Synthesis, screening and biological activity of potent thiosemicarbazone compounds as a tyrosinase inhibitor

Article abstract of DOI:10.1039/c9nj02360g

Tyrosinase plays an essential role in melanogenesis. Tyrosinase inhibition may not only be a means to alleviate skin hyperpigmentation, but also the most effective method for fruit and vegetable preservation. Thiosemicarbazone compounds have received enormous attention because of their potential therapeutic activities due to their antitumoral, antibacterial and antimalarial properties. In this study, we focused on the screening, toxicity, interaction and inhibitory activities of potent thiosemicarbazone compounds as novel tyrosinase inhibitors. The results showed that the inhibitor effectiveness was drastically reduced by the absence of the ortho-hydroxyl group and was improved by changing the substituents of benzaldehyde. To develop a pharmacophore model, we explored the binding mode of thiosemicarbazone compounds in the active site of tyrosinase. Docking analysis using an AUTODOCK program was conducted to explain the flexible thiosemicarbazone molecules binding with the active site of tyrosinase, because of their perfect match with both the active site and the substrate channel of tyrosinase in both size and hydrophobicity. Their binding was mainly stabilized via both the coordination of the sulfur and nitrogen atoms in the inhibitors to binuclear copper in the tyrosinase active site. Further complementary studies on these types of inhibitors, such as for potential drug candidates for treating abnormal melanin pigmentation and inhibiting food or fruit browning, are still needed.

Full text of DOI:10.1039/c9nj02360g

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Synthesis, screen and biological activity of potent thiosemicarbazone compoundVsiew Article Online
as a tyrosinase inhibitor
DOI: 10.1039/C9NJ02360G
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Penggen Cai †, Yi Xiong †, Yao Yao, Wu Chen*, Xiongwei Dong*
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National Local Joint Engineering Laboratory for Advanced Textile Processing and Clean
Production, Wuhan Textile University, Wuhan 430200, China
E-mail: xwdong@wtu.edu.cn;
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† These authors contributed equally
Abstract
Tyrosinase plays an essential role in melanogenesis. Tyrosinase inhibition may be not only a means
to alleviate skin hyperpigmentation, but also the most effective method for fruit and vegetable
preservation. Thiosemicarbazone compounds have received enormous attentions because of their
potential therapeutic activities due to their antitumoral, antibacterial and antimalarial properties. In
this study, we focused on the screen, toxicity, interaction and inhibitory activities of potent
thiosemicarbazone compounds as novel tyrosinase inhibitors. The results showed that the inhibitor
effectiveness was drastically reduced by the absence of ortho-hydroxyl group and was improved
through changing the substituents of benzaldehyde. To develop a pharmacophore model, we
explored the binding mode of thiosemicarbazones compounds in the active site of tyrosinase.
Docking analysis using an AUTODOCK program was conducted to explain the flexible
thiosemicarbazone molecules binding with the active site of tyrosinase, because of their perfect
match with both the active site and the substrate channel of tyrosinase in both size and
hydrophobicity. Their binding was mainly stabilized via both the coordination of the sulfur and
nitrogen atoms in the inhibitors to binuclear copper in the tyrosinase active site. Further
complementary studies on these types of inhibitors, such as for potential drug candidates for treating
abnormal melanin pigmentation and inhibiting food or fruit browning, are still needed.
Keywords: screen; thiosemicarbazone; tyrosinase; inhibitor
Introduction
Tyrosinase inhibition by using the enzyme inactivation to limit the pigmentation process，is the
most common method for skin hyperpigmentation treatment and fruit or vegetable preservation. For
the past few decades, many researchers have investigated the natural or synthetic tyrosinase
inhibitors. Thiosemicarbazone acting as a tyrosinase inhibitor has attracted enormous attentions in
biological research [1-2]. Thiosemicarbazone compounds consisting of thiourea moieties play an
important role in medical field with various pharmacological activities such as antimicrobial [3],
antiviral and anticancer features [4]. Thiourea-based salicylaldehyde ligands have drawn intensive
interests in recent years due to their capacity of coordinating with metal ions [5, 6], they have lots
of pharmacological and biological properties as metal complexes and free ligands. The position of
the substitutions determines the activity of the compounds. Meanwhile, the thiosemicarbazone
contains a strongly copperchelating and a H-bonding moiety –C(SH)–NH–, which can be applied

