenylacetamide derivatives: 6a-n
13
14
15
16
17
18
19
20
In a round bottom flask, 10 mmol aniline derivitives (4a-n) and 20 mmol potassium carbonate were
suspended in 15 ml DMF and cooled to 0 °C. Then 12 mmol chloroacethylchloride was added. The reaction
mixture was then stirred at room temperature for 20 min; quenched with saturated sodium hydrogen
carbonate (10 ml) and washed with water. The organic layer was separated and dried over sodium sulfate.
The organic layer was evaporated under reduced pressure, and the crude reaction mixture was purified by
column chromatography using chloroform and methanol as a mobile phase to obtain a pure compound [37-
40]. The CAS-numbers and yield of synthesized compounds were reported in the supplementary file (Table
S1).
21
22
4.1.3. Procedure for the synthesis2-(4-((4-formylphenoxy) methyl)-1H-1,2,3-triazol-1-yl)-N-
phenylacetamide derivatives: 7a-n
22

1
2
3
4
5
6
The target compounds 7a-n were synthesized via click reaction. At first, 2 mmol of different aryl acetamide
derivative (6a-n) and 2 mmol sodium azide in the presence of TEA were dissolved in DMF/H2O and the
mixture was refluxed for 3h and then 2 mmol of compound 3 and catalytic amount of CuSO4,5H2O and
sodium ascorbate were added [30]. After 24 h the production was formed and completion of the reaction
was monitored by TLC. The yield of synthesized compounds was reported in the supplementary file (Table
S2).
7
2-(4-((4-formylphenoxy) methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxyphenyl) phenylacetamide (7h)
8
9
Beige solid; isolated yield: 74%; mp 134-136 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 10.43 (s, 1H,
H5), 9.89 (s, 1H, H1), 8.32 (s, 1H, H3), 7.9 (d, J = 8.8 Hz, 2H, Ar-H), 7.68 (d, J = 9.1 Hz, 2H, Ar-H), 7.49
(d, J = 8.8 Hz, 2H, Ar-H), 6.93 (d, J= 9.1 Hz, 2H, Ar-H ), 5.36 (s, 2H, H4), 5.23 (s, 2H, H2), 3.85 (s, 3H,
H6).
10
11
12
13
4.1.4.2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-
phenylacetamide derivatives: 9a-n
14
15
16
17
1 mmol from 7a-n and 1 mmol thiosemicarbazide were refluxed in acetic acid for around 2h. After
completion of the reaction (monitored by TLC), water was added and the resulting participate was filtered
and purified by recrystallization in methanol. The yield and melting point of 9a-n compounds were reported
in the supplementary file (Table S3).
18
19
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-
phenylacetamide (9a):
20
21
22
23
24
25
26
White solid; isolated yield: 85%; mp 189-191 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 1H,
H2), 10.41 (s, 1H, H7), 8.28 (s, 1H, H3), 8.16 – 8.10 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.90 (m, 1H, H1),
7.78 (m, 1H, Ar-H), 7.48 (m, 3H, Ar-H), 7.17 – 7.09 (m, 4H, Ar-H), 5.35 (s, 2H, H6), 5.25 (s, 2H, H4; 13C
NMR (DMSO-d6, 76 MHz): δc(ppm): 178, 172.5, 164.6, 159.9, 142.7, 138.7, 132.2, 130.4, 127.4, 126.8,
124.3, 119.7, 115.6, 115.3, 61.5, 52.6; MS (EI), m/z (%): 409 (11), 280 (9), 105 (24), 91 (56), 55 (100), 41
(24); IR (KBr) v(cm-1): 3434 (s), 3255 (m), 3132 (w), 1681 (s) 1604 (s), 1545 (m), 1240 (m), 1011 (w);
Anal Calcd for C19H19N7O2S, C, 55.73, H, 4.68, N, 23.95 found C, 55.76, H, 4.65, N, 23.91.
