Inhibitory properties of aromatic thiosemicarbazones on mushroom tyrosinase: Synthesis, kinetic studies, molecular docking and effectiveness in melanogenesis inhibition

Article abstract of DOI:10.1016/j.bioorg.2018.09.003

The group of 19 thiosemicarbazones (TSCs) were synthesized and its inhibitory activity toward mushroom tyrosinase and ability to inhibition of melanogenesis in B16 cells were investigated. Moreover, molecular docking of these compounds to the active site of the enzyme was performed. The obtained results allowed to make the structure-activity relationship (SAR) analysis. Kinetic studies revealed that TSCs 1, 2, 11 and 18 have better inhibitory properties than kojic acid, a reference compound, with the best inhibitory constant (K_i) value of 0.38 μM for TSC 2. According to SAR analysis, the smaller and less branched molecules exhibit higher affinity to the enzyme. Melanin production in B16 cells was inhibited by all investigated compounds at micromolar level. Most of compounds studied in this work can be considered as potent inhibitors of tyrosinase and melanogenesis. They may have broad application in food preservatives and cosmetics. Combined results of molecular docking and SAR analysis can be helpful in designing novel tyrosinase inhibitors of desired properties.

Full text of DOI:10.1016/j.bioorg.2018.09.003

Accepted Manuscript
Inhibitory properties of aromatic thiosemicarbazones on mushroom tyrosinase:
Synthesis, kinetic studies, molecular docking and effectiveness in melanogen-
esis inhibition
K. Hałdys, W. Goldeman, M. Jewgiński, E. Wolińska, N. Anger, J. Rossowska,
R. Latajka
PII:
S0045-2068(18)30591-1
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.003
YBIOO 2500
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 June 2018
5 September 2018
6 September 2018
Please cite this article as: K. Hałdys, W. Goldeman, M. Jewgiński, E. Wolińska, N. Anger, J. Rossowska, R. Latajka,
Inhibitory properties of aromatic thiosemicarbazones on mushroom tyrosinase: Synthesis, kinetic studies, molecular
docking and effectiveness in melanogenesis inhibition, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.09.003
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibitory properties of aromatic thiosemicarbazones on
mushroom tyrosinase: Synthesis, kinetic studies, molecular
docking and effectiveness in melanogenesis inhibition
K. Hałdys1*, W. Goldeman2, M. Jewgiński1, E. Wolińska1, N. Anger3, J. Rossowska3, R.
Latajka1
1Department of Organic and Pharmaceutical Technology, Wrocław University of Science and
Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
2Department of Organic Chemistry, Wrocław University of Science and Technology,
Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland
3Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of
Science, Rudolfa Weigla 12, 53-114 Wrocław, Poland
Abstract
The group of 19 thiosemicarbazones (TSCs) were synthesized and its inhibitory activity
toward mushroom tyrosinase and ability to inhibition of melanogenesis in B16 cells were
investigated. Moreover, molecular docking of these compounds to the active site of the
enzyme was performed. The obtained results allowed to make the structure-activity
relationship (SAR) analysis. Kinetic studies revealed that TSCs 1, 2, 11 and 18 have better
inhibitory properties than kojic acid, a reference compound, with the best inhibitory constant
(Ki) value of 0,38 µM for TSC 2. According to SAR analysis, the smaller and less branched
molecules exhibit higher affinity to the enzyme. Melanin production in B16 cells was
inhibited by all investigated compounds at micromolar level. Most of compounds studied in
this work can be considered as potent inhibitors of tyrosinase and melanogenesis. They may
have broad application in food preservatives and cosmetics. Combined results of molecular
docking and SAR analysis can be helpful in designing novel tyrosinase inhibitors of desired
properties.
katarzyna.haldys@pwr.edu.pl; waldemar.goldeman@pwr.edu.pl;
michal.jewginski@pwr.edu.pl; ewa.mrozinska@pwr.edu.pl; natalia.anger@iitd.pan.wroc.pl;
joanna@iitd.pan.wroc.pl; rafal.latajka@pwr.edu.pl
Keywords: tyrosinase, thiosemicarbazones, kinetic studies, SAR, molecular docking, B16F10
cell line.

1. Introduction
Tyrosinase (EC 1.14.18.1) is a type III copper protein with two copper ions in its active
site. The enzyme catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase
activity) and subsequent oxidation of the o-diphenols to the corresponding o-quinones
(diphenolase activity). When L-tyrosine is the substrate, the product of tyrosinase catalyzed
reaction is dopaquinone which is then converted to melanins [1].
Melanins are the family of pigments known for their ability to protect the mammalian skin
from damage caused by ultraviolet radiation (UV). Melanin absorbs UV sunlight and removes
reactive oxygen species (ROS). Melanocytes are special cells within the basal layer of the
dermis, secreting the pigment. The amount, type and distribution of melanins in surrounding
keratinocytes determines the color of the human skin [2].
Hyperactivity of tyrosinase leads to overproduction of melanin which is accumulated in
the skin causing many dermatological disorders. Such hyperpigmentation problems include
freckles, melasma, post inflammatory melanoderma, age spots and melanoma [3].
There is a growing trend for lighter skin in the whole world with Asia as the largest skin
whitening products market. Around 15% of the world population, including both men and
women, buys skin lightening cosmetics. Global Industry Analysts, Inc. predicts that the global
market for skin lightening agents will reach US$23 billion by 2020 [4].
Moreover, a lot of disorders in melanin biosynthesis pathway are related to some
neurodegenerative diseases, such as, Alzheimer's as well as Parkinson's and Huntington's
diseases [5].
Tyrosinase is also responsible for enzymatic browning in plant-derived foods. Formation
of brown or black pigment in fruits and vegetables is the main factor affecting the shelf life of
fresh-cut products, leading to decreased sensory properties. Browning spoils the senses and
taste. Furthermore, some of o-quinone compounds can combine with protein residue groups
(e.g. amino, sulfhydryl) to change the nutrition characteristics of the protein [6].
Because of their biological and pharmacological properties, aromatic thiosemicarbazones
have been recently the compounds of great interest to medicinal chemists. They have been
used in antimicrobial [7], antimalarial [8] and anticancer [9,10] treatment.
For the past ten years thiosemicarbazones have been also intensively studied as tyrosinase
inhibitors [11-29]. Derivatives of benzaldehyde thiosemicarbazone [11-18], acetophenone
thiosemicarbazone [19-21] and aromatic heterocycle thiosemicarbazone [22-28] exhibited
potent inhibitory activity.
As they possess thiourea moiety, thiosemicarbazone derivatives are potential donor
ligands for transition metal ions [30]. It was reported that thiosemicarbazone derivatives are
potent tyrosinase inhibitors because the sulfur and nitrogen atoms of the thiourea moiety are
able to chelate copper ions in the active site of tyrosinase [21].
Many potent tyrosinase inhibitors already described in the literature bear phenyl group
[31,32] what refers to the natural tyrosinase substrates, L-tyrosine and L-dopa. High affinity
of such compounds to the enzyme is the result of the van der Waals interactions of phenyl
group with the residues present in the hydrophobic tyrosinase cavity [21].
The aim of the present paper is, to study the inhibitory effect of a series of aromatic
thiosemicarbazone analogues on the mushroom tyrosinase activity and evaluate the kinetic
parameters and the inhibition mechanisms. Based on structural information of tyrosinase, 19
thisosemicarbazones with different hydrophobic substituents were synthesized. Cytotoxicity
of presented compounds against murine melanoma B16F10 cell line and their effectiveness in
inhibition of melanin production has been also investigated . The molecular docking have
been also performed to compare the wet lab results with theoretical calculations. These
outcomes could be used to provide the basis for developing novel effective tyrosinase

inhibitors and searching for new whitening agents in cosmetic preparations or anti-browning
agents for food products.
2. Results and discussion
2.1. Chemistry
The synthesis of the title compounds is shown in Scheme 1. All the thiosemicarbazones
(TSCs) were prepared in the reaction of carbonyl compounds (ketones or aldehydes) with
thiosemicarbazide or N(4)-substituted thiosemicarbazides. The reaction was carried out in
boiling ethanol in the presence of acetic acid or p-toluenesulfonic acid as catalyst.
S
R1
S
R1
R3
N
H+
N
N
+
R3
H2N
H
H
N
N
O
R2
EtOH, reflux
H
H
R2
Scheme 1. General procedure for the synthesis of investigated thiosemibcarbazones
Crude TSCs were precipitated with aqueous sodium bicarbonate solution, separated by
filtration and subsequently recrystallized. In the case of semisolid/oily TSCs (8-10) they were
extracted with dichloromethane. In the case of N(4)-substituted TSCs, N(4)-substituted
thiosemicarbazides were first prepared by the reaction of appropriate isothiocyanates with
1
hydrazine hydrate in isopropyl alcohol. All thiosemicarbazones were characterized by H
NMR, 13C NMR and HRMS.
From the all prepared TSCs only phenyl isopropyl ketone thiosemicarbazone 4 was
obtained as a mixture of E and Z isomers, while other TSCs were obtained as single isomers.
It is noteworthy that in many cases of thiosemicarbazones described in the literature, the
problem of formation of E and/or Z isomers is completely omitted. However, it is proved that
in the case of typical aromatic aldehydes and aryl alkyl ketones E isomer is predominantly
preferred [33-36], while formation of Z isomer can become favorable by intramolecular
hydrogen bonding [37, 38, 39, 40], especially in the case of N(4) disubstituted
1
thiosemicarbazones [41]. Both isomers can be readily distinguish on the basis of H NMR.
The diagnostic signal in the analysis of the geometry of the C=N double bond of TSCs is the
signal of N2H proton (C=N-NH-) [36, 42, 43]. For E thiosemicarbazones derived from the
aryl alkyl ketones the signal of N2H proton is in the range of ~10.2-10.6 ppm [20,44,45],
while in case of aromatic aldehydes derivatives the corresponding signal is usually shifted
downfield to over 11 ppm [36, 44].
In the case of aldehydes-derived TSCs 1 and 19 the N2H signals were observed at 11.44
and 11.66 ppm, respectively. The aryl alkyl ketone derivatives (TSCs 2-3, 5-12 and 14-18)
1
exhibit the signals of N2H protons at 10.23-10.60 ppm. Therefore, the H NMR studies in
DMSO-d6 solution together with literature data confirm that thiosemicarbazones 1-3, 5-12 and
14-19 exist as E isomers. Due to the Cahn-Ingold-Prelog priority rules, the same orientation
of substituents attached to the C=N carbon in the case of 2,2,2-trifluoroacetophenone
thiosemicarbazone 13 translates to formally Z isomer.
In the case of thiosemicarbazone 4 which exhibits two sets of signals, chemical shift of
the N2H signal of the minor isomer (10.29 ppm) suggests E configuration, that might indicate