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DOI: 10.1039/C9NJ02360G
as a metal-chelating inhibitor.
The thiosemicarbazone compounds have potential applications in chemotherapeutics, and
thiosemicarbazone compounds with salicylaldehyde moieties demonstrate a wide range of
pharmacological activities, such as antibacterial, antifungal, and tyrosinase inhibitory activities. The
approach of using thiosemicarbazone compounds as tyrosinase inhibitors has been developed in the
field of melanogenesis inhibition agents for skin-whitening cosmetics and anti-browning agents for
fruit or fungi enzymes [7].
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This inhibition comes from its strong metal-chelating ability to form a stable chelating copper in
enzyme, e.g. superoxide dismutase (ref. 4) and tyrosinase. More importantly, the phenolic hydroxyl
further facilitates the coordination of copper [8]. Tyrosinase contains two copper ions that
coordinate histidine residues in the active site, and the dinuclear coppers are also critical for catalytic
activities of tyrosinase which are flexible during catalysis [9]. The tyrosinase inhibitors have been
a great concern due to the key role of tyrosinase in both mammalian melanogenesis [10] and food
or fruit browning [11, 12]. Tyrosinase inhibitors usually chelate the coppers within the tyrosinase
active site, obstruct the substrate–enzyme interaction or prevent oxidation via an electrochemical
process [13].
The recent research by V. Namasivayam and others researchers [14, 15] indicated that amine group
was necessary for determining the tyrosinase activation activity, thiosemicarbazone moiety was
crucial for the tyrosinase inhibitory activity because of the coordination between sulfur atom and
the dicopper nucleus in the active site, and tyrosinase would lose its catalyzing ability after forming
complex with metal chelators in its active center [16]. Besides, thiosemicarbazones with oxygenate
substituents in the aromatic ring exhibited strong inhibition (IC50:～0.3-1.2 mM) on the activity of
mushroom tyrosinase [17], which might be caused by the higher affinity of hydroxyl groups than
sulfur/nitrogen groups for Cu2+ ions. However, benzaldehyde thiosemicarbazone was also reported
as potent tyrosinase inhibitor. The high affinity of them to tyrosinase was caused by the van der
Waals interactions of aromatic ring with the hydrophobic residues in the tyrosinase cavity (IC50:～
0.84-9μM) [7,18]. Furthermore, Jimenez’s work indicated that the substituent position at phenyl
ring also influenced the inhibitory potency of tyrosinase [19]. In most cases, the substitution of
phenyl ring reduced inhibitory properties of the investigated compounds, except for the substitutions
of hydroxyl groups in ortho and para positions at the same time.
However, some of tyrosinase inhibitors displayed weak or moderate inhibitory activity [20], or
could not be used in vivo because of their toxicity or instability. Thus, it is still important to screen
further classes of the known molecules in order to develop potent tyrosinase inhibitors with good
bioavailability and low toxicity [21, 22]. In this study, we focus on the screen, toxicity, mechanism
and their inhibitory activities against tyrosinase. Furthermore, docking simulation using
AUTODOCK 4.2 was performed to position screened compounds into the active site of tyrosinase
to determine the probable binding conformations. Meanwhile, the structure–activity relationships
of these compounds were also discussed. The aim of the present study is to discover safe and
efficient compounds as food additives or food preservatives, and this research will also provide the
references for designing and synthesizing on novel tyrosinase inhibitors.
Experimental