23

1
2
N-(3-bromophenyl)-2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-
yl)acetamide (9b):
3
4
5
6
7
8
9
White solid; isolated yield: 95%; mp 242-244 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm):11.34 (s, 1H,
H2), 10.41 (s, 1H, H7), 8.28 (s, 1H, H3), 8.16 – 8.10 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.90 (m, 1H, H1),
7.78 (m, 1H, H6"), 7.48 (m, 3H, H2", H3", H4" ), 7.17 – 7.09 (m, 4H, Ar-H), 5.35 (s, 2H, H6), 5.25 (s, 2H,
H4); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1, 167.6, 165.2, 159.9, 143, 142.7, 142.6, 135.9, 133.3,
129.3, 128.6, 127.5, 126.8, 126.5, 115.4, 61.6, 50.9; IR (KBr) v(cm-1): 3437 (s), 3299 (w), 3253 (w), 1681
(m), 1508 (s), 1238 (m), 1012 (w); Anal Calcd for C19H18BrN7O2S, C, 46.73 H, 3.72, N, 20.08 found C,
46.78 H, 3.69, N, 20.01.
10
11
N-(4-bromophenyl)-2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-
yl)acetamide (9c):
12
13
14
15
16
17
18
White solid; isolated yield: 87%; mp 201-203 °; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.35 (s, 1H,
H2), 10.63 (s, 1H, H7), 8.29 (s, 1H, H3), 8.18 – 8.10 (m, 1H, H1), 8.04 (s, 1H, H5), 8.00 – 7.90 (m, 1H, H1),
7.77 (d, J = 8.5 Hz, 2H, Ar-H), 7.63 – 7.50 (m, 4H, Ar-H), 7.11 (d, J = 8.5 Hz, Ar-H), 5.39 (s, 2H, H6), 5.25
(s, 2H, H4); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178, 172.5, 164.8, 159.9, 142.6, 138.2, 132.2, 129.3,
127.5, 126.8, 121.6, 121.6, 115.9, 115.3, 61.6, 52.7; IR (KBr) v(cm-1): 3437 (m), 3299 (w), 3253 (w), 1681
(s), 1508 (s), 1238 (m), 1012 (w); Anal Calcd for C19H18BrN7O2S, C, 46.73, H, 3.72, N, 20.08 found C,
46.67, H, 3.73, N, 20.05.
19
20
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-
chlorophenyl)acetamide (9d):
21
22
23
24
25
26
27
White solid; isolated yield: 92%; mp 178-180 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 1H,
H2), 10.41 (s, 1H, H7), 8.28 (s, 1H, H3), 8.16 – 8.10 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.90 (m, 1H, H1),
7.78 (m, 1H, Ar-H), 7.48 (m, 3H, Ar-H), 7.17 – 7.09 (m, 4H, Ar-H), 5.35 (s, 2H, H6), 5.25 (s, 2H, H4), 13C
NMR (DMSO d6, 76 MHz): δc(ppm): 178.1, 165.3, 159.9, 142.7, 142.5, 134.6, 133.9, 130.1, 129.3, 128,
127.5, 127.2, 126.8, 126.3, 115.3, 61.6, 52.4; IR (KBr) v(cm-1): 3437 (m), 3299 (w), 1681 (s), 1508 (s),
1238 (m), 1012 (w); Anal Calcd for C19H18ClN7O2S, C, 51.41, H, 4.09, N, 22.09 found C, 51.37, H, 4.02,
N, 22.07.