that the second N2H signal of the major isomer (8.20 ppm) corresponds to the Z isomer. To
verify this hypothesis, 2D NMR NOESY (Nuclear Overhauser Effect Spectroscopy)
experiment was performed. There correlation between N2H signal at 10.29 ppm and methine
proton of the isopropyl group at ~3.5 ppm was observed, what confirms that the minor isomer
of TSC 4 has E configuration. While, the NOE interaction of the N2H signal at 8.20 ppm with
the ortho protons of the phenyl group is a strong evidence for Z configuration of the major
isomer. The discrepancies between the chemical shifts of the signals for N2H proton in 4
assigned as Z isomer (8.20 ppm) and the chemical shifts of typical Z thiosemicarbazones (14-
15 ppm) may be due to the fact that known examples are heterocyclic and the Z conformation
is promoted by intramolecular hydrogen bond between the N2H and heterocyclic nitrogen
while in the case of 4 it seems that the steric effect of isopropyl group determines geometry.
1
Thiosemicarbazone 4 is described in the literature as a single isomer and the given H NMR
data corresponds to Z isomer [46]. Furthermore, thiosemicarbazone 4 is also described in
other papers, while without any NMR data or information on E/Z isomer ratio [45, 47].
2.2. Kinetic studies
Among different types of tyrosinase, mushroom tyrosinase has the highest homology
with the mammalian tyrosinase [48]. Moreover, because of convenient assay method, the high
activity and commercial availability mushroom tyrosinase has become a model system for
studying tyrosinase activity and inhibition.
Buitrago et al. [29] reported kinetics of interaction with mushroom tyrosinase for
benzaldehyde thiosemicarbazone. This compound revealed strong inhibition potency with Ki
of 0,93 µM. Based on this result, we decided to test other 18 thiosemicarbazone analogues as
the inhibitors of oxidation of L-dopa by tyrosinase from mushroom. Kojic acid, a well-known
tyrosinase inhibitor [49,50] was used as positive control. The inhibition type and the
inhibition constants (Ki and αKi) were determined using methods as described by Copeland
[51]. The results are shown in Table 1.
Table 1. Structures and results of kinetic studies of all target compounds.
KI (µM)
αKI(µM)
Compound R1
R2
R3
Inhibition type
noncompetitive
competitive
mixed
TSC 1
-H
-H
3.7 ± 0.1
3.7 ± 0.1
-
TSC 2
-CH3
-Et
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
0.38 ± 0.1
TSC 3
20.0 ± 0.2
85.5 ± 0.8
454.6 ± 0.3
577.5 ± 0.3
TSC 4
i-Pr
mixed
507.5 ± 0.5
>1000
TSC 5
n-Pr
mixed
TSC 6
i-Bu
No inhibition effect
826.3 ± 0.2
TSC 7
n-Bu
mixed
uncompetitive
mixed
>1000
TSC 8
n-pentyl
n-hexyl
n-undecyl
26.2 ±1.0
330.4 ± 0.2
TSC 9
59.9 ± 0.3
No inhibition effect
0.58 ± 0.07
TSC 10
TSC 11
TSC 12
TSC 13
mixed
mixed
2.6 ± 0.2
No inhibition effect
293.1 ± 0.2
-CF3
889.4 ± 0.4

TSC 14
TSC 15
-CH3
-CH3
-CH3
competitive
202.3 ± 2.1
10.9 ± 0.5
-
noncompetitive
10.9 ± 0.5
TSC 16
TSC 17
-CH3
-CH3
uncompetitive
mixed
-
52.4 ± 0.2
602.8 ± 3.7
432.3 ± 3.4
TSC 18
TSC 19
-CH3
-H
-H
mixed
0.83 ± 0.16
1.4 ± 0.2
-H
competitive
mixed
37.5 ± 0.7
10.2 ± 0.2
-
Kojic acid
138.7 ± 1.7
2.2.1. Type of inhibition
The inhibition type was determined by the Lineweaver-Burk plot based on the results of
inhibitory effect of the compounds on the diphenolase activity of tyrosinase.
TSC 3-5, 7, 9, 11, 13, 17, 18 revealed mixed pattern of inhibition what means that they
have affinity to both, free enzyme and enzyme-substrate complex. In all cases of mixed-type
inhibitors affinity to the free enzyme is much higher than to the enzyme-substrate complex (Ki
values are lower then αKi ) so Ki value will be used for further comparison of these compound
in this work.
Compounds 1 and 15 turned out to be non-competitive inhibitors (equal affinity to the
free enzyme and enzyme-substrate complex). Compounds 2, 14 and 19 inhibit tyrosinase
competitively, binding to its active site and compounds 8 and 16 have affinity only to the
enzyme-substrate complex.
2.2.2. Inhibition constants
Inhibition constants Ki were determined by the secondary plots of apparent Km versus the
inhibitor concentration for competitive inhibition and by the secondary plots of the slope of
the lines from the Lineweaver-Burk plot as a function of the inhibitor concentration for non-
competitive and mixed-type inhibition. Inhibition constant αKi was determined by the
secondary plots of 1/Vmax as the function of the inhibitor concentration for uncompetitive and
mixed-type inhibition.
Kojic acid, well known and the most intensively studied tyrosinase inhibitor is often used
as a positive standard when the new inhibitors are investigated [49, 50]. All inhibition
constants of investigated compounds were compared with the one for kojic acid. Compounds
1, 2, 11 and 18 revealed better inhibitory properties than kojic acid (Ki = 10,2 µM).
Compounds 2, 11 and 18 are the most potent tyrosinase inhibitors out of investigated
thiosemicarbazones as their inhibition constants are lower than 1 µM. Compound 2 is 27
times stronger inhibitor than kojic acid, compound 11 18 times stronger and compound 18 is
12 times stronger.
In our investigations, benzaldehyde thiosemicarbazone (TSC 1) exhibited Ki value of 3,7
µM what is 4 times higher value than the one reported in [29] but still we can consider TSC 1
as a potent inhibitor (3 times better inhibitory activity than kojic acid).
Fig. 1. presents Lineweaver-Burk plots and secondary plots for two most potent
tyrosinase inhibitors investigated in this study (TSC 2 and TSC 11).

Fig. 1. Lineweaver-Burk plots for inhibition of TSC 2 (A) and TSC 11 (C) on mushroom
tyrosinase with L-DOPA as substrate. The concentrations of TSC 2 for curves 1-5 are 0.0, 1.0,
2.0, 3.0 and 4.0 µM, respectively. The concentrations of TSC 11 for curves 1-5 are 0.0, 0.3,
0.6, 0.9 and 1.2, respectively. B, D and E are secondary plots of Km, slopes (from
Lineweaver-Burk plots) and 1/Vmax, respectively, versus inhibitor concentrations for
determination of inhibitory constants.
2.2.3. Inhibition mechanism of TSC 2 and TSC 11 on the diphenolase activity of tyrosinase
Inhibition mechanism indicates if investigated compound inhibits the enzyme activity in
reversible or irreversible mode.
Reversible and irreversible inhibitors can be distinguished by plotting reaction velocity
versus enzyme concentration or total units of enzyme activity added to the assay. For a
reversible inhibition, curves representing “plus inhibitor” have smaller slopes than the control
curve and goes through the origin. For irreversible inhibition, the “plus inhibitor” curves have
the same slope as the control curve (they are parallel) but intersect the ‘X’ axis at a position
equivalent to the amount of enzyme that is irreversibly inactivated.
The inhibition mechanism of TSC 2 and TSC 11 on tyrosinase for diphenolase activity
was tested using standard graphical method that is usually used for determining inhibition
mechanism [13,14,25]. Both compounds revealed the same manner of inhibitory activity. As
shown in Fig. 2. the plots representing the remaining tyrosinase activity versus the enzyme
concentration at different inhibitor concentrations (with constant substrate concentration - L-
DOPA) gave a family of straight lines which all passed through the origin of the coordinate
system.