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General methods. All reagents were purchased from commercial sources (e.g., Sigma) and directly
used unless stated otherwise. All of solutions and buffers were prepared using deionized water that
was obtained a Millipore-Q ultrapurification system. Elementary analysis was carried out on a
Perkin–Elmer 2400 analyser. UV-Vis spectra were recorded on an Analytik Jena Specord 210
spectrophotometer (Analytic Jena AG). 1H NMR spectra were measured on a Varian Mercury 600
spectrometer. The MS data was obtained using a Quattro II ESI mass spectrometer (Waters, USA).
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Synthesis of compounds. General procedure: The reaction equation is shown in Scheme 1.
Thiosemicarbazide (10 mmol, in 10 ml water) were mixed with corresponding salicylaldehyde or
its derivatives in 1:1 molar ratio, and then a catalytic amount of acetic acid in alcohol (30 ml) was
added. The mixture was refluxed for 3-6 h, cooled, and the solvent was evaporated. The
thiosemicarbazone compounds were isolated by column chromatography or purified by
recrystallization.
Data for Thio-1: yield 1.32 g (69%); white crystals; ESI-MS: m/z 196.20 (M-H+), calculated:196.24;
Elem. Anal. calcd for C8H9N3OS: C 49.22, H 4.65, N 21.52, S 16.42; found: C 49.12, H 4.78, N
21.59, S 16.37. 1H NMR (600 MHz, DMSO) δ11.39 (s, 1H), 9.89 (s, 1H), 8.37 (s, 1H), 8.12 (s, 1H),
7.92 (d, J = 7.0 Hz, 2H), 7.20 (d, J = 7.2 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 6.80 (t, J = 7.5 Hz, 1H).
13C NMR (400 MHz, DMSO) δ 177.71, 156.53, 140.14, 131.34, 126.97, 120.35, 119.50, 116.23.
Data for Thio-2: yield 1.14 g (59%); light yellow solid; ESI-MS: m/z 212.05 (M-H+), calculated:
212.24; Elem. Anal. calcd for C8H9N3O2S: C 45.49, H 4.29, N 19.89, S 15.18; found: C 45.55, H
1
4.24, N 19.93, S 15.23. H NMR (600 MHz, DMSO) δ 11.28 (s, 1H), 9.12 (s, 1H), 8.73 (s, 1H),
8.30 (s, 1H), 8.02 (s,1H), 7.74 (s, 1H), 7.19 (dd, J = 8.6 Hz, 1H), 6.68 (d, J = 8.8 Hz, 2H). 13C NMR
(400 MHz, DMSO) δ 177.66, 155.86, 139.24, 130.14, 125.88, 122.14, 121.77, 117.19.
Data for Thio-3: yield 1.41 g (63%); yellow solid ； ESI-MS: m/z 241.05 (M-H+), calculated:
241.24; Elem. Anal. calcd for C8H8N4O3S: C 40.00, H 3.36, N 23.32, S 13.35; found: C 39.89, H
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3.38, N 23.40, S 13.29. H NMR (600 MHz, DMSO) δ 11.47 (s, 2H), 8.82 (s, 1H), 8.37 (s, 1H),
8.21 (s, 1H), 8.16 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 9.1 Hz, 1H). 13C NMR (400 MHz,
DMSO) δ 178.42, 158.33, 141.26, 131.56, 125.10, 121.34, 120.33, 116.89.
Data for Thio-4: yield 1.66 g (73%); white crystals; ESI-MS: m/z 230.15 (M-H+), calculated：
230.69; Elem. Anal. calcd for C8H8ClN3OS: C 41.84, H 3.51, N 18.30, S 13.96; found: C 41.93, H
1
3.50, N 18.23, S 13.89. H NMR (600 MHz, DMSO)δ 11.47 (s, 1H), 10.23 (s, 1H), 8.35 (s, 1H),
8.18 (s, 2H), 8.12 (d, J = 2.6 Hz, 1H), 7.24 (dd, J = 8.7 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H). 13C NMR
(400 MHz, DMSO) δ 177.87, 155.16, 137.50, 130.42, 125.48, 123.51, 122.35, 117.72.
Data for Thio-5: yield 1.39 g (66%); white crystal; ESI-MS: m/z 210.05 (M-H+), calculated: 210.27;
Elem. Anal. calcd for C9H11N3OS: C 51.66, H 5.30, N 20.08, S 15.32; found: C 51.60, H 5.26, N
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20.11, S 15.24. H NMR (600 MHz, DMSO) δ 11.28 (s, 1H), 9.55 (s, 1H), 8.33 (s, 1H), 8.03 (s,
1H), 7.85 (s,1H), 7.70 (s, 1H), 7.01 (d, J = 6.8 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 2.21 (s, 4H). 13C
NMR (400 MHz, DMSO) δ 162.24, 148.17, 142.15, 124.79, 113.15, 99.67, 91.62, 85.89, 24.24.
Data for Thio-6: yield 1.35 g (61%); white solid; ESI-MS: m/z 226.08 (M-H+), calculated:226.27;
Elem. Anal. calcd for C9H11N3O2S: C 47.99, H 4.92, N 18.65, S 14.23; found: C 47.84, H 4.98, N
1
18.54, S 14.20. H NMR (600 MHz, DMSO) δ 11.18 (s, 1H), 9.90 (s, 1H), 8.27 (s, 1H), 7.92 (s,
1H), 7.76 (s,2H), 6.46 – 6.39 (m, 2H), 3.73 (s, 3H). 13C NMR (400 MHz, DMSO) δ 176.12, 152.14,
150.85, 141.02, 120.79, 118.27, 116.87, 112.02, 40.82.
Data for Thio-7: yield 1.78 g (69%); light brown solid; ESI-MS: m/z 267.15 (M-H+), calculated:

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267.36; Elem. Anal. calcd for C12H18N4OS: C 54.11, H 6.81, N 21.03, S 12.04; found: C 54.14, H
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6.79, N 21.10, S 12.09. H NMR (600 MHz, DMSO) δ 11.02 (s, 1H), 9.47 (s, 1H), 8.18 (s, 1H),
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6H). 13C NMR (400 MHz, DMSO) δ 176.31, 154.94, 138.02, 131.26, 125.98, 123.86, 122.86,
117.49, 55.92, 18.05.
Data for Thio-8: yield 1.60 g (71%); white crystals; ESI-MS: m/z 230.00 (M-H+), calculated: 230.68;
Elem. Anal. calcd for C8H8ClN3OS: C 41.84, H 3.51, N 18.30, S 13.96; found: C 41.75, H 3.59, N
18.25, S 14.03. 1H NMR (600 MHz, DMSO) δ 11.35 (s, 1H), 10.37 (s, 1H), 8.31 (s, 1H), 8.05 (s,
1H), 7.97 (d, J = 7.3 Hz, 1H), 7.91 (s, 1H), 6.92–6.83 (m, 2H). 13C NMR (400 MHz, DMSO) δ
176.25, 154.50, 140.18, 132.48, 128.26, 120.20, 118.16, 116.64.
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Data for Thio-9: yield 1.32 g (69%); white solid; ESI-MS: m/z 238.06 (M-H+)，calculated：238.28;
Elem. Anal. calcd for C9H11N3OS: C 51.66, H 5.30, N 20.08, S 15.32; found: C 51.62, H 5.25, N
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20.12, S 15.29. H NMR (600 MHz, DMSO) δ 11.23 (s, 1H), 8.01 (s, 1H), 7.91 (d, J = 16.3 Hz,
2H), 7.39 (s, 1H), 7.19 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 8.3 Hz, 1H), 4.26 (d, J = 4.1 Hz, 4H). 13
C
NMR (400 MHz, DMSO) δ 176.96, 152.41, 150.42, 140.92, 120.31, 118.14, 116.08, 112.60, 40.57.
Measurement of inhibitory activity against tyrosinase
The commercial mushroom tyrosinase was purchased from Sigma. Tyrosinase activity was
determined through measuring the oxidation of 3,4-dihydroxyphenylalanine (L-DOPA, Sigma),
which was constructed by Pomerantz et al.:[23, 24] 50 μL of enzyme extract was added to 450 μL
of 0.05 M phosphate buffer (pH 6.8) containing 50 μM chelators, and the mixture was preincubated
at 30 ℃ for 10 min, 500 μL of 5 mM L-DOPA solution was added to the mixture. The increase in
absorption at 475 nm (ε = 3600 M cm-1) due to the formation of DOPA chrome was monitored as a
function of time. The initial rate was used to estimate the tyrosinase activity and the control was a
blank containing 1% DMSO.
Determination of acidity and stability constants. Acidity constants (pKa) of the compounds and
stability constant (pK) of the M2+-complexes (M = Cu, Fe and Zn) were determined by
potentiometric titrations. Potentiometric titrations were performed with a Metrohm 877 Titrino Plus
automated titrator equipped with a Metrohm 6.0262.100 glass electrode was calibrated against
standard buffers. The water-jacketed titration vessel was maintained at 25.0 ℃ (± 0.5 ℃). To
estimate pKa values, compounds were predissolved in excess of HCl (1.0 mM) and diluted with
water [10% (vol/vol) DMSO] in the titration vessel (25.0 mL), the final concentrations of HCl and
chelators were 100 μM. All measurements were conducted in the presence of 100 mM KCl to
maintain constant ionic strength. Solutions were titrated with 75.5 mM NaOH, which was prepared
with degassed water and standardized with Potassium biphthalate prior to each titration. Data
analysis was carried out with the program HyperQuad 2013 (Protonic Software, UK) [25, 26] The
stability constant of M2+-complex was determined by the titrations performed in the presence of 1.0
equiv of 100.0 μM metal chlorides using the pKa values determined above. Species distribution plots
and titration simulations were built with the program HySS2009 (Protonic Software) [27, 28]. The
data represented here were the mean value with standard deviation of the results obtained from three
independent measurements.
Electrochemical measurement. Cyclic voltammograms were recorded with a CH instruments

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Electrochemical Analyser was equipped with Chi600E software, and a glassy carbon was DeOmI:p1l0o.1y0e3d9/C9NJ02360G
as working electrode, platinum wire as auxiliary electrode, and Ag/AgCl as reference electrode. The
compounds were firstly incubated with metal ions with a ratio of 1:1 in DMF for 8 h, and then the
cyclic voltammetry measurements were performed. All measurements were carried out in DMF and
2 mM of analyte was dissolved in the 0.1 M sodium perchlorate solution. Each solution was purged
with nitrogen prior to analysis and measured at ambient temperature. Each sample was referenced
to an internal reference of ferrocene, which was taken as having a E1/2 = 0.56 V in DMF versus SCE.
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Molecular docking simulation. The molecular simulation on docking ligands into the 3D structures
(http://www.rcsb.org) of the tyrosinase (PDB: 4P6T) was carried out using the program suite
AutoDock 4.2.0 (http://autodock.scripps.edu). The widely used Lamarckian Genetic Algorithm
(LGA) [29, 30] was chosen for the docking calculation. AutoDockTools (ADT 1.4.6) was performed
to setup each ligand-protein interaction, where all hydrogen atoms were added, Gasteiger charges
were calculated and nonpolar hydrogen atoms were merged to carbon atoms. The solvent molecules
were removed from the protein 3D structures to obtain the docking grid, and the active site was
defined using AutoGrid. The grid size was set to 80 *80 * 80 points with grid spacing of 0.375 Å,
van der Waals well depth of 0.100 kcal/mol, an iteration of 200[31] and a population size of 100.
The grid box was centered on the center of the ligand from the corresponding protein structures.
Formal structures of the ligands were assigned by the program SYBYL, and the best ranked pose
was selected from the ChemScore. The conformation with the lowest binding energy was used to
analyze ligand placement.
Cytotoxicity Assay. Cytotoxicity of the compounds was evaluated by MTT assay. These studies
were performed in 1% (w/w) DMSO/H2O solvent mixture due to the low solubility of the compound
in pure water. Briefly, after Hela cells reached 80% confluence in 96-well plates, the FBS-
containing medium was replaced with FBS-free medium. After incubation for 24 h in the medium,
the chelators were added (varied concentrations) into the wells (6 parallel wells tested). Following
further incubation for 72 h at 37 ℃, 20 μL of MTT (5 mg/mL) in PBS was added to the wells. After
incubated for 4h, the MTT-containing medium was replaced by DMSO. The 96-well plates were
oscillated for 10 min to fully dissolve the formazan crystal formed by living cells. The absorbance
of formazan generated by MTT was determined at 590 nm using a Biotek SynergyTM 2 Multi-
detection Microplate Reader. HeLa cells (from the China Center of Typical Culture Collection) were
treated and their viability was determined as described above. Non-treated cells (in DMEM) were
used as a control and the relative cell viability of each of the LD-treated cells (mean% ± SD, n=3)
was expressed as ODsample / OD control * 100%.
Results and discussion
Design strategy
Tyrosinase contains a strongly coupled binuclear copper active site but performs different biological
functions. Polyphenol compounds have been attractive targets for tyrosinase inhibitors because of
their structural similarity with tyrosine and antioxidant properties [32, 33].When the copper-
chelating inhibitors of tyrosinase are designed based on the copper coordination chemistry and
catalytic circle in the active site of the enzyme [34, 35], the pivotal factors to be considered

