A Series of Benzylidenes Linked to Hydrazine-1-carbothioamide as Tyrosinase Inhibitors: Synthesis, Biological Evaluation and Structure?Activity Relationship

Article abstract of DOI:10.1002/cbdv.202000285

Tyrosinase is a type 3 copper enzyme responsible for skin pigmentation disorders, skin cancer, and enzymatic browning of vegetables and fruits. In the present article, 12 small molecules of 2-benzylidenehydrazine-1-carbothioamide were designed, synthesized and evaluated for their anti-tyrosinase activities followed by molecular docking and pharmacophore-based screening. Among synthesized thiosemicarbazone derivatives, one compound, (2E)-2-[(4-nitrophenyl)methylidene]hydrazine-1-carbothioamide, is the strongest inhibitor of mushroom tyrosinase with IC₅₀ of 0.05 μM which demonstrated a 128-fold increase in potency compared to the positive control. Kinetic studies also revealed mix type inhibition by this compound. Docking studies confirmed the complete fitting of the synthesized compounds into the tyrosinase active site. The results underline the potential of 2-benzylidenehydrazine-1-carbothioamides as potent pharmacophore to extend the tyrosinase inhibition in drug discovery.

Full text of DOI:10.1002/cbdv.202000285

Title: A series of benzylidene linked to hydrazinecarbothioamide as
tyrosinase inhibitors
Synthesis, biological evaluation and structure-activity relationship
Authors: Hona Hosseinpoor, Aida Iraji, Najmeh Edraki, Somayeh
Pirhadi, Mahshid Attarroshan, Mahsima Khoshneviszadeh,
and Mehdi Khoshneviszadeh
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
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to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.202000285
Link to VoR: https://doi.org/10.1002/cbdv.202000285

10.1002/cbdv.202000285
Chemistry & Biodiversity
A series of benzylidene linked to hydrazinecarbothioamide as tyrosinase inhibitors
Synthesis, biological evaluation and structure-activity relationship
Hona Hosseinpoora,b, Aida Irajib,c, Najmeh Edrakib, Somayeh Pirhadib, Mahshid Attarroshanb,
Mahsima Khoshneviszadehb, Mehdi Khoshneviszadeha,b*
a Medicinal and Natural Products Chemistry Research Ceter, Shiraz University of Medical Sciences,
71348 Shiraz, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences,
71345 Shiraz, Iran
c Central Research Laboratory, Shiraz University of Medical Sciences, 71468 Shiraz, Iran
*Corresponding author:
Mehdi Khoshneviszadeh, PharmD, PhD
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz,
Iran
Tel: +98 713 230 3872; Fax: +98 713 230 2225
E-mail: M.khoshneviszadeh@gmail.com
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10.1002/cbdv.202000285
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Abstract
Tyrosinase is a type 3 copper enzyme responsible for skin pigmentation disorders, skin cancer,
and enzymatic browning of vegetables and fruits. In the present article, 12 small molecules of
benzylidene-hydrazinecarbothioamide were designed, synthesized and evaluated for their anti-
tyrosinase activities followed by molecular docking and pharmacophore-based screening. Among
synthesized thiosemicarbazone derivatives, 3d is the strongest inhibitor of mushroom tyrosinase
with IC50 of 0.05 µM which demonstrated a 128 fold increase in potency compared to the positive
control. Kinetic studies also revealed mix type inhibition by 3d. Docking studies confirmed the
complete fitting of the synthesized compounds into the tyrosinase active site. The results underline
the potential of benzylidene hydrazinecarbothioamides as potent pharmacophore to extend the
tyrosinase inhibition in drug discovery.
Keywords
Tyrosinase inhibitor, Organic synthesis, Antioxidant assay, Pharmacophore modeling, Molecular
docking
Highlight
•
•
12 derivatives of benzylidene-hydrazinecarbothioamide were designed and synthesized.
Inhibition of tyrosinase enzyme in the presence of two substrates L-dopa and L-tyrosine
were tested.
•
•
•
3d efficiently inhibited tyrosinase with IC50 of 0.05 µM in the in vitro assay.
Kinetic studies were carried out revealing mix type inhibition by 3d.
Additionally, pharmacophore modeling and docking analyses were evaluated.
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10.1002/cbdv.202000285
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Introduction
Tyrosinase (TYR) is a binuclear copper oxidoreductase mainly responsible for two catalytic
activities. First, in monophenolase action, monophenols such as L-tyrosine hydroxylate to o-
diphenolic compound (e.g. L-dopa). Second, during diphenolase activity, o-diphenols oxidizes to
o-quinones such as o-dopaquinone. In general, TYR structure is divided into three domains,
namely, the central, the N-terminal, and the C-terminal domains. The central domain regarded as
the active site of TYR, comprises six histidine residues and two copper ions (CuA and CuB).