24

1
2
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-
chlorophenyl)acetamide (9e):
3
4
White solid; isolated yield: 88%; mp 191-193 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.35 (s, 1H,
H2), 10.64 (s, 1H, H7), 8.29 (s, 1H, H3), 8.19 – 8.08 (m, 1H, H1), 8.04 (s, 1H, H5), 7.99 – 7.89 (m, 1H, H1),
7.77 (d, J = 8.6 Hz, 2H, Ar-H), 7.63 (d, J = 8.8 Hz, 2H, Ar-H), 7.41 (d, J = 8.8 Hz, 2H, Ar-H), 7.11 (d, J =
5
13
6
8.6 Hz, 2H, Ar-H), 5.39 (s, 2H, H6), 5.26 (s, 2H, H4); C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1,
7
172.5, 164.8, 159.9, 142.7, 142.6, 137.8, 127.8, 127.5, 126.8, 121.2, 115.3, 61.6, 52.6; MS (EI), m/z (%):
443 (1.8), 149 (16), 60 (69), 43 (100); IR (KBr) v(cm-1): 3437 (m), 3299 (w), 3253 (w), 1681 (s), 1508 (s),
1238 (m), 1012 (w); Anal Calcd for C19H18ClN7O2S, C, 51.41, H, 4.09, N, 22.09 found C, 51.38, H, 4.07,
N, 22.04.
8
9
10
11
12
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-
fluorophenyl)acetamide (9f):
13
14
15
16
17
18
19
White solid; isolated yield: 88%; mp 182-184°C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.33 (s, 1H,
H2), 10.39 (s, 1H, H7), 8.27 (s, 1H, H3), 8.14 – 8.07 (m, 1H, H1), 8.04 (s, 1H, H5), 7.98 – 7.90 (m, 1H, H1),
7.76 (d, J = 8.7 Hz, 2H, Ar-H), 7.50 (d, J = 9.0 Hz, 2H, Ar-H), 7.10 (d, J = 8.8 Hz, 2H, Ar-H), 6.91 (d, J =
9.1 Hz, 2H, Ar-H), 5.34 (s, 2H, H6), 5.23 (s, 2H, H6); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1,
172.9, 163.9, 160.3, 157.4, 145, 142.8, 135.9, 129.3, 126.8, 125.5, 119.5, 115.3, 60.3, 50.1; IR (KBr) v(cm-
1): 3442 (w), 3214 (w), 3147 (w), 1677 (m), 1508 (s), 1222 (m), 1002 (w); Anal Calcd for C19H18FN7O2S,
C, 53.39, H, 4.24, N, 22.94 found C, 53.43, H, 4.28, N, 22.90.
20
21
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-
methoxyphenyl)acetamide (9g):
22
23
24
25
26
27
28
White solid; isolated yield: 94%, mp 206-208 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 1H,
H2), 10.41 (s, 1H, H7), 8.28 (s, 1H, H3), 8.16 – 8.10 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.90 (m, 1H, H1),
7.78 (m, 1H, H6"), 7.48 (m, 3H, H2", H3", H4"), 7.17 – 7.09 (m, 4H, Ar-H), 5.35 (s, 2H, H6), 5.25 (s, 2H, H4),
3.74 (s, 3H, H8); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1, 172.4, 164.3, 159.9, 142.7, 142.6, 136.3,
133.2, 132.2, 129.7, 129.3, 127.5, 126.8, 119.7, 115.3, 61.6, 56, 52.6; IR (KBr) v(cm-1): 3432 (w), 3233
(w), 1674 (s), 1509 (s), 1238 (s), 1011 (m); Anal Calcd for C20H21N7O3S, C, 54.66, H, 4.82, N, 22.31 found
C, 54.64, H, 4.83, N, 22.37.
25

1
2
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-
methoxyphenyl)acetamide (9h):
3
4
5
6
7
8
9
White solid; isolated yield: 93%; mp 194-196 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.36 (s, 1H,
H2), 10.37 (s, 1H, H7), 8.29 (s, 1H, H3), 8.17 – 8.11 (m, 1H, H1), 8.03 (s, 1H, H5), 7.99 – 7.94 (m, 1H, H1),
7.78 (d, J = 8.8 Hz, 2H, Ar-H ), 7.52 (d, J = 9.1 Hz, 2H, Ar-H), 7.11 (d, J = 8.8 Hz, 2H, Ar-H), 6.93 (d, J
= 9.1 Hz, 2H, Ar-H ), 5.34 (s, 2H, H6), 5.25 (s, 2H, H4), 3.74 (s, 3H, H8); 13C NMR (DMSO d6, 76 MHz):
δc(ppm): 178.1, 172.4, 164, 159.9, 156, 142.7, 131.9, 129.3, 127.5, 126.7, 121.3, 115.3, 114.5, 61.6, 55.6,
52.6; IR (KBr) v(cm-1): 3433 (m), 3223 (m), 3065 (w), 1673 (s), 1548 (s), 1238 (s), 1038 (m); Anal Calcd
for C20H21N7O3S, C, 54.66, H, 4.82, N, 22.31 found C, 54.62, H, 4.76, N, 22.36.