TSC 2 and TSC 11 are reversible inhibitors of tyrosinase. The presence of inhibitor did
not bring down the amount of active enzyme but just led to a decrease in the enzyme activity
(velocity).
Fig. 2. The plots of inhibitory mechanism of TSC 2 (A) and TSC 11 (B) on mushroom
tyrosinase with L-DOPA as substrate. The concentrations of TSC 2 for curves 1-5 are 4.0, 3.0,
2.0, 1.0 and 0.0 µM, respectively. The concentrations of TSC 11 for curves 1-5 are 1.2, 0.9,
0.6, 0.3 and 0.0, respectively.
2.2.4. Structure-activity relationship
The general structure of thiosemicarbazones examined in this work is shown in Fig. 3.
Fig. 3. The general structure of thiosemicarbazone derivatives
Effect of R1, R2 and R3 substituents on the biological activity is discussed as follows.
Substitution of phenyl group with naphthyl group in position R1 increased Ki value from
0,38 (compound 2) to 0,83 (compound 18) but still the inhibitor is considered as a very potent
one. Substitution of phenyl group with bigger anthracenyl group increased Ki value 10 times
what can be related with the size of anthracenyl group and lower ability of fitting to the active
site of the enzyme.
There is no clear relationship between elongation of alkyl chain in position R2 and the
strength of interaction with the enzyme. Generally we can say that the shorter chain effects in
the more potent inhibitor (proton, methyl and ethyl groups have Ki 3,7 and 0,38 and 20,0 µM,
respectively. Elongation of alkyl chain to C-3 and C-4 increased Ki values more than 10 times
and for iso-butyl there is no inhibition effect. What is interesting, compound 8 (with n-pentyl
group) has relatively low value of inhibition constant but it can interact only with enzyme-
substrate complex and compound 9 (with n-hexyl group) also exhibited stronger potency than
TSCs with propyl and butyl substituents. Elongation of the chain to linear C-11 results in no
inhibitory activity of the investigated compound. Phenyl group is almost as potent as methyl

group giving Ki of 0,58 µM but substitution of phenyl group with benzyl one led to total loss
of inhibitory activity what suggests that the distance of aromatic ring in position R2 from the
rest of the TSC molecule is crucial for its inhibition ability. The try of substitution of
hydrophobic methyl group with more hydrophilic trifluoromethyl group increased Ki value
almost 800 times.
Proton seems to be the best substituent in position R3. Substitution with methyl group
increased the Ki value about 500 times. Phenyl group in this position exhibits significant
impact on inhibitory ability in comparison with methyl group but still is worse than proton.
An increase the distance of aromatic ring from the thiosemicarbazide moiety decreased the
inhibitory activity of these compounds.
2.3. Molecular docking
The understanding of the physical nature of ligand-receptor interaction is crucial for
understanding molecular assembly of this complex and for understanding mode of inhibitory
action.
Optimized conformations of the inhibitors were obtained using the Gaussian09 at the
B3LYP/6-311g(d,p) level of theory [52] with the solvent model and consequently they were
docked to the active site of tyrosinase, available from Protein Data Bank [53] using AutoDock
software [54]. To perform accurate docking procedure in AutoDock good assignment of
charges on ligand is required. Therefore they have been obtained from the ab initio
calculations with Merz-Singh-Kollman scheme [55]. Structure of tyrosinase was protonated at
pH 6.8 using H+ web server [56,57]. In the case of uncompetitive and mixed inhibitors,
docking to the structure of tyrosinase has been performed with the predocked L-dopa.
As a result of docking process the clusters representing different binding manner have
been obtained for each ligand. The most probable assignments of investigated ligands in the
tyrosinase active site based on docking simulation are reported in Table S1. Multiple clusters
have been specified for the thiosemicarbazones with the aromatic ring in position R1 or R2
localized in the active site in close contact to catalytic copper ions. The most important
information is delivered by combining the cluster size and the binding energy between the
inhibitor molecule and the enzyme active site. From the obtained results it could be
concluded, that the best inhibitory properties are found for the following compounds TSC 19,
TSC 2, TSC 3 and TSC 18.
The magnitude of interaction of these inhibitors in the enzyme active site is described by
the highest total absolute value of the binding energy for the biggest clusters.
The docking of these molecules in the active site is shown in Fig. 4. According to this
Figure, inhibitor-enzyme complexes shown on A, C, D, E, G, H, I and J diagrams are
stabilized by at least two intermolecular hydrogen bonds. Moreover, in the case of TSC 2,
strong π-π interaction between phenyl ring of inhibitor and side chain of His263 stabilizes the
inhibitor arrangement in the enzymatic active pocket (see Fig. 4. D). Only cluster 2 found for
TSC 11 presents orientation of the inhibitor which could allow to complex the copper ions by
the thiourea moiety (see Fig. 4. J) what was reported for such compounds by Xie et al. [24]
and Buitrago et al. [29]. Based on manual docking and following molecular dynamic
simulation, Buitrago et al., suggest that TSC 1 is able to decrease the enzyme activity due to
interaction between thiosemicarbazide moiety of inhibitor and catalytic copper ions of
tyrosinase [29]. Results of simulation of docking process in current work reveal a different
way of interaction with the enzyme active site. Achieved results show that TSC 1 is able to
inhibit tyrosinase, indeed. However, the found alignment of the inhibitor is similar to the one
of the enzyme’s substrate (cluster 2 – see Fig. 4. B). This scheme of interaction is not the
most probable and most favorable one, though. This example is represented by cluster 1 (see

Fig. 4. B) which includes almost 50% of all docked structures and is characterized by the
mean binding energy lower than for cluster 2 of about 0.2 kcal/mol.
Fig. 4. Orientation of the selected inhibitors in the active site of tyrosinase (copper ions are
represented by orange spheres and interatomic hydrogen bonds by dashed lines); A) TSC 1
cluster 1; B) TSC 1 cluster 2; C) TSC 2; D) TSC 2 with marked π-π interactions; E) TSC 19
cluster 1; F) TSC 19 cluster 2; G) TSC 3 cluster 1; H) TSC 3 cluster 2; I) TSC 11 cluster 1; J)
TSC 11 cluster 2; K) TSC 18 cluster 1; L) TSC 18 cluster 2.
2.4. Effectiveness of TSCs in melanogenesis inhibition
For the purpose of screening of antimelanogenic effects, B16F10 melanoma cells are
widely used, because they are relatively easy to culture in vitro, and they share most of the
melanogenic mechanisms of normal human melanocytes [58].
In order to test the inhibitory effect of the studied TSCs on melanogenesis, B16 mouse
melanoma cells were stimulated with α-MSH, which is physiological stimulator of the
pigmentation. During 48 hours of hormonal stimulation, cells were treated with the inhibitors
at various concentration (1-100 μM). Kojic acid was used as a positive control. B16 cell
viability was also estimated in the presence of the tested inhibitors.
Fig. 6. represents percentage inhibition of melanin production in B16 cells. TSC 2, 3, 6,
8, 9, 10, 13 and 18 fully inhibit this process even at the inhibitor concentration of 10 µM.
Only TSC 1, 14 and 17 exhibit value of inhibition lower than 50%. All investigated
thiosemicarbazones reveal stronger inhibition in comparison with kojic acid.
Melanin production in B16 cells was inhibited by TSC 1-19 in dose-dependent manner.
Table 2. shows IC50 values of this process for comparison to results presented in the Fig. 5.
All of investigated compounds showed strong potency of melanogenesis inhibition (low IC50
values in comparison with positive control – kojic acid). Compounds with IC50 value between
1 and 5 µM (TSC 2, 4, 7, 11, 12, 13, 16, 18 and 19) were classified as strong inhibitors and
exhibited more than 5 times stronger inhibition ability than kojic acid. In turn, compounds

with IC50 value less than 1 µM (TSC 3,5,6, 8-10) could be considered as very strong
melanogenesis inhibitors (more than 30 times stronger inhibition potency than kojic acid).
These results were verified with the effect of tested inhibitors on proliferation of B16
cells (see Table 2.). The decrease in melanin production might be caused by high toxicity of
the inhibitors which, in turn, can lead to the inhibition of cell proliferation.
Cells treated with inhibitors: 10, 15, 18 and 19 produced low amount of melanin.
However, these results might be caused by high toxicity of the inhibitors (IC50<10 µM) which
led to the inhibition of cell proliferation. Inhibitors which revealed strong melanogenesis
inhibitory effect and did not cause high inhibition of proliferation may be considered as
potent, non-toxic inhibitors of melanin biosynthesis pathway in B16 cells (TSC 2, 3, 4, 5, 6, 7,
8, 9, 11, 12, 13, 16).
Fig. 5. Percentage inhibition of melanin production in B16 cells at inhibitor concentrations of
40 and 10 µM. Kojic acid was positive control, whereas DMSO was control of TSC solvent.
With respect to the TSC 19 the proper absorbance measurement at 40 µM was unachievable
because of the compound color.
Table 2. Inhibition of melanin production and cell proliferation in B16 cells.
Inhibition of melanin
production IC50
(µM)
Inhibition of cell
proliferation IC50 95% CI IC50
(µM)
95% CI
IC50
Compound
TSC 1
TSC 2
TSC 3
TSC 4
TSC 5
TSC 6
TSC 7
TSC 8
TSC 9
TSC 10
TSC 11
10.2
2.0
<1.0
2.3
<1.0
<1.0
1.2
<1.0
<1.0
<1.0
1.4
9.1 - 11.4
1.4 - 2.9
0.7 - 1.9
1.4 - 3.6
0.05 - 9.1
0.01 - 5.6
0.7 - 1.8
0.2 - 1.4
NA
54.6
19.6
32.3
28.5
25.5
27.3
36.8
34.6
10.8
2.5
45.9 - 64.8
13.7 - 28.1
19.2 - 54.3
22.3 - 36.5
18.6 - 35.1
19.1 - 39.2
27.4 - 49.5
24.4 - 49.1
7.0 - 16.8
1.6 - 4.0
NA
1.0 - 1.8
43.6
33.7 - 56.4