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include: (1) the stability constants of their copper complexes, (2) their coordination tDoOIc: o10p.1p0e39r/,C9NJ02360G
which could block the cyclic redox reactions of tyrosinase. Thiosemicarbazone as an efficient
metal chelator, and their coordination chemistry was well developed [36, 37], especially for the
transition-metal ions, e.g. copper (II) [38]. A high affinity to certain metal ions also maked them
useful for competitive inhibitor designs (Fig.1a). The sulfur atom of the compound binds to both
copper ions in the active site of the enzyme and blocks enzyme activity [39].
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a
OH
OH
NH2
H
S
N
R
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R
O
+
N
S
N
NH2
H
Salicylic aldehyde Groups
Metal-coordinating moiety
Metal-chelating inhibitors
b
Fig. 1 The design strategy of chelator’s structure (a) and Cu2+-coordinating hypothesis in tyrosinase’s active site (b).
In order to verify this design, by virtue of the tyrosinase structural data (PDB: 4P6T), molecular
docking simulations were carried out using the AutoDock 4.2 program (Fig. 1b). The results indicate
that the flexible thiosemicarbazone molecules was localized into the active site in tyrosinase due to
their perfect match with both the active site and the substrate channel of tyrosinase on both size and
hydrophobicity. This localization was largely stabilized via the coordination of the sulfur and
nitrogen atoms in the inhibitors to binuclear copper in the tyrosinase active site. The coordination
effect might not only hinder the access of anion to the active site copper ions, but also disrupt the
noncovalent interactions between the coppers and residues. As the design strategy was supported
by the molecular docking simulations, a series of thiosemicarbazone inhibitors for the inhibition of
tyrosinase activity were synthesized and characterized. The thiosemicarbazone inhibitors were
prepared according to Scheme 1, using the thiosemicarbazide and respective substituted
salicylaldehydes.
H
R3
R2
R3
R2
N
NH2
S
O
N
H2N
+
N
NH2
S
R1
R1
H

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Scheme 1. Synthetic route for thiosemicarbazone compounds synthesis.
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Coordination of the inhibitors to copper
The acidity constants of the inhibitors and the stability constants of their complexes with three
transition-metal ions (Cu2+, Zn2+, Fe2+) were determined via potentiometric titrations at 25 ℃. The
stability constants of the inhibitors are listed in Table 1.
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Table 1. The data for coordination with different divalent metal ions and pH range for CuL complexes.
Ligand
Thio-1 Thio-2 Thio-3 Thio-4 Thio-5 Thio-6 Thio-7 Thio-8
logKCu2+, n=1, M-1
logKZn2+, n=1, M-1
logKFe2+, n=1, M-1
pH range for Cu2+ complexes
18.62±0.22
13.39±0.31
14.24±0.24
18.83±0.14
13.53±0.11
14.18±0.15
19.10±0.15
13.69±0.12
14.48±0.09
19.87±0.30
14.13±0.26
14.39±0.21
19.15±0.11
13.13±0.21
13.87±0.24
18.95±0.10
13.56±0.17
13.95±0.31
20.15±0.14
14.07±0.37
14.31±0.12
19.43±0.22
12.51±0.20
13.54±0.22
3 - 10
3 - 9
4 - 9
3 - 10
3 - 9
3 - 9
3 - 10
3 - 10
The calculations showed that the Cu2+ complexes had large stability constants (KCu2+ ≥1018 M-1, i.e.,
2+
Kd,Cu ≤ 10-18 M, Table 1). Evidently, these results demonstrate that the chelate-inhibitors can
compete for the Cu2+ ion in the tyrosinase active site via chelation.
The acidity constants (pKa1 and pKa2) of chelate-inhibitors were determined by potentiometric
titrations at 25 ℃ (I = 0.1 M KCl), and the acidity constants were indicated by equation pKa =
logβ(HnL) ‒ logβ(Hn-1L). The larger pKa values corresponded to the deprotonation of amines or
sulfhydryls, and the small pKa value was assigned to the deprotonation of phenols.
The species distribution curve in Fig. 2 showed that for 1:1 feeds of inhibitors and Cu2+, Zn2+, Fe2+
at low pH, the complexes mainly exist in the form of MLH (M = Cu2+, Zn2+, Fe2+) . The inhibitor
ligands also have protons attached. As the pH increases, protons will be removed, and the structures
of CuL, ZnL, and FeL appear at pH = 3 ~ 10. All of the binding constants of the thiosemicarbazone
inhibitors to metal ions were higher than 1018 M-1. These results demonstrate that the binding
abilities of thiosemicarbazone inhibitors to Cu2+ are greater than tyrosinase to Cu2+ (KCu2+ ~1013 M-
1) [32]. The ability to capture Cu2+ and stabilize its presence in complex suggested that it is possible
to achieve chelation of tyrosinase central metal ions in vitro and to inhibit tyrosinase activity. In
addition, the stability constants of the Cu2+ complexes were 4–5 orders of magnitude higher than
those of the complexes with the other divalent metal ions tested (Zn2+, Fe2+, Table 1), indicating that
the chelators had notable selectivity for Cu2+ over other divalent metal ions tested. The results
obtained here were in agreement with those obtained with previous results reported in literature
concerned [40].