10
11
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-
nitrophenyl)acetamide (9i):
12
13
14
15
16
17
18
White solid; isolated yield: 93%, mp: 169-171 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm):11.33 (s, 1H,
H2), 10.39 (s, 1H, H7), 8.27 (s, 1H, H3), 8.14 – 8.07 (m, 1H, H1), 8.04 (s, 1H, H5), 7.98 – 7.90 (m, 1H, H1),
7.76 (d, J = 8.7 Hz, 2H, Ar-H), 7.50 (d, J = 9.0 Hz, 2H, Ar-H), 7.10 (d, J = 8.8 Hz, 2H, Ar-H), 6.91 (d, J =
13
9.1 Hz, 2H, Ar-H), 5.34 (s, 2H, H6), 5.23 (s, 2H, H4); C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1,
165.9, 159.9, 142.5, 135.4, 129.9, 129.8, 129.3, 127.5, 126.7, 115.7, 115.4, 115.3, 61.6, 52; IR (KBr) v(cm-
1): 3442 (w), 3214 (w), 1677 (s), 1508 (s), 1222 (m), 1002 (w); Anal Calcd for C19H18N8O4S, C, 50.21, H,
3.99, N, 24.66 found C, 50.18, H, 3.96, N, 24.61.
19
20
N-benzyl-2-(4-((4-((carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide
(9j):
21
22
23
24
25
26
27
White solid; isolated yield: 90%; mp 183-185 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.35 (s, 1H,
H7), 8.87 (s, 1H, H2), 8.24 (s, 1H, H3), 8.18 – 8.09 (m, 1H, H1), 8.03 (s, 1H, H5), 7.98 – 7.91 (m, 1H, H1),
7.77 (d, J = 8.8 Hz, 2H, Ar-H), 7.40 – 7.24 (m, 5H, Ar-H), 7.11 (d, J = 8.8 Hz, 2H, Ar-H), 5.34 – 5.10 (m,
4H, H4, H5), 4.36 (d, J = 5.8 ,2H, H8); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1, 165.8, 159.9, 146.6,
142.6, 139.1, 133.6, 129.3, 128.8, 127.8, 127.8, 127.4, 126.6, 115.3, 61.6, 52.1, 42.8; IR (KBr) v(cm-1):
3491.1 (w), 3299 (w), 1680 (s), 1604 (s), 1509 (s), 1248 (s), 1171 (m); Anal Calcd for C20H21N7O2S, C,
56.72, H, 5.00, N, 23.15 found C, 56.73, H, 5.05, N, 23.12.
26

1
2
N-benzyl-2-(4-((4-((carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-
fluorophenyl)acetamide (9k):
3
4
5
6
7
8
9
White solid; isolated yield: 88%; mp: 182-184°C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 2H,
H2, H7), 8.30 (d, J = 5.2 Hz, 2H, Ar-H ), 8.26 (s, 1H, H3), 8.10 – 8.16 (m, 1H, H1), 8.03 (s, 1H, H5), 7.91 –
7.97 (m, 1H, H1), 7.86 (d, J = 9.3 Hz, 2H, Ar-H), 7.78 (d, J = 8.8 Hz, 2H, Ar-H), 7.11 (d, J = 8.8 Hz, 2H,
Ar-H), 5.48 (s, 2H, H6), 5.26 (s, 2H, H4), 4.52 (s, 2H, H8); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1,
172.9, 163.9, 160.3, 157.4, 145, 142.8, 135.9, 129.3, 126.8, 125.5, 119.5, 115.3, 60.3, 50.1, 43.3; IR (KBr)
v(cm-1): 3491.1 (w), 3437 (w), 3267 (w), 1680 (s), 1604 (s), 1542 (s), 1248 (s), 1171 (m); Anal Calcd for
C20H20FN7O2S, C, 54.41, H, 4.57, N, 22.21 found C, 54.44, H, 4.52, N, 22.25.