TSC 12
TSC 13
TSC 14
TSC 15
TSC 16
TSC 17
TSC 18
TSC 19
DMSO
1.3
4.7
14.8
5.2
4.7
11.6
1.8
1.0
98.9
32.2
0.7 - 2.4
3.2 - 6.8
12.0 - 18.4
4.7 - 5.9
4.4 - 5.1
9.5 -14.2
1.5 - 2.3
0.3 - 3.8
63.8 – 153.2
26.3 – 39.5
49.5
131.8
62.4
7.5
14.4
21.1
2.7
5.7
252.5
181.3
41.2 - 59.5
97.3 - 178.7
37.0 - 105.3
5.4 - 10.2
11.5 - 18.2
18.2 - 24.4
1.9 - 3.9
4.1 - 7.9
235.1 – 271.1
150.6 – 218.3
Kojic acid
3. Conclusions
In the frame of this work, 19 thiosemicarbazones were synthesized and its inhibitory
activity toward mushroom tyrosinase was examined. Moreover, molecular docking of these
compounds to the active site of the enzyme was performed and ability to inhibition of
melanogenesis in B16 cells was checked.
Kinetic studies of inhibition of diphenolase activity of tyrosinase revealed that there is a
correlation between structure of investigated molecules and its inhibitory activity. The
influence of substituents in R1, R2 and R3 positions on thiosemicarbazone inhibitory abilities
was investigated. Phenyl ring in R1 enables the best inhibition of enzyme in comparison with
naphthyl and anthracenyl substituents. In R2, methyl and phenyl groups are favorable.
Elongation and branching of alkyl chain diminishes the inhibitory potential of TSCs. With
respect to R3, the replacement of proton decreases the activity of inhibitors. Abovementioned
observations indicate that the smaller and less branched molecules exhibit higher affinity to
enzyme what might be associated with the steric constraints. The fact that TSCs demonstrate
different types of inhibition (competitive, noncompetitive, uncompetitive and mixed type)
proves their affinity to both: free enzyme and substrate-enzyme complex.
Molecular docking studies show that in the majority of the cases TSCs interact rather
with tyrosinase active site via aromatic ring than thiosemicarbazide moiety what can be
helpful information in designing novel tyrosinase inhibitors.
Most of investigated compounds reveals very good inhibitory activity against melanin
production in B16 cells. There is no obvious relationship between inhibition of melanogenesis
in melanoma cells and enzyme inhibition. Moreover, in the case of melanogenesis, elongation
of alkyl chain in R2 position seems to have a positive impact on inhibition. It can be predicted
then that most of melanogenesis inhibitors described above inhibit the pathway also on the
other stages than the step catalyzed by tyrosinase, for example affecting activity of other key
enzymes in melanin biosynthesis like dopachrome tautomerase, microphthalmia associated
transcription factor or tyrosinase related protein 1 [59].
Results of our studies show that thiosemicarbazones can be considered as potent inhibitors
of tyrosinase and melanogenesis.
4. Experimental

4.1. Chemistry
1
13
General information: The H and C NMR spectra were recorded on a 600 MHz Bruker
Avance and Jeol ECZ 400S spectrometers in DMSO - d6 as solvent. Chemical shifts (δ) are
given in parts per million (ppm) relative to solvent signals (2.50 ppm and 39.52 ppm in
1H NMR and in 13C NMR spectra, respectively). Multiplicity is reported as follows: s =
singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, hept = heptet, m =
multiplet, br = broad. High-resolution Mass Spectra were recorded on an LCT Premier XE
Waters apparatus, on ESI+ mode. All solvents were of commercial quality and purchased
from a local supplier. Starting carbonyl compound, thiosemicarbazide, methyl isothiocyanate,
phenyl isothiocyanate, phenethyl isothiosyanate and other reagents were purchased from
Sigma-Aldrich. Benzyl isothiocyanate was prepared by literature procedure [60]. No
procedures or isolations of products were optimized.
4.1.1. General Procedure for preparation of N4-substituted thiosemicarbazide
Hydrazine monohydrate (0.12 mol, ~6 mL) was added dropwise to a stirred solution of
o
the appropriate isothiocyanate (0.10 mol) in 2-propanol (75 mL) in 15-20 minutes at 0 C
o
(water-ice bath). The resulting suspension was left for ~ 30 minutes at 0 C and ~1 hour at
room temperature. Then the product was filtered, washed with 2-propanol (4 × 25 mL) and
diethyl ether (2 × 50 mL) and dried on air. Obtained crude N4-substituted thiosemicarbazides
1
were sufficiently pure (based on H NMR) and used in the next step without further
purification.
4.1.1.1. 4-Methylthiosemicarbazide
1
Yield 85%; H NMR (600MHz, DMSO-d6) δ: 2.90 (s, 3H, CH3), 4.42 (bs, 2H, NH2),
7.81 (bs, 1H, NH), 8.56 (s, 1H, NH).
4.1.1.2. 4-Phenylthiosemicarbazide
Yield 91%; 1H NMR (600MHz, DMSO-d6) δ: 4.76 (bs, 2H, NH2), 7.11 (t, 1H, J=7.3 Hz,
ArH), 7.31 (t, 2H, J=8.1 Hz, ArH), 7.64 (bd, 2H, J not resolved, ArH), 9.07 (s, 1H, NH), 9.65
(bs, 1H, NH). NMR spectrum is in agreement with data reported in literature [61].
4.1.1.3. 4-Benzylthiosemicarbazide
Yield 61%; 1H NMR (600MHz, DMSO-d6) δ: 4.52 (bs, 2H, NH2), 4.72 (d, 1H, J=6.2 Hz,
CH2), 7.22-7.25 (m, 1H, ArH), 7.29-7.33 (m, 4H, ArH), 8.38 (bs, 1H, NH), 8.75 (s, 1H, NH).
NMR spectrum is in agreement with data reported in literature [62]
4.1.1.4. 4-Phenethylthiosemicarbazide
1
Yield 86%; H NMR (600MHz, DMSO-d6) δ: 2.83 (t, 2H, J=7.5 Hz, CH2), 3.68 (dt, 2H,
J=6.6 Hz, J=7.6 Hz, NCH2), 4.44 (s, 2H, NH2), 4.72 (d, 1H, J=6.2 Hz, CH2), 7.21 (t, 1H,
J=7.2 Hz, ArH), 7.25 (d, 2H, J=7.4 Hz, ArH), 7.31 (t, 2H, J=7.4 Hz, ArH), 7.88 (bs, 1H, NH),
8.65 (s, 1H, NH). NMR spectrum is in agreement with data reported in literature [63].
4.1.2. General Procedure for preparation of thiosemicarbazones 1-19 (TSCs 1-19).
The mixture of carbonyl compound (0.020 mol) and thiosemicarbazide (1.9 g, 0.021 mol
for TSCs 1-8 and 18-19 or 5.5 g, 0.060 mol for TSCs 9-13) or the appropriate N4-substituted
thiosemicarbazide (0.021 mol for 14-17) and acid catalyst (~2 mL of acetic acid for 1-8, 19 or
p-toluenesulfonic acid monohydrate, 3.8g, 0.02 mol for 9-18) in 96% ethanol (50 mL) was
refluxed for about 8 hours. After cooling to room temperature, the mixture was added in few
portions to vigorously stirred 10% sodium bicarbonate solution (150 mL) and stirring was

continued for about 30 min. The resulting solid was filtered, washed with water (2 × 50 mL,
10 × 20 mL) and dried on air giving a crude TSC. Analytical samples were obtained by
crystallization from appropriate solvent: 96% EtOH for 1- 3, 7, 11-17 and 19; 75% aq. EtOH
for 4 and 6; anh. EtOH: petroleum ether (2:3, V/V) for 5; MeOH for 18. In case of 8-10, the
suspension in aq. NaHCO3, was extracted with dichloromethane (1 × 50 mL, 2 × 25 mL). The
combined organic phase was washed with water (5 × 30mL), dried over anh. Na2SO4, and
concentrated under reduced pressure, to give the products 8-10 as thick oils, which partially
solidified after standing at room temperature. These TSC were used without further
purification.
4.1.2.1. (E)-Benzaldehyde thiosemicarbazone (TSC 1).
1
Yield 31 %; H NMR (600MHz, DMSO-d6) δ: 7.38-7.43 (m, 3H, ArH), 7.79-7.81 (m,
13
2H, ArH), 8.00 (s, 1H, NH), 8.06 (s, 1H, CH=N), 8.21 (s, 1H, NH), 11.44 (s, 1H, NH). C
NMR (100 MHz, DMSO-d6) δ: 127.82, 129.18,134.70, 134.70, 142.79 (C=N), 178.51 (C=S).
HRMS (ESI+): m/z calcd for C8H10N3S (M+H)+ 180.0595, found 180.0592. NMR spectra are
in agreement with data reported in literature [64,65].
4.1.2.2. (E)-Acetophenone thiosemicarbazone (TSC 2).
Yield 69%; 1H NMR (600MHz, DMSO-d6) δ: 2.31 (s, 3H, CH3), 7.38-7.40 (m, 3H, ArH),
13
7.92-7.94 (m, 3H, NH + ArH), 8.29 (bs, 1H, NH), 10.23 (s, 1H, NH). C NMR (100 MHz,
DMSO-d6) δ: 14.54 (CH3), 127.11, 128.76, 129.73, 138.16, 148.38 (C=N), 179.45 (C=S).
HRMS (ESI+): m/z calcd for C9H12N3S (M+H)+ 194.0752, found 194.0782. NMR spectra are
in agreement with data reported in literature [44,66].
4.1.2.3. (E)-Propiophenone thiosemicarbazone (TSC 3).
1
Yield 78%; H NMR (600MHz, DMSO-d6) δ: 1.03 (t, 3H, J=7.8 Hz, CH3), 2.88 (q, 2H,
J=7.8 Hz, CH2), 7.39-7.41 (m, 3H, ArH), 7.90-7.93 (m, 3H, NH + ArH), 8.29 (bs, 1H, NH),
13
10.35 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 11.50 (CH3), 19.77, 127.21, 128.90,
129.68, 136.92, 152.30 (C=N), 179.49 (C=S). HRMS (ESI+): m/z calcd for C10H14N3S
1
(M+H)+ 208.0909, found 208.0949. H NMR spectrum is in agreement with data reported in
literature [46,67].
4.1.2.4. Mixture of Z and E isomers (in 89:11 molar ratio) of isobutyrophenone
thiosemicarbazone (TSC 4).
Yield 82%; 1H NMR (600MHz, DMSO-d6) δ: 1.08 (d, J=6.8 Hz, CH3, Z isomer) and 1.12
(d, J=7.0 Hz, CH3, E isomer) [total integration 6H], 2.83 (hept, J=6.8 Hz, CH, Z isomer) and
3.56 (hept, J=7.0 Hz, CH, E isomer) [total integration 1H], 7.27 (d, J=8.1 Hz, ArH, Z isomer)
and 7.38 (m, ArH, E isomer) [total integration 2H], 7.52 (m, ArH, E + Z isomers) and 7.58 (t,
ArH, J=7.3 Hz, Z isomer) [total integration 3H], 7.66 (bs, NH, E isomer) and 7.79 (bs, NH, Z
isomer) [total integration 1H], 8.24 (bs, 1H, NH, E + Z isomers). 8.45 (s, 1H, NH, Z isomer)
and 10.33 (s, 1H, NH, E isomer) [total integration 1H]. 13C NMR (100 MHz, DMSO-d6) δ:
major, Z isomer: 20.39, 36.04, 127.70, 129.99, 158.38, 178.37*; minor E isomer: 20.28,
27.64, 128.46, 128.51, 128.87, 137.57, 157.48, 179.64; * one aromatic CH carbon signal is
missing, probably due to coalescence and overlaps. HRMS (ESI+): m/z calcd for C11H15N3S
(M+H)+ 222.1065, found 222.1066. Note: the crude and recrystallized samples shown
practically the same ratio of E and Z isomers.
4.1.2.5. (E)-Butyrophenone thiosemicarbazone (TSC 5).
1
Yield 73%; H NMR (600MHz, DMSO-d6) δ: 0.95 (t, 3H, J=7.5 Hz, CH3), 1.43 (sextet,
2H, J=7.5 Hz, CH3CH2), 2.86 (t, 2H, J=7.5 Hz, CH2), 7.39 (m, 3H, ArH), 7.91 (m, 3H, NH +