Tyrosinase has a critical role in melanogenesis activities responsible for the formation of the color
of the skin and hair. However, numerous studies confirmed the role of tyrosinase with some skin
pigmentation disorders, skin cancer,[1] and enzymatic browning of vegetables and fruits.[2] On the
other hand, increase amounts of dopaquinone result in neuronal damage and cell death. It is
proposed that tyrosinase is responsible for neurodegenerative disorders.[3-5] As a result, TYR
inhibitors have gained high attention in the therapies of skin pathologies and as well as in dermo-
cosmetic treatments and central nervous system (CNS) disorders. Several tyrosinase inhibitors
(TYRI) from natural and synthetic origin have been reported in recent years. Different chemical
classes of natural compounds from several sources, such as flavonoids,[6] chalcone, stilbene,[7]
[7]
ascorbic acid,[8] sulphates,[9] kojic acid, cinnamoyl amide,[10] tropolone derivatives have been
investigated. Searching for further TYRIs from synthetic source introduced notable example
including thiazole,[11, 12] triazole,[13] pyrimidine,[14] benzonitriles,[15] xanthone,[16] carbazole.[17]
Among them, tyrosinase inhibitors with the phenolic structure containing hydrazide linker have
been characterized by high potency.[18] It seems that this scaffold could provide new approaches
to the discovery of TYRIs.
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10.1002/cbdv.202000285
Chemistry & Biodiversity
Therefore, to further find new TYR inhibitors with potential antioxidant activity, in this study,
we attempted to connect thiosemicarbazone with the different aryl moieties to design a series of
new benzylidenehydrazinecarbothioamide anti-TYR agents.
Results and discussion
Design of novel aryl thiosemicarbazones
L-tyrosine and L-dopa have been regarded as a natural tyrosinase ligand and generally used as
tyrosinase pharmacological substrates for the in vitro reaction conditions (compound A and B,
Fig1).
We had previously reported 4-hydroxy-N'-methylenebenzohydrazide as small synthetic
compounds as a new class of tyrosinase inhibitors, and some of them displayed even better
potency than that of kojic acid as a reference. One of the most active derivatives in these series
demonstrated an IC50 value of 9.9 μM with competitive-type inhibitory activity (compound C,
Fig. 1.). Structure-activity relationship (SAR) studies confirmed that all compounds containing
the hydrazide linker bounded to 4-hydroxyl of benzyl was active TYRI. The SAR analysis was
consistent with the molecular docking study suggesting a crucial role of 4-hydroxyl of benzyl
ring via H-bound interaction with critical histidine residue.[19]
Due to the prevalent presence of 4- hydroxyl phenyl motif in tyrosinase natural ligand and even
in most of the potent tyrosinase inhibitors, it seems that the presence of substitutions with H-
bound interaction ability at para position of the benzene ring increases the chance of ligands
to reach to the region of tyrosinase active site and improve the inhibitory potency.
Recently, Soares et al reported thiosemicarbazones derivatives (compound D, Fig. 1.) as a
strong inhibitor of the mushroom tyrosinase. The most active compound in this series exhibited
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10.1002/cbdv.202000285
Chemistry & Biodiversity
IC50= 0.35 μM with competitive inhibition. They concluded that the amino groups of the
thiosemicarbazone linker can interact via hydrogen bonding with the oxygen and nitrogen of
Gly170 and Val171 residues.[20]
 Considering the above mentioned points, the aromatic benzene ring was kept in the
structure while substitution patterns at the benzyl ring were evaluated via different
electron-donating and electron-withdrawing groups with different bulkiness.
 The other structural modification involved the replacement of the aceto hydrazide
linker (C) with thiosemicarbazide through single bioisosteric modifications while
maintaining the linker length constant. From a biological viewpoint, the thio-
substituents modified and improved the tyrosinase inhibitory potential and control
important metal-chelating function.[20]
 Based on the previous studies, different derivatives of benzaldehyde thiosemicarbazone
demonstrated promising IC50 values that vary from 0.84 to 9 μM.[21] It seems that
phenyl ring with thiosemicarbazone regarded as a very popular scaffold for developing
novel tyrosinase inhibitors. As a result, aimed strategies were based on synthesizing
new derivatives of thiosemicarbazone and high similarities of designed structure with
L-dopa and L-tyrosine, (Fig. 1, A, B) as the natural ligands of tyrosinase. Besides, for
the first time, tyrosinase inhibition in the presence of two substrates was evaluated. It
was assumed that the supposed structure reduces the probability of oxidation and
increases structural stability.
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Fig. 1. Rational design and structural modifications of benzylidenehydrazinecarbothioamide
scaffold
Chemistry
A series of aryl thiosemicarbazone analogs were synthesized as depicted in Schemes 1.