10
11
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2,3-
dichlorophenyl)acetamide (9l):
12
13
14
15
16
17
18
White solid; isolated yield: 94%; mp 219-221 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.35 (s, 1H,
H2), 10.27 (s, 1H, H7), 8.30 (s, 1H, H3), 8.18 – 8.10 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.90 (m, 1H, H1),
7.80 – 7.73 (m, 3H, Ar-H), 7.50 (d, J = 7.2 Hz, 2H, Ar-H), 7.11 (d, J = 8.6 Hz, 2H, Ar-H), 5.51 (s, 2H, H6),
5.25 (s, 2H, H4); 13C NMR (DMSO d6, 76 MHz): δc(ppm): 178, 172.5, 165.5, 159.9, 142.7, 142.6, 136.6,
132.4, 129.3, 128.6, 127.5, 126.8, 125.5, 124.9, 115.3, 61.5, 52.4; MS (EI), m/z (%): 43 (100), 57 (20), 69
(19), 91 (11), 121 (6), 478 (3); IR (KBr) v(cm-1): 3491 (w), 3281 (w), 1680 (s), 1586 (s), 1244 (s), 1052
(m); Anal Calcd for C19H17Cl2N7O2S, C, 47.71, H, 3.58, N, 20.50 found C, 47.68, H, 3.54, N, 20.56.
19
20
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-chloro-2-
methylphenyl)acetamide (9m):
21
22
23
24
25
26
27
White solid; isolated yield: 89%; mp 240-242 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 1H,
H2), 10.27 (s, 1H, H7), 8.30 (s, 1H, H3), 8.17 – 8.08 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.88 (m, 1H, H1),
7.82 – 7.70 (m, 4H, Ar-H), 7.47 (t, J = 8.0 Hz, 1H, H3"), 7.11 (d, J = 8.5 Hz, 2H, H2", H4"), 5.48 (s, 2H, H6),
13
5.25 (s, 2H, H4), 2.32 (s, 3H, H8); C NMR (DMSO d6, 76 MHz): δc(ppm): 165.4, 159.9, 157.3, 151.4,
142.7, 142.6, 137.7, 130.2, 129.4, 127.5, 127.3, 126.9, 126.8, 121.6, 115.3, 61.6, 52.3, 14.2; IR (KBr) v(cm-
1): 3428 (w), 3256 (w), 1673 (s), 1542 (s), 1602 (s), 1247 (s), 1010 (m); Anal Calcd for C20H20ClN7O2S, C,
52.46, H, 4.40, N, 21.41 found C, 52.43, H, 4.47, N, 21.45.
27

1
2
2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-methyl-3-
nitrophenyl)acetamide (9n):
3
4
White solid; isolated yield: 84%; mp 166-168 °C; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 1H,
H2), 10.27 (s, 1H, H7), 8.30 (s, 1H, H3), 8.17 – 8.08 (m, 1H, H1), 8.03 (s, 1H, H5), 7.97 – 7.88 (m, 1H, H1),
7.82 – 7.70 (m, 4H, Ar-H), 7.47 (t, J = 8.0 Hz, 1H, H3"), 7.11 (d, J = 8.5 Hz, 2H, H2", H4"), 5.48 (s, 2H, H6),
5
13
6
5.25 (s, 2H, H4), 2.32 (s, 3H, H8); C NMR (DMSO d6, 76 MHz): δc(ppm): 178.1, 165.4, 159.9, 151.3,
7
142.7, 142.59, 137.7, 130.2, 129.3, 127.5, 127.3, 126.9, 126.8, 121.6, 115.3, 61.6, 52.3, 21.5; MS (EI), m/z
(%): 43 (100), 57 (20), 69 (19), 91 (11), 236 (5), 468 (3); IR (KBr) v(cm-1): 3422 (w), 3508 (w), 3263 (w),
1669 (s), 1509 (s), 1235 (s), 1008 (m); Anal Calcd for C20H20N8O4S, C, 51.27, H, 4.30, N, 23.92 found C,
51.24, H, 4.25, N, 23.90.