ArH), 8.28 (bs, 1H,NH), 10.41 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 14.23, 20.12,
28.11, 127.24, 128.86, 129.62, 137.35, 151.14 (C=N), 179.45 (C=S). HRMS (ESI+): m/z
calcd for C11H16N3S (M+H)+ 222.1065, found 222.1059.
4.1.2.6. (E)-Isovalerophenone thiosemicarbazone (TSC 6).
1
Yield 81%; H NMR (400MHz, DMSO-d6) δ: 0.82 (d, 6H, J=6.7 Hz, (CH3)2CH), 1.78
(hept, 1H, J=6.7 Hz, CH), 2.80 (d, 2H, J=7.3 Hz, CH2), 7.33 (m, 3H, ArH), 7.84-7.86 (m, 3H,
NH + ArH), 8.23 (bs, 1H, NH), 10.30 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 22.42,
26.98, 34.29, 127.44, 128.79, 129.54, 137.84, 150.93 (C=N), 179.34 (C=S). HRMS (ESI+):
m/z calcd for C12H18N3S (M+H)+ 236.1221, found 236.1225.
4.1.2.7. (E)-Valerophenone thiosemicarbazone (TSC 7).
Yield 87%; 1H NMR (600MHz, DMSO-d6) δ: 0.88 (t, 3H, J=7.6 Hz, CH3), 1.38 (m, 4H,
CH3(CH2)2), 2.88 (t, 2H, J=7.5 Hz, CH2), 7.39 (m, 3H, ArH), 7.89-7.91 (m, 3H, NH + ArH),
8.28 (bs, 1H, NH), 10.38 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 14.34 (CH3), 22.57,
26.22, 28.86, 127.22. 128.86, 129.62, 137.34, 151.37 (C=N), 179.45 (C=S). HRMS (ESI+):
1
m/z calcd for C12H18N3S (M+H)+ 236.1221, found 236.1226. H NMR spectrum is in
agreement with data reported in literature [46].
4.1.2.8. (E)-Hexanophenone thiosemicarbazone (TSC 8).
Yield 80%; 1H NMR (400MHz, DMSO-d6) δ: 0.80 (t, 3H, J=7.0 Hz, CH3), 1.20-1.37 (m,
6H, CH3(CH2)3), 2.82 (t, 2H, J=7.8 Hz, CH2), 7.32-7.35 (m, 3H, ArH), 7.82-7.86 (m, 3H, NH
13
+ ArH), 8.24 (bs, 1H, NH), 10.34 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 14.43,
22.51, 26.34, 26.39, 31.54, 127.22, 128.86, 129.62, 137.33, 151.38 (C=N), 179.44 (C=S).
HRMS (ESI+): m/z calcd for C13H20N3S (M+H)+ 250.1378, found 250.1390.
4.1.2.9. (E)-Heptanophenone thiosemicarbazone (TSC 9).
Yield 96%;1H NMR (400MHz, DMSO-d6) δ: 0.80 (t, 3H, J=7.0 Hz, CH3), 1.19-1.25 (m,
4H, CH3(CH2)2),1.25-1.40 (m, 4H, CH3(CH2)2(CH2)2), 2.82 (t, 2H, J=7.0 Hz, CH2), 7.30-7.35
(m, 3H, ArH), 7.80-7.88 (m, 3H, NH + ArH), 8.23 (bs, 1H, NH), 10.34 (s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6) δ: 14.45, 22.60, 26.40, 26.67, 29.00, 31.59, 127.22, 128.86, 129.60,
137.33, 151.33 (C=N), 179.42 (C=S). HRMS (ESI+): m/z calcd for C14H22N3S (M+H)+
264.1534, found 264.1540.
4.1.2.10. (E)-Dodecanophenone thiosemicarbazone (TSC 10).
Yield 96%; 1H NMR (400MHz, DMSO-d6) δ: 0.79 (t, 3H, J=7.0 Hz, CH3), 1.10-1.25 (m,
14H, CH3(CH2)7),1.25-1.39 (m, 4H, CH3(CH2)7(CH2)2), 2.81 (t, 2H, J=6.7 Hz, CH2), 7.30-
7.35 (m, 3H, ArH), 7.80-7.87 (m, 3H, NH + ArH), 8.24 (bs, 1H, NH), 10.34 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6) δ: 14.47, 22.65, 26.42, 26.68, 29.28, 29.36, 29.41, 29.55, 29.56,
29.57, 31.85, 127.17, 128.81, 129.55, 137.35, 151.23 (C=N), 179.47 (C=S). HRMS (ESI+):
m/z calcd for C19H32N3S (M+H)+ 334.2317, found 334.2324.
4.1.2.11. Benzophenone thiosemicarbazone (TSC 11).
1
Yield 94%; H NMR (600MHz, DMSO-d6) δ: 7.33-7.40 (m, 4H, ArH), 7.41-7.44 (m,
1H, ArH), 7.61-7.65 (m, 1H, ArH), 7.65-7.70 (m, 4H, ArH), 8.38 (bs, 1H, NH), 8.41 (bs, 1H,
13
NH), 8.65 (bs, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 128.11, 128.83, 128.89, 130,30,
130,38, 130,52, 131.74, 136.83, 149.61 (C=N), 178.37 (C=S). HRMS (ESI+): m/z calcd for
C14H14N3S (M+H)+ 256.0909, found 256.0913. NMR spectra are in agreement with data
reported in literature [44,68].

4.1.2.12. (E)-2-Phenylacetophenone thiosemicarbazone (TSC 12).
Yield 96%; 1H NMR (600MHz, DMSO-d6) δ: 4.39 (s, 2H, CH2), 7.14 (d, 2H, J = 7.8 Hz,
ArH), 7.19 (t, 1H, J = 7.3 Hz, ArH), 7.29 (t, 2H, J = 7.8 Hz, ArH), 7.36 (m, 3H, ArH), 7.96
13
(m, 2H, ArH), 8.07 (bs, 1H, NH), 8.40 (bs, 1H, NH), 10.52 (bs, 1H, NH). C NMR (100
MHz, DMSO-d6) δ: 32.24 (CH2), 126.87, 127.56, 128.61, 128.83, 129.21, 129.70, 136.92,
137.42, 148.45 (C=N), 179.63 (C=S). HRMS (ESI+): m/z calcd for C15H16N3S (M+H)+
270.1065, found 270.1064. NMR spectra are in agreement with data reported in literature
[20].
4.1.2.13. (Z)-2,2,2-Trifluoroacetophenone thiosemicarbazone (TSC 13).
Yield 98%;1H NMR (600MHz, DMSO-d6) δ: 7.46 (m, 2H, ArH), 7.61 (m, 3H, ArH),
13
8.10 (bs, 1H, NH), 8.78 (bs, 1H, NH), 9.87 (s, 1H, NH). C NMR (150 MHz, DMSO-d6) δ:
2
121.14 (q, 1JC-F = 272.8 Hz, CF3), 126.74, 129.04, 130.08, 131.54, 136.20 (q, JC-F = 33.8 Hz
C=N), 180.21 (C=S). HRMS (ESI+): m/z calcd for C9H9F3N3S (M+H)+ 248.0469, found
248.0474.
4.1.2.14. (E)-Acetophenone N(4)-methylthiosemicarbazone (TSC 14).
Yield 77%; 1H NMR (600MHz, DMSO-d6) δ: 2.31 (s, 3H, CH3), 3.06 (s, 3H, NCH3), 7.40 (m,
13
3H, ArH), 7.93-7.95 (m, 2H, ArH), 8.46 (bs, 1H, NH), 10.23 (s, 1H, NH). C NMR (100
MHz, DMSO-d6) δ: 14.54(CH3), 31.65(NCH3), 127.09, 128.75, 129.67 138.23, 148.04
(C=N), 179.23 (C=S). HRMS (ESI+): m/z calcd for C10H14N3S (M+H)+ 208.0909, found
208.0932. 1H NMR spectrum is in agreement with data reported in literature [21].
4.1.2.15. (E)-Acetophenone N(4)-phenylthiosemicarbazone (TSC 15).
Yield 42%; 1H NMR (600MHz, DMSO-d6) δ: 2.40 (s, 3H, CH3), 7.22 (tt, 1H, J = 7.4 Hz, J =
1.4 Hz, ArH), 7.38 (m, 2H, ArH), 7.42-7.44 (m, 3H, ArH), 7.58 (m, 2H, ArH), 8.00-8.03 (m,
13
2H, ArH), 10.05 (bs, 1H, NH), 10.60 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 14.97
(CH3), 125.88, 126.36, 127.39, 128.61, 128.79, 129.95, 138.02, 139.70, 149.50 (C=N), 177.56
1
(C=S). HRMS (ESI+): m/z calcd for C15H16N3S (M+H)+ 270.1065, found 270.1069. H NMR
spectrum is in agreement with data reported in literature [21]
4.1.2.16. (E)-Acetophenone N(4)-benzylthiosemicarbazone (TSC 16).
1
Yield 79%; H NMR (600MHz, DMSO-d6) δ: 2.33 (s, 3H, CH3), 4.88 (d, 2H, J= 6.4 Hz,
CH2), 7.24 (tt, 1H, J = 7.0 Hz, J = 1.9 Hz, ArH), 7.31-7.36 (m, 4H, ArH), 7.39-7.41 (m, 3H,
13
ArH), 7.92-7.95 (m, 2H, ArH), 9.02 (bt, J= 6.4 Hz, 1H, NH), 10.31 (s, 1H, NH). C NMR
(100 MHz, DMSO-d6) δ: 14.70(CH3), 47.30 (CH2), 127.17, 127.21, 127.70, 128.69, 128.78,
129.77, 138.18, 140.00,148.64 (C=N), 179.14 (C=S). HRMS (ESI+): m/z calcd for C16H18N3S
(M+H)+ 284.1221, found 284.1219.
4.1.2.17. (E)-Acetophenone N(4)-phenethylthiosemicarbazone (TSC 17).
1
Yield 86%; H NMR (600MHz, DMSO-d6) δ: 2.31 (s, 3H, CH3), 2.94 (bt, 2H, J= 7.7 Hz,
PhCH2), 3.81 (m, 2H, NCH2), 7.25 (tt, 1H, J = 7.0 Hz, J = 1.5 Hz, ArH), 7.29-7.35 (m, 4H,
ArH), 7.41-7.43 (m, 3H, ArH), 7.83-7.86 (m, 2H, ArH), 8.46 (bt, J= 5.8 Hz, 1H, NH), 10.31
13
(s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ: 14.64 (CH3), 35.36 (CH2), 45.62 (CH2),
126.76, 127.02, 128.80, 129.03, 129.17, 129.76, 139.77, 140.00,148.26 (C=N), 178.43 (C=S).
HRMS (ESI+): m/z calcd for C17H20N3S (M+H)+ 298.1378, found 298.1378.
4.1.2.18. (E)-2-Acetonaphthone thiosemicarbazone (TSC 18).
Yield 84%; 1H NMR (600MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 7.54 (m, 2H, ArH), 7.88 (d,
1H, J=8.9Hz, ArH), 7.93 (m, 1H, ArH), 7.99 (m, 1H, ArH), 8.07 (bs, 1H, NH), 8.31 (dd, 1H,