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Scheme 1. Synthesis of benzylidenehydrazinecarbothioamide derivatives 3a-l. Reagents and conditions:
(a) EtOH, reflux, 4ꢀh
All substituted thiosemicarbazones were prepared by the treatment of thiosemicarbazide with
various aldehydes. Briefly, 1 mmol thiosemicarbazide (compound 1, scheme 1) was reacted with
1 mmol of selected aldehyde in refluxing ethanol to afford desirable compounds.[22] Completion of
the reaction was monitored by TLC. The reaction mixture was cooled. The condensate was filtered
and then purified by recrystallization from methanol. Structural determination and signal
assignments of the final products 3a–l (42.63–91.29 %) were accomplished by the application of
IR, Mass and NMR experiments. Some of the physical properties of synthesized derivatives are
presented in Table 1. The proposed mechanism for this reaction is illustrated in Scheme 2. The
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10.1002/cbdv.202000285
Chemistry & Biodiversity
reaction starts by the nucleophilic attack of the lone pair of electrons of the basic nitrogen of the
thiosemicarbazides to the carbonyl of different derivatives followed by the elimination of a
molecule of water and caused E isomers (confirmed by X-ray crystallography). The
electrophilicity of the carbonyl carbon is the main character controlling the yield of the reaction.
NO2 motif induces a strong electron-withdrawing group via sigma-bond from the aromatic ring
and exerts an inductive electron withdrawal resonance properties. As a result, NO2 containing
compounds showed exceptional reactivity through increasing the positive partial charge on carbon
of carbonyl. This information confirmed the high yield of NO2 containing derivatives.
Table1. Chemical structures and physical properties of the synthesized compounds
Compound
R
Yield (%) [a]
Melting point [a]
53.4 %
297
3a
3b
48.6 %
42.62 %
71 %
332
376
343
341
3c
3d
3e
71.62 %
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10.1002/cbdv.202000285
Chemistry & Biodiversity
90.09 %
43.85 %
331
351
3f
3g
57.94 %
91.29 %
357
343
3h
3i
76.65 %
60.17 %
343
348
3j
3k
68.88 %
348
3l
[a] Yield of isolated purified compounds
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Scheme 2. A suggested mechanism for synthesis of benzylidenehydrazinecarbothioamide derivatives
Structure-activity relationship studies
The anti-tyrosinase potential of the synthesized compounds was evaluated by determining IC50
values against mushroom tyrosinase. All data of tyrosinase inhibitory activity of
benzylidenehydrazinecarbothioamide derivatives with the use of L-dopa and L-tyrosine as
substrate are summarized in Table 2 and 3, respectively.
Tyrosinase inhibition with use of L-dopa as a substrate
To find the influence of the type of substitution of the aryl ring on the potency of compounds the
SAR analyses have been performed. Based on the observed results, all of the compounds exhibited
remarkable anti-tyrosinase activity displaying IC50 values in the range of 0.05–17.77ꢀµM in the
case of L-dopa as the substrate and four compounds including 3b, 3c, 3d, and 3j demonstrated
inhibition at submicromolar concentrations.
 The SAR of this set of benzylidenehydrazinecarbothioamide analogs seemed to be very
straightforward as the anti-tyrosinase potency was directly correlated to the position of the
motif on benzyl ring. Substitutions at the para position of benzyl ring (3b, 3c, 3d, 3e and
3h) result in an overall increase in potency with respect to the unsubstituted one. The most
potent ligand identified in this study was 3d (4-nitro, IC50= 0.05 µM) demonstrated more
than 128 fold increase in IC50 compared to the positive control. The activity of para analogs
changes in the following order: NO2> Br > Cl> N(CH3)2.
 The introduction of halogenated atom into 3a, resulting in 3b, 3c and 3g. One substituted
halogenated derivatives (3b, 3c) exhibited approximately similar potency against
tyrosinase cause 30 fold decrease in IC50 compared to the unsubstituted one. However,
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10.1002/cbdv.202000285
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adding extra halogen atom to the mentioned structure results in significantly drop in the
tyrosine inhibitory activity (3g, IC50= 5.09 µM).
 The effects of ring size bonded to thiosemicarbazide were also evaluated. The SAR study
indicated that decreasing ring size form six-membered to five units provided 3k with IC50=
17.77 µM. While 3l bearing naphthalene as two fused aromatic moieties demonstrated
IC50= 3.38 µM.
Taking the in vitro data together, compound 3d as the most potent compound was selected to
determine the type of inhibition and evaluate via molecular dynamic studies.