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Fig. 2 Species distribution plots measured with potentiometric titrations for the Cu2+, Zn2+ and Fe2+ -chelators
systems. Potentiometric titrations were performed for the solutions containing the chelators and equimolar amount
metal ions at 25 ℃ (I = 0.1 M potassium ion).
Direct comparison of the stability constants of the thiosemicarbazone complexes consisting with the
different metal ions (Table 1) reveals the following stability order: Cu2+ >> Fe2+ > Zn2+. However,
despite the high log β values, there is an obvious tendency of certain metal ions to hydrolyze at
physiological pH, which will lead to decreased stability. Thiosemicarbazone ligands provides an
(O–, N1, S–) binding mode with the formation of a six- and a five-membered ring system between
metal atom and ligand. Especially, the stability constant of Thio-7 with Cu2+ is up to 20.15. Thio-7
might be an efficiently potential inhibitor of tyrosinase. Overall, all the present thiosemicarbazone
compounds may inhibit the activity of tyrosinase by competitively binding to Cu2+ in the active

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center of tyrosinase.
Indeed, there might be two binding modes of the inhibitors and tyrosinase, such as pulling-out Cu2+
from tyrosinase or just only disturbing the coordination bonds formed by Cu2+ with amino acid
residues. Regardless of the mechanism of action, the inhibitors could also effectively inhibit the
activity of tyrosinase. In order to examine whether the coordination of the inhibitors can make the
Cu2+ become redox-inactive, we observed the redox behavior of the Cu2+ complexes with cyclic
voltammograms. The cyclic voltammograms of all of the complexes showed only a distinct cathode
peak at ~ -0.6 V in anhydrous DMF at a glassy carbon working electrode (Fig. 3), indicating that
the Cu2+ in the complexes does not undergo a reversible redox process. These results confirm the
anticipation that the coordination of the inhibitors can block the Cu2+Cu+ redox cycle that is
essential for the catalytic activity of tyrosinase. These inhibitors can effectively inhibit tyrosinase
activity through blocking the redox cycle of Cu2+ in active center.
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Fig. 3 Cyclic voltammograms of the Cu2+-inhibitor complexes at 2.0 mM in anhydrous deoxygenated DMF
containing 0.1 M NaNO3 as the supporting electrolyte at a scan rate of 0.05 V/s. Working electrode: glassy carbon;
counter electrode: Pt wire; reference electrode: Ag/AgCl.
Cytotoxicity Assay
For comparison, the relative viability was firstly evaluated for the HeLa cells incubated with the
thiosemicarbazone compounds treated after 72h, respectively. The data obtained by MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) measurements indicated that all the species
tested did not possess significant toxicity on the types of Hela cells. To facilitate the comparison of
cytotoxicity data, we selected a comparative analysis of the concentrations of different chelators
when the Hela cells survival rate was maintained above 80% (Fig 4). All the effective concentrations
of thiosemicarbazone compounds were measured up to 50 μM.

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Effective Concentrations were 50 M
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Fig 4. Estimation of cytotoxicity on HeLa cells after 72h, the viability estimated by MTT for 50 μM
thiosemicarbazone compounds, respectively. Here, at least, measurements were performed in triplicate, and the
control was a blank containing 1% DMSO.
Inhibition of tyrosinase activity
Inhibitory effects of the synthesized thiosemicarbazone compounds on mushroom tyrosinase
activity have been evaluated. The concentrations to inhibit 50% of the tyrosinase activity (IC50) are
summarized in Table 2. It is well known that tyrosinase can be inhibited by aromatic aldehydes.
Thus, benzaldehyde and salicylic aldehyde were two typical tyrosinase inhibitors, which were
selected as positive control compounds with inhibitory activity already reported previously[42]
(benzaldehyde IC50 is 820 μM, salicylic aldehyde IC50 is 3300 μM). The analysis indicated that this
inhibition comes from its strong copper-chelating ability to form a stable complex by chelating
copper in the enzyme.