J=8.9Hz, J=1.6Hz, ArH), 8.35 (d, 1H, J=1.6Hz, ArH), 8.36 (bs, 1H, NH), 10.30 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6) δ: 14.34 (CH3), 124.63, 126.87, 127.03, 127.30, 127.96,
128.06, 129.05, 133.28, 133.76, 135.62, 148.13 (C=N), 179.46 (C=S). HRMS (ESI+): m/z
calcd for C13H14N3S (M+H)+ 244.0909, found 244.0897. NMR spectra are in agreement with
data reported in literature [44,46].
4.1.2.19. (E)-9-Anthrylcarboxyaldehyde thiosemicarbazone (TSC 19).
Yield 75% 1H NMR (600MHz, DMSO-d6) δ: 7.59 (m, 2H, ArH), 7.65 (m, 2H, ArH), 7.73 (bs,
1H, NH), 8.16 (d, 2H, J=8.8Hz, ArH), 8.34 (bs, 1H, NH), 8.58 (d, 2H, J=8.8Hz, ArH), 8.72
13
(s, 1H, CH=N), 9.34 (s, 1H, ArH), 11.66 (s, 1H, NH). C NMR (100 MHz, DMSO-d6) δ:
125.31, 125.51, 126.11, 127.85, 129.48, 129.98, 130,16, 131,39, 142.68 (C=N), 178.54
1
(C=S). HRMS (ESI+): m/z calcd for C16H14N3S (M+H)+ 280.0909, found 280.0919. H NMR
spectrum is in agreement with data reported in literature [69,70].
4.2. Enzyme preparation
The enzyme was isolated from mushrooms (Agaricus bisporus) purchased from a local
supplier in Wrocław according to the procedure described by Gąsowska et al. [71] with slight
modifications. The full description of enzyme preparation is included in electronic
supplementary information.
4.3. Enzyme activity assay
The diphenolase activity of tyrosinase was checked using L-dopa as substrate. The
activity assay was performed as described by Xie et al. [24] with slight modifications. Briefly,
all investigated thiosemicarbazones were dissolved in DMSO and then diluted in 50 mM
Na2HPO4-NaH2PO4 buffer (pH 6.8) to desired test concentrations (final concentration of
DMSO in reaction mixture was below 1%). Enzyme solution (0.5 mg/ml, 40 000 U/mg) was
diluted 5 times in 50 mM Na2HPO4-NaH2PO4 buffer (pH 6,8). 10 µl of diluted enzyme
solution was first pre-incubated with the compounds in 50 mM Na2HPO4-NaH2PO4 buffer
(pH 6.8) for 5 min at 25°C. After the solution of L-dopa was added the reaction was
monitored by measuring the change in absorbance of colour product (dopachrome) for 5 min
(λ = 475 nm, 25 °C) using Molecular Devices SpectraMax Plus 384 Microplate Reader.
Control sample contained the same reagents as the test samples except for the substrate. Kojic
acid was used as a positive control and was treated the same way as the other inhibitors.
4.4. Molecular docking
The structures of all the ligands were optimized in program Gaussian09 at the B3LYP/6-
311g (d,p) level of theory [52] with the PCM solvent model using water as the solvent.
Additionally, Merz-Singh-Kollman scheme [55] has been used for the determination of the
atomic charges. The simulation of the docking process was performed using AutoDock v.4.2
program [54]. The starting geometry of the ligands as well as ligand charges has been
assigned based on the ab initio calculations. The structure of tyrosinase was taken from the
crystal structure of Agaricus bisporus deposited in Protein Data Bank [53]. Lacking protons
and charges were added to the protein using H++ server [56, 57] according to pH value 6.8.
During the docking process stereochemistry of C-N double bond has been fixed, whereas the
rest of single bonds has been left as routable. The coordinates of the copper ions were taken as

a grid center in the docking process and the chosen grid had size 80x80x80 points. Each
docking procedure using Lackmarkian Genetic Algorithm (LGA) was repeated until obtaining
200 conformations. Several possible structures of ligand-enzyme complex were gathered in
the clusters for each type of ligand.
4.5. Cell proliferation assay
B16F10 cells were cultured in DMEM (Biowest) medium supplemented with 100 U/ml
penicillin (Sigma-Aldrich), 100 U/ml streptomycin (Sigma-Aldrich) and 10% fetal bovine
serum (Sigma-Aldrich). Cells were maintained in 5% CO2 at 37°C.
Cells viability was estimated using colorimetric 3-(4,5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) assay as described [72]. B16 cells were seeded into 96-
well plate (4x103 cells/well) and incubated in the presence of tyrosinase inhibitors in various
concentration (1000, 400, 100, 40, 10, 1, 0.1 μM) for 48 hours. As a control, DMSO and kojic
acid were used. For the last 4 hours MTT was added into wells (0.625 mg/ml, Sigma-
Aldrich). After that, lysing buffer was added in order to dissolve insoluble formazan. The
absorbance was measured at 570 nm using Thermo Lab Multiscan RC microplate reader.
4.6. Measurement of melanin production
Measurement of melanin production in B16 cells was performed as described by Bellei et
al. [72]. B16 cells were seeded into 96-well plate (5x103 cells/well) and stimulated with α-
MSH (100 nM, Sigma-Aldrich). After 24 hours cells were treated with tyrosinase inhibitors as
well as DMSO and kojic acid at a final concentration of 100, 40, 10, 4 and 1 μM. After 48
hours of incubation with inhibitors, cells were solubilized in 1N sodium hydroxide at 60°C for
2 hours. In order to measure melanin content, absorbance was measured at 405 nm using
microplate reader.
Acknowledgements
The authors acknowledge the support from Wroclaw Centre of Biotechnology,
programme The Leading National Research Center (KNOW) for years 2014-2018 and the
Polish Ministry of Science and Higher Education (PMSHE) for the Faculty of Chemistry of
Wrocław University of Science and Technology.
The molecular modeling was carried out using hardware and software resources of The
Supercomputing and Networking Center in Wroclaw (grant no. 197).
References
[1] X. Lai, H.J. Wichers, M. Soler-Lopez, B.W. Dijkstra, Structure and Function of Human
Tyrosinase and Tyrosinase-Related Proteins, Chem. Eur. J. 24 (2018) 47–55.
[2] P. Pongkai, T. Saisavoey, P. Sangtanoo, P. Sangvanich, A. Karnchanatat, Aphichart,
Effects of protein hydrolysate from chicken feather meal on tyrosinase activity and melanin
formation in B16F10 murine melanoma cells, Food Sci. Biotechnol. 26 (2018) 1199-1208.
[3] H. Hridya, A. Amrita, S. Mohan, M. Gopalakrishnan, T. K. Dakshinamurthy, G.P. Doss,
R. Siva, Functionality study of santalin as tyrosinase inhibitor: A potential depigmentation
agent, Int. J. Biol. Macromol. 86 (2016) 383–389.
[4] T. Pillaiyar, M. Manickam, V. Namasivayam, Skin whitening agents: medicinal chemistry
perspective of tyrosinase inhibitors, J. Enzym. Inhib. Med. Ch. 32 (2017) 403-425.