Table 2. In vitro anti-tyrosinase activity of benzylidenehydrazinecarbothioamide analogs in the
tyrosinase-catalyzed oxidation of L-dopa
% Inhibition % Inhibition % Inhibition % Inhibition
Compounds
3a
at 0.05 µM [a]
at 0.1 µM [a]
at 10 µM [a]
at 50 µM [a]
IC50 (µM) [a]
9.7±4.04
16.7±3.03
67.71±0.8
83.85±1.41
6.4±1.15
18±5.98
32.85±1.5
51.42±0.1
53.85±7.07
42.42±5.5
30.42±1.2
76.42±2.76
85.14±0.8
78.57±5.25
83.42±3.23
76.28±2.62
91.14±2.62
91.66±4.44
92.14±1.01
87.71±1.21
84.28±2.01
0.26±0.38
0.1±0.56
3b
3c
3d
3e
3f
41.28±2.22
52.85±4.44
0.05±0.64
1.13±1.35
4.25±1.27
41.71±7.03
13.71±6.06
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30.42±1.02
25.14±1.37
21.14±6.46
26.85±4.02
0
33.85±1.01
33.57±7.27
29.28±3.83
45.42±3.9
0
69±6.26
73.71±1.21
71.57±2.62
89.71±0.8
44.42±2.4
79.71±1.21
84±0.20
86.57±1.41
85.57±1.81
90.57±1.21
73.14±4.8
85±3.8
5.09±2.04
2.182±1.76
3.95±1.56
0.5±0.94
3g
3h
3i
3j
17.77±1.9
3k
3l
5.85±3.03
37.1±6.8
3.38±2.16
9.28±1.15
Kojic acid[b]
[a] Data presented here are the mean ± S.E.M. of three to five independent experiments
[b] Standard tyrosinase inhibitor
Tyrosinase inhibition with use of L-tyrosine as the substrate
The in vitro anti-tyrosinase activity of the compounds 3a-l was evaluated using a mushroom
tyrosinase in the presence of L-tyrosine as a substrate based on methods already described.
As is obvious in Table 3, the aromatic ring substituents had a significant effect on tyrosinase
inhibition. Synthetic derivatives showed IC50 values in the range of 0.027–2.26ꢀµM.
 The strongest inhibitory activity was observed for di-substituted derivatives such as 3h
(3,4-di dimethoxy, IC50=0.027 µM), 3g (2-chloro-3-fluoro, IC50= 0.046 µM) and 3f (2,3-
hydroxy, IC50= 0.085 µM). It seems that the increase the bulkiness of substituted compound
in two different positions of phenyl ring a significantly reduce the IC50. This may be due
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10.1002/cbdv.202000285
Chemistry & Biodiversity
to the increasing of occupation of a key pocket within the active site and prevent interaction
with the substrate.
 In the case of compounds containing NO2 motif, surprisingly, the substitution of nitro at
the para position of benzyl ring (3d) led to a reduction in the potency compare to
unsubstituted benzyl. In particular, the order of potency in nitro series was as follows ortho
> meta > para which is unlike inhibition in the presence of L-dopa.
 It was interesting to note that benzylidenehydrazinecarbothioamide derivatives showed
higher inhibitory activity toward tyrosinase in the presence of L-tyrosine as a substrate
compared to L-dopa. In other words, synthetic compounds exhibited much better
monophenolase inhibitory potential than diphenolase inhibitory activity.
Table 3. Tyrosinase inhibitory activity of benzylidenehydrazinecarbothioamide in
presence of L-tyrosine as substrate
% Inhibition
at 0.05 µM [a]
% Inhibition
at 0.1 µM [a]
% Inhibition
at 10 µM [a]
% Inhibition
at 50 µM [a]
Compounds
3a
IC50 (µM) [a]
33.87±7.5
38.61±3.06
19.72±1.59
19.52±1.44
39.79±0.6
39.38±0.15
28.08±6.78
19.86±0.67
85.71±0.625
73.35±2.16
77.73±1.77
59.89±7.2
92.71±4.38
75.04±2.45
89.72±1.45
87.56±4.6
0.15±1.05
1.011±2.16
2.26±0.76
1.74±1.64
3b
3c
3d
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13.89±4.76
23.1±2.1
25.09±3.79
32.1±5.05
47.5±6.13
35.7±2.16
39.03±2.41
33.67±3.13
39.59±3.1
41.63±3.7
78.37±5.05
37.7±0.72
93.4±1.09
87.4±3.5
69.49±6.23
95.4±2.73
97.5±7.09
98.8±0.27
88.9±5.05
92.00±1.25
84.77±1.25
88.85±1.73
2.26±2.7
0.085±2.98
0.046±2.09
0.027±1.43
0.21±0.97
0.36±1.37
2.23±3.01
0.99±1.56
9.28±1.15
3e
3f
44.1±2.52
33.3±4.32
37.7±4.32
30.67±1.01
34.69±8.1
36.73±3.1
3g
3h
45.1±0.3
3i
90.81±0.45
77.55±1.56
87.75±0.38
3j
3k
3l
Kojic acid [b]
[a] Data presented here are the mean ± S.