8
9
10
11
4.2 Tyrosinase inhibition assay
12
13
14
15
16
17
18
The anti-TYR activity of synthesized compounds was achieved according to our previous study. Briefly,
10 µL of 0.5 mg/ml of tyrosinase enzyme was mixed with 160 µL of 50 mM phosphate buffer at pH= 6.8
in 96-well microplate and 10 µL of the test sample (different concentrations) was added to each well. The
plate was incubated for 20 min at 28 °C and 20 µL of L-dopa or L-tyrosine solutions as substrates were
added to each well and then the absorbance of the mixtures was measured at 475 nm. DMSO without test
compounds was used as the control, and kojic acid was used as a positive control [41]. Percentage of
tyrosinase inhibition was calculated as
[
(
)
] × ퟏퟎퟎ
푨풃풔푫푴푺푶
―
푨풃풔풔풂풎풑풍풆 ― 푨풃풔풃풍풂풏풌
% Tyrosinase inhibition =
19
푨풃풔푫푴푺푶
20
21
22
23
24
where Abssample is the absorbance of buffer + tyrosinase + substrate (L-dopa or L-tyrosine); Absblank is the
absorbance of buffer + sample/standard; AbsDMSO is the absorbance of DMSO as positive control+ substrate
(L-dopa or L-tyrosine). IC50 values, as the concentration yielding 50% of tyrosinase inhibition, were
calculated by best-fit equations of the % tyrosinase inhibition vs concentration curve using Curve Expert
v1.3 statistical program.
25
26
4.3 Kinetic assay
28

1
2
3
4
The kinetic study for the inhibition of TYR by compound 9j was carried out according to the colorimetric
method used for the inhibition assay. The inhibition was evaluated by a Lineweaver-Burk plot for
compound 9j (0, 100, 172, 500 nM) and variable concentrations of L-dopa (0.25, 0.5, 0.75 and 1 mM). The
Lineweaver–Burk reciprocal plot was provided by plotting 1/V against 1/[S] according to this equation,
1
5
=
+
1/V [S] Km/Vm 1/Vm
6
7
8
Where V is the initial reaction rate; KM, also known as the Michaelis-Menten constant, is the substrate
concentration at which the reaction velocity reaches half of the maximum; and Vm is the enzyme's maximum
rate in the absence of inhibitor [42].
9
10
4.4 Docking Analysis
11
12
13
14
15
16
17
18
19
20
21
MarvinSketch 18.20.0 was used to sketch all compounds and the steepest descent algorithm was applied to
optimize the energy of compounds by Open Babel 2.4.0. All dockings were carried out by the GOLD
docking program. PDB code of 2Y9X was applied for doing docking analyses which comprises the
Agaricus bisporus tyrosinase having tropolone as the native ligand in the binding site. Two Cu2+ metal ions
are present in the binding site of tyrosinase which all were included for docking calculations. The protein
structure was prepared using Discovery studio client, adding all hydrogens. The binding site of the enzyme
was detected automatically using the coordinates of the native ligand. GOLD program was applied for
doing all docking analyses for all compounds, and all available scoring functions in GOLD namely
CHEMPLP, ChemScore, ASP, and GoldScore were tried to find the most appropriate one for analyses. To
choose and validate the docking algorithm, tropolone inside the 2Y9X was redocked using all the mentioned
scoring functions.
22
23
24
25
4.5 Cell culture
Fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) and L-DOPA were
purchased from Gibco-Invitrogen (USA). B16/F10 melanoma ATCCR CRL-6475TM cells were prepared
from Pasteur Institute (Iran). Streptomycin -penicillin, and trypsin were provided from Biosera, England.
29