[5] T. Pillaiyar, M. Manickamb, S.-H. Jung, Recent development of signaling pathways
inhibitors of melanogenesis, Cellular Signalling 40 (2017) 99-115.
[6] H. Xu, X. Zhang, E. Karangwa, S. Xia, Correlating enzymatic browning inhibition and
antioxidant ability of Maillard reaction products derived from different amino acids, J. Sci.
Food Agric. 97 (2017) 4210-4218.
[7] M. Yusuf, P. Jain, Synthesis and biological significances of 1,3,4-thiadiazolines and
related heterocyclic compounds, Arab. J. Chem. 7 (2014) 525-552.
[8] P. Chellan, S. Nasser, L. Vivas, K. Chibale, G.S. Smith, Cyclopalladated complexes
containing tridentate thiosemicarbazone ligands of biological significance: Synthesis,
structure and antimalarial activity, J. Organomet. Chem. 695 (2010) 2225-2232.
[9] H.C. Zhao, Y.P. Shi, Y.M. Liu, C.W. Li, L.N. Xuan, P. Wang, K. Zhang, B.Q. Chen,
Synthesis and antitumor-evaluation of 1,3-selenazole-containing 1,3,4-thiadiazole derivatives,
Bioorg. Med. Chem. Lett. 23 (2013) 6577-6579.
[10] Y. Yu , Y. Suryo Rahmanto, D.R. Richardson, Bp44mT: an orally active iron chelator of
the thiosemicarbazone class with potent anti-tumour efficacy, Br. J. Pharmacol. 165 (2012)
148–166.
[11] M.-H. Yang, C.-M. Chen. Y.-H. Hu, C.-Y. Zheng, Z.-C. Li, L.-L. Ni, L. Sun, Q.-X.
Chen, Inhibitory kinetics of DABT and DABPT as novel tyrosinase inhibitors, J. Biosci
Bioeng. 115 (2013) 514-517.
[12] Z.-C. Li, L.-H. Chen, X.-J. Yu, Y.-H. Hu, K.K. Song, X.W. Zhou, Q.-X. Chen, Inhibition
Kinetics of Chlorobenzaldehyde Thiosemicarbazones on Mushroom Tyrosinase, J. Agric.
Food Chem. 58 (2010) 12537–12540.
[13]L.-H. Chen, Y.-H. Hu, W. Song, K.-K. Song, X. Liu, Y.-L. Jia, J.-X. Zhuang, Q.-X.
Chen, Synthesis and Antityrosinase Mechanism of Benzaldehyde Thiosemicarbazones: Novel
Tyrosinase Inhibitors, J. Agric.Food Chem. 60 (2012) 1542−1547.
[14] C.-B. Xue, L. Zhang, W.-C. Luo, X.-Y. Xie, L. Jiang, T. Xiao, 3D-QSAR and molecular
docking studies of benzaldehyde thiosemicarbazone, benzaldehyde, benzoic acid, and their
derivatives as phenoloxidase inhibitors, Bioorgan. Med. Chem. 15 (2007) 2006–2015.
[15] W. Yi, C. Dubois, S. Yahiaoui, R. Haudecoeur, C. Belle, H. Song, R. Hardré b, M.
Réglier, A. Boumendjel, Refinement of arylthiosemicarbazone pharmacophore in inhibition
of mushroom tyrosinase, Eur. J. Med. Chem. 46 (2011) 4330-4335.
[16] M.A. Soares, M.A. Almeida, C. Marins-Goulart, O.A. Chaves, A. Echevarria, M.C.C. de
Oliveira, Thiosemicarbazones as inhibitors of tyrosinase enzyme, Bioorg. Med. Chem. Lett.
27 (2017) 3546-3550
[17] W. Yi, R.-H. Cao, Z.-Y. Chen, L. Yu, L. Ma, H.-C. Song, Design, Synthesis and
Biological
Evaluation
of
Hydroxy-
or
Methoxy-Substituted
Phenylmethylenethiosemicarbazones as Tyrosinase Inhibitors, Chem. Pharm. Bull. 57 (2009)
1273-1277.
[18] W. Yi, R.-H. Cao, Z.-Y. Chen, L. Yu, H. Wen, Q. Yan, L. Ma, and H. Song, Rational
Design and Synthesis of 4-O-Substituted Phenylmethylenethiosemicarbazones as Novel
Tyrosinase Inhibitors, Chem. Pharm. Bull. 58 (2010) 752-754.
[19] A. You, J. Zhou, S. Song, G. Zhu, H. Song, W. Yi, Structure-based modification of 3-/4-
aminoacetophenones giving a profound change of activity on tyrosinase: From potent
activators to highly efficient inhibitors, European Journal of Medicinal Chemistry 93 (2015)
255-262.
[20] S. Song, A. You, Z. Chen, G. Zhu, H. Wen, H. Song, W. Yi, Study on the design,
synthesis and structure-activity relationships of new thiosemicarbazone compounds as
tyrosinase inhibitors, Eur. J. Med Chem. 139 (2017) 815-825.

[21] T.-H. Zhu, S.-W. Cao, Y.-Y. Yu, Synthesis, characterization and biological evaluation of
paeonol thiosemicarbazone analogues as mushroom tyrosinase inhibitors, Int. J. Biol.
Macromol. 62 (2013) 589-595.
[22] H. Dong, J. Liu, X. Liu, Y. Yu, S. Cao, Combining molecular docking and QSAR
studies for modeling the anti-tyrosinase activity of aromatic heterocycle thiosemicarbazone
analogues, J. Mol. Struct. 1151 (2018) 353-365.
[23] H. Dong, J. Liu, X. Liu, Y. Yu, S. Cao, Molecular docking and QSAR analyses of
aromatic heterocycle thiosemicarbazone analogues for finding novel tyrosinase inhibitors,
Bioorg. Chem. 75 (2017) 106–117.
[24] J. Xie, H. Dong, Y. Yu, S. Cao, Inhibitory effect of synthetic aromatic heterocycle
1
thiosemicarbazone derivatives on mushroom tyrosinase: Insights from fluorescence, H NMR
titration and molecular docking studies, Food Chemistry 190 (2016) 709–716.
[25] J. Liu, M. Li, Y. Yu, S. Cao, Novel inhibitors of tyrosinase produced by the 4-
substitution of TCT (П), Int. J. Biol. Macromol. 103 (2017) 1096–1106.
[26] J. Xu, J. Liu, X. Zhu, Y. Yu, S. Cao, Novel inhibitors of tyrosinase produced by the 4-
substitution of TCT, Food Chem. 221 (2017) 1530–1538.
[27] M.M. El-Sadek, S.Y. Hassan, H.E. Abdelwahab, G.A. Yacout, Synthesis of New 1,3,4-
Thiadiazole and 1,2,3,4-Oxathiadiazole Derivatives from Carbohydrate Precursors and Study
of Their Effect on Tyrosinase Enzyme, Molecules 17 (2012) 8378-8396.
[28] A. You, J. Zhou, S. Song, G. Zhu, H. Song, W. Yi, Rational design, synthesis and
structure–activity relationships of 4-alkoxy- and 4-acyloxy-phenylethylenethiosemicarbazone
analogues as novel tyrosinase inhibitors, Bioorganic & Medicinal Chemistry 23 (2015) 924–
931.
[29] E. Buitrago, A. Vuillamy, A. Boumendjel, W. Yi, G. Gellon, R. Hardré, C. Philouze, G.
Serratrice, H. Jamet, M. Regliér, C. Belle, Exploring the Interaction of N/S Compounds with a
Dicopper Center: Tyrosinase Inhibition and Model Studies, Inorg. Chem. (2014), 53, 12848-
12858.
[30] H. Arslan, N. Duran, G. Borekci, C.K. Ozer, C. Akbay, Antimicrobial Activity of Some
Thiourea Derivatives and Their Nickel and Copper Complexes, Molecules 14 (2009) 519-527.
[31] S. Parvez, M. Kang, H.-S. Chung, H. Bae, Naturally Occurring Tyrosinase Inhibitors:
Mechanism and Applications in Skin Health, Cosmetics and Agriculture Industries Phytother.
Res. 21 (2007) 805–816.
[32] Y.J. Kima, H. Uyamab, Tyrosinase inhibitors from natural and synthetic sources:
structure, inhibition mechanism and perspective for the future, Cell. Mol. Life Sci. 62 (2005)
1707-1723.
[33] M. Serda, J.G. Małecki, A. Mrozek-Wilkiewicz, R. Musioł, J. Polański, Microwave
assisted synthesis, X-ray crystallography and DFT calculationsof selected aromatic
thiosemicarbazones, J. Mol. Struct. 1037 (2013) 63–72.
[34] T.K. Venkatachalam, G.K. Pierens, D.C. Reutens, Synthesis, NMR structural
characterization and molecular modeling of substituted thiosemicarbazones and
semicarbazones using DFT calculations to prove the syn/anti isomer formation, Magn. Reson.
Chem. 52 (2014) 98–105.
[35] J. Song, L.M. Jones, G.D. Kishore Kumar, E.S. Conner, L. Bayeh, G.E. Chavarria, A.K.
Charlton-Sevcik, S.-E. Chen, D.J. Chaplin, M.L. Trawick, K.G. Pinney, Synthesis and
Biochemical Evaluation of Thiochromanone Thiosemicarbazone Analogues as Inhibitors of
Cathepsin LACS, Med. Chem. Lett. 3 (2012) 450−453.
[36] P. Makam, R. Kankanala, A. Prakash, T. Kannan, 2-(2-Hydrazinyl)thiazole derivatives:
Design, synthesis and in vitro antimycobacterial studies, Eur. J. Med. Chem. 69 (2013) 564-
576.