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Fig 5. The effect of representative Thio-7 inhibited tyrosinase activity by UV-L-DOPA method.
The results showed that most of the screened thiosemicarbazone compounds had relatively high
activities. Especially, Thio-7 exhibited the most potent tyrosinase inhibitory activity.
Table 2 Concentrations of thiosemicarbazone compounds and positive control compounds leading to 50% activity
loss (IC50) of tyrosinase and substrate L-DOPA.
Substituents
R2
Compounds
IC50 ± SD (μM)
R1
R3
Thio-1
Thio-2
Thio-3
Thio-4
Thio-5
Thio-6
Thio-7
Thio-8
Thio-9
Thio-10
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-H
-H
-H
-H
-OH
-NO2
-Cl
14.25 ± 0.21
4.75 ± 0.11
9.88 ± 0.09
1.75 ± 0.12
1.88 ± 0.05
1.12 ± 0.08
0.60 ± 0.04
1.68 ± 0.12
41.20 ± 0.22
440 [2]
-H
-H
-H
-CH3
-H
-OCH3
-N(C2H5)2
-Cl
-H
-H
-OCH2-
-OCH3
-OCH2-
-H
-H
Salicylaldehyde
methoxy para substituted
30 [41]
derivative
820 [42]
Benzaldehyde
3300 [42]
Salicylaldehyde
The thiosemicarbazone compounds were prepared according to Scheme 1, and the inhibitory effects
on mushroom tyrosinase activity have been evaluated. Calculations using the titration data showed
that all the Cu2+ complexes have large stability constants (Table 1), ranging from 18.83 to 20.15
2+
2+
(KCu ≥1018 M-1, i.e., Kd,Cu ≤10-18 M). The result reveals that the Cu2+ affinity of all the designed

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chelators is much greater than that of tyrosinase [29, 39]. It was suggested that thiosemicarbazonVeiew Article Online
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compounds could compete for the Cu2+ in the active center of tyrosinase via coordination. Thus, we
determined the inhibition of mushroom tyrosinase activity by this competitively binding for Cu2+.
The concentrations to inhibit 50% of the tyrosinase activity (IC50) are summarized in Table 2. As
can be seen, most of the screened thiosemicarbazone compounds had relatively high activities, with
IC50 values between 0.60 and 41.20 μM. Particularly, the IC50 value of Thio-7 reached ~ nM scales
(0.60 μM), which was much lower than those of the reported inhibitors of tyrosinase. The lowest
IC50 value of Thio-7 was consistent with the maximum stability constants of this Cu2+ complexes.
All of these indicated that Thio-7 could strongly bind to Cu2+ and function as an effective tyrosinase
inhibitor.
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After fully analyzing the ortho-substitution of benzaldehyde in all designed inhibitors, it could be
concluded that the absence of ortho-hydroxyl group drastically reduced the inhibitor effectiveness,
and Thio-10 almost had no visible inhibitory effects (IC50 = 440 μM). Therefore, the ortho-hydroxyl
group of salicylaldehyde should be an essential factor to inhibit the activity of tyrosinase. By
comparing the meta-substituents of Thio-1, 2, 3, 4 and 5, it was found that both the meta-methyl
and chlorine substitutions significantly increased the inhibitor effectiveness. It should be noted that
para-chlorine (Thio-8) or para-methoxyl (Thio-6) substituents group could slightly increase the
effectiveness of inhibitor relative to the absence of para-substitution group. For Thio-7, the
presence of para-diethylamine group significantly increased its inhibitor effectiveness. Overall, the
ortho-hydroxyl group might be advantageous for the salicylaldehyde tyrosinase inhibitors, and the
inhibitor effectiveness could be improved through changing the para- and meta-substituents.
Docking simulation
In an attempt to theoretically describe the difference of the synthesized thiosemicarbazone
compounds, the possible interactions between the tyrosinase molecule and the inhibitors were
explored by the molecular docking simulation using the AutoDock 4.2 program. The dinuclear
copper center of tyrosinase is found in the center of a hydrophobic pocket lined by the side chains
of histidine residues (Fig. 7). The simulation suggested that the inhibitor molecules approach the
copper active site in tyrosinase likely via hydrophobic and other non-covalent interactions with the
interior of the enzyme. The bonding analysis of all compounds against tyrosinase indicates that all
screened inhibitors could directly interact with active residues of targeted protein.
The optimized cluster (200 occurrences) was ranked by energy level in the best conformation of
inhibitor–tyrosinase modeled structures, where the binding energies of the amino acid residues with
the corresponding thiosemicarbazone species Thio-1–10 were calculated (Fig. 8). This procedure
yielded modeled structures with much better docking scores, those whose docking scores were
better than -4.0 were then submitted to an energy minimization process in order to improve the
interactions at the binding sites. This optimization process was carefully designed to evaluate the
different schemes of partial minimizations of model structures.
According to the minimum energy principle, the lower value implies more stable complex formed
between the ligand and target protein. The difference of binding energies may provide useful
information for an understanding of the tyrosinase inhibitory activity of thiosemicarbazone
compounds. The results suggest that Thio-7 is the most active inhibitor with a better binding energy
value (-5.38 kcal/mol) and the lowest intermolecular energy (−7.47 kcal/mol) compared to others