[37] P.V. Bernhardt, M. Martínez, C. Rodríguez, M. Vazquez, Biologically active
thiosemicarbazone Fe chelators and their reactions with ferrioxamine B and ferric EDTA; a
kinetic study, Dalton Trans. 41 (2012) 2122-2130.
[38] T.K. Venkatachalam, P.V. Bernhardt, C.J. Noble, N. Fletcher, G.K. Pierens, K.J.
Thurecht, D.C. Reutens, Synthesis, characterization and biological activities of
semicarbazones and their copper complexes, J. Inorg. Biochem. 162 (2016) 295–308.
[39] L.A. Lessa, D.C. Reis, J.G. Da Silva, L.T. Paradizzi, N.F. da Silva, Mde F Carvalho,
S.A. Siqueira, H. Beraldo, Coordination of Thiosemicarbazones and Bis(thiosemicarbazones)
to Bismuth(III) as a Strategy for the Design of Metal-Based Antibacterial Agents, Chem.
Biodivers. 9 (2012) 1955-1966.
[40] O. Dömötör, N.V. May, K. Pelivan, T. Kiss, B.K. Keppler, C.R. Kowol, É. A. Enyedy, A
comparative study of α-N-pyridyl thiosemicarbazones: Spectroscopic properties, solution
stability and copper(II) complexation, Inorganica Chim. Acta 472 (2018) 264–275.
[41] D.R. Richardson, D.S. Kalinowski, V. Richardson, P.C. Sharpe, D.B. Lovejoy, M. Islam,
P.V. Bernhardt, 2-Acetylpyridine Thiosemicarbazones are Potent Iron Chelators and
Antiproliferative Agents: Redox Activity, Iron Complexation and Characterization of their
Antitumor Activity, J. Med. Chem. 52 (2009) 1459–1470.
[42] V. Opletalová, D.S. Kalinowski, M. Vejsová, J. Kuneš, M. Pour, J. Jampílek, V. Buchta,
D.S. Richardson, Identification and Characterization of Thiosemicarbazones with Antifungal
and Antitumor Effects: Cellular Iron Chelation Mediating Cytotoxic, Activity Chem. Res.
Toxicol. 21 (2008) 1878–1889.
[43] A.A. Ali, H. Nimir, C. Aktas, V. Huch, U. Rauch, K.H. Sch fer, M. Veith,
Organoplatinum(II) Complexes with 2-Acetylthiophene Thiosemicarbazone: Synthesis,
Characterization, Crystal Structures, and in Vitro Antitumor Activity, Organometallics 31
(2012) 2256−2262.
[44] H.R. Fatondji, S. Kpoviessi, F. Gbaguidi, J. Bero, V. Hannaert, J. Quetin-Leclercq, J.
Poupaert, M. Moudachirou, G.C Accrombessi, Structure–activity relationship study of
thiosemicarbazones on an African trypanosome: Trypanosoma brucei brucei, Med. Chem.
Res. 22 (2013) 2151–2162.
[45] P. Thanigaimalai, K.C. Lee, V.K. Sharma, E. Roh, Y. Kim, S.H. Jung,
Ketonethiosemicarbazones: Structure–activity relationships for their melanogenesis inhibition
Bioorganic Med. Chem. Lett. 21 (2011) 3527–3530.
[46] C. Murakata, Y. Yamashita, R. Nakai, K. Akasaka, Y. Ino, K. Kato, Y. Kitamura, M-
stage Kinesin inhibitor, European Patent Application EP616866 2006 (see page 65)].
[47] J.R. Dimmock, J.M. McColl, S.L. Wonko, R.S. Thayer, D.S. Hancock, Evaluation of the
thiosemicarbazones of some aryl alkyl ketones and related compounds for anticonvulsant
activities, Eur. J. Med. Chem. 26 (1991) 529-534.
[48] K. Bagherzadeh, F.S. Talari, A. Sharifi, M.R. Ganjali, A.A. Saboury, M. Amanlou, A
new insight into mushroom tyrosinase inhibitors: docking, pharmacophore-based virtual
screening, and molecular modeling studies, J. Biomol. Struct. Dyn. 33 (2015) 487–501.
[49] T.-S. Chang, An Updated Review of Tyrosinase Inhibitors, Int. J. Mol. Sci. 10, (2009),
2440-2475.
[50] J.S. Chen , C.I. Wei, M. R. Marshall, Inhibition mechanism of kojic acid on polyphenol
oxidase, J. Agric. Food Chem. 39 (1991) 1897–1901.
[51] R.A Copeland, Enzymes: A Practical Introduction to Structure, Mechanism, and Data
Analysis, second ed., Viley-VCH, New York, 2000.
[52] Gaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H.
Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L.
Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima,

Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M.
Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J.
Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N.
Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R.
Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S.
Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox,
Gaussian, Inc., Wallingford CT, 2013.
[53] W.T. Ismaya, H.J. Rozeboom, A. Weijn, F. Fusetti, H.J. Wichers, B.W. Dijkstra, Crystal
structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and
interaction with tropolone, Biochemistry 50 (2011) 5477-5486.
[54] G. M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S Goodsell, A.J.
Olson, Autodock4 and AutoDockTools4: automated docking with selective receptor
flexibility, J. Comput. Chem. 16 (2009) 2785-2791.
[55]B. H. Besler, K. M. Merz Jr., P. A. Kollman, Atomic charges derived from semiempirical
methods, J. Comput. Chem. 11 (1990) 431-439.
[56] http://biophysics.cs.vt.edu/H++
[57] R. Anandakrishnan, B. Aguilar, A.V. Onufriev, H++ 3.0: automating pK prediction and
the preparation of biomolecular structures for atomistic molecular modeling and simulation,
Nucleic Acids Res. 40 (2012) 537-541.
[58]T.-S. Chang, Natural Melanogenesis Inhibitors Acting Through the Down-Regulation of
Tyrosinase Activity, Materials, 5 (2012) 1661-1685.
[59] A. Lehraiki, P. Abbe, M. Cerezo, F. Rouaud, C. Regazzetti, B. Chignon-Sicard, T.
Passeron, C. Bertolotto, R. Ballotti, S. Rocchi, Inhibition of Melanogenesis by the
Antidiabetic Metformin, J. Invest. Dermatol. 134 (2014) 2589-2597.
[60] N. Sun, B. Li, J.P. Shao, W.M. Mo, B.X. Hu, Z.L. Shen, X.Q. Hu, A general and facile
one-pot process of isothiocyanates from amines under aqueous conditions, Beilstein J. Org.
Chem. 8 (2012) 61-70.
[61] B. Hu, B. Wang, B. Zhao, Q. Guo, Z.H. Li, X.H. Zhang, G.Y. Liu, Y. Liu, Y. Tang, F.
Luo, Y. Du, Y.X. Chen, L.Y. Ma, H.M. Liu, Thiosemicarbazone-based selective proliferation
inactivators inhibit gastric cancer cell growth, invasion, and migration, Med. Chem. Commun.
8 (2017) 2173-2180.
[62] S.J. Yang, J.H. Choe, Y.D. Gong, Solid-Phase Synthesis of 1,3,4-Thiadiazole Derivatives
via Desulfurative Cyclization of Thiosemicarbazide Intermediate Resin, ACS Comb. Sci. 18
(2016) 499–506.
[63] A.R. Katritzky, N.M. Khashab, A.V. Gromova, Preparations of diversely substituted
thiosemicarbazides and N-hydroxythioureas, Arkivoc 3 (2006) 226-236.
[64] A.G. Mal’kina, V.V Nosyreva, A.I. Albanov, A.V. Afonin, A.V. Vashchenko, S.V.
Amosova, B.A. Trofimov, Regioselective N(2)-H-functionalization of thiosemicarbazones of
aromatic and heteroaromatic aldehydes with acrylonitrile Synth. Commun. 47 (2017) 159–
168.
[65] K.H. Markiewicz, P. Zembko, K. Półtorak, I. Misztalewska, S. Wojtulewski, A.M.
Majcher, E. Fornal, A.Z. Wilczewska, Magnetic nanoparticles with chelating shells prepared
by RAFT/MADIX polymerization, New J. Chem. 40 (2016) 9223-9231.
[66] L. Blau, R.F. Menegon, G.H.G. Trossini, J.V.D. Molino, D.G. Vital, R.M.B. Cicarelli,
G.D. Passerini, P.L. Bosquesi, C.M. Chin, Design, synthesis and biological evaluation of new
aryl thiosemicarbazone as antichagasic candidates, Eur. J. Med. Chem. 67 (2013) 142-151.
[67] X.H. Du, C. Guo, E. Hansell, P.S. Doyle, C.R. Caffrey, T.P. Holler, J.H. McKerrow, F.E.
Cohen, Synthesis and Structure−Activity Relationship Study of Potent Trypanocidal Thio

Semicarbazone Inhibitors of the Trypanosomal Cysteine Protease Cruzain, J. Med. Chem. 45
(2002) 2695–2707.
[68] G. D. K. Kumar, G.E. Chavarria, A.K. Charlton-Sevcik, G.K. Yoo, J. Song, T.E.
Strecker, B.G. Siim, D.J. Chaplin, M.L. Trawicka, G.K. Pinneya, Functionalized
benzophenone, thiophene, pyridine, and fluorene thiosemicarbazone derivatives as inhibitors
of cathepsin L G, Bioorganic Med. Chem. Lett. 20 (2010) 6610-6615.
[69] J. Bai, R..H. Wang, Y. Qiao, A. Wang, C.J. Fang, Schiff base derived from
thiosemicarbazone and anthracene showed high potential in overcoming multidrug resistance
in vitro with low drug resistance index, Drug Des Devel Ther. 11 (2017) 2227–2237.
[70] I.E. Tolpygin, Thiosemicarbazones as Effective Fluorescent Sensors for Cations and
Anions Russ. J. Gen. Chem. 82 (2012) 1533–1536.
[71] B. Gąsowska, P. Kafarski, H. Wojtasek, Interaction of mushroom tyrosinase with
aromatic amines, o-diamines and o-aminophenols, BBA-Gen Subjects 1673 (2004) 170-177.
[72] B. Bellei , A. Pitisci, E. Izzo, M. Picardo, Inhibition of Melanogenesis by the Pyridinyl
Imidazole Class of Compounds: Possible Involvement of the Wnt/β-Catenin Signaling
Pathway, PLoS ONE 7 (2012) e33021.

Highlights
- Thiosemicarbazones can be considered as potent tyrosinase inhibitors
- Small groups on thiosemicarbazone skeleton are preferable for tyrosinase inhibition
- Thiosemicarbazones reveals very well inhibitory activity against melanogenesis

Ki = 0.38 µM
Favourable arrangement of substituents on thiosemicarbazone skeleton for
inhibition of mushroom tyrosinase.