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thiosemicarbazone compounds. The other candidate compounds also possess good binding energy
values of around -5.0 kcal/mol, respectively. It is in good agreement with our experimental results.
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Intermolecular Energy
Binding Energy
-10
Fig. 6 The binding energy and the lowest intermolecular energy of Thio-1–10 and Salicylaldehyde in the modeled
molecules of tyrosinase–inhibitor complexes.
Access to the catalytic copper center is controlled by the hydrophobic pocket (Fig. 1) formed by
Met61, Gly196, Phe197, Pro201, Asn205, Arg209, Gly216, Val217 and Val218, locating in the loop
region connecting two α helices. The inhibitor molecules must alter their conformation to adapt to
the shape of the metal active site and to form the H-bonds, respectively, between its certain groups
and the tyrosinase residues Asn205, Glu195, Met215 or Val217 (see the H-bonding interactions
indicated in Fig. 7d, 7e). Upon binding of thiosemicarbazone the phenyl ring of Phe197 and the
imidazole ring of histidine undergo a conformational change to form hydrophobic interactions with
the aromatic ring of the inhibitor. This may be caused by the decrease transfer efficiency of the
electron transport chain. The thiosemicarbazone compounds blocked the electron transport pathway
in tyrosinase active site,resulting in tyrosinase loses its activity. Therefore, the specific inhibiting
activity for tyrosinase could be caused by the coordinated actions of the above mechanisms.
In addition to the H-bonding interactions with the interacted residues, van der Waals interactions
between the residues lining the hydrophobic cavity (His60, Met61, Phe197, His204, His208, Arg209,
Gly216 and Val218) may contribute to the high affinity of the thiosemicarbazone to the enzyme.
Here, it is noticeable that most of candidates compounds bind in the active binding regions but little
fluctuated with their conformational positions. The orders of these binding free energies were in
good agreement with the orders of their IC50 values. These simulations confirm that the different
position of substituent at salicylaldehyde is important for the inhibitory activities against tyrosinase.

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Fig. 7 The proposed interactions between a tyrosinase molecule (PDB: 4P6T) and the inhibitor Thio-1 (a, d), Thio-7
(b, e) or Salicyladehyde (c, f) based on the molecular docking simulations.
In contrast, no hydrogen bond was found between Salicyladehyde and the amino acid residues of the
enzyme active site (Fig. 7c, 7f). It suggests the subtle different interactions of these inhibitors with the
active pocket of tyrosinase in the presence of the different substrates, tyrosine and L-DOPA.
In addition, the binding energy and the lowest intermolecular energy in the modeled structure of
Thio-7 were slightly lower than the corresponding values observed in the other positive control
compounds, which may be due to the strong intermolecular hydrogen bond formed between the
chelating ligand and the active site of the enzyme. The structure activity relationship analysis shows
that Thio-7 builds two hydrogen bonds at specific residues Asn205 and Met215 against target
protein. The values of binding energies proved that Thio-7 is more stable ligand receptor complex
with target protein.
Conclusion
The inhibitory activity of all the obtained thiosemicarbazone compounds have been tested in vitro
against tyrosinase. These thiosemicarbazone species possess good inhibitory activities. Calculations
using the titration data show that all the Cu2+ complexes have large stability constants and
demonstrate that the binding abilities of thiosemicarbazone compounds to Cu2+ is greater than those
of tyrosinase. The cyclic voltammograms confirm that the coordination of the inhibitors can block
the Cu2+Cu+ redox cycle that is essential for the catalytic activity of tyrosinase. After thoroughly
analyzing the ortho-hydroxyl group of benzaldehyde in all designed inhibitors, it is concluded that
the absence of ortho-hydroxyl group drastically reduces the inhibitor effectiveness, and the inhibitor
effectiveness could be improved through changing the para- and meta-substituents. Especially, the
IC50 value of Thio-7 (para-diethylamine) reached nM scales (0.60 μM). The docking simulation
described herein suggests that the thiosemicarbazone species possess good potential as tyrosinase

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inhibitors. Further investigations into the mechanisms of tyrosinase inhibitory activityare in
progress.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 21701128);
Hubei Provincial Natural Science Foundation of China (No. 2017CFB206); Educational
Commission of Hubei Province of China under grants (No. Q20171601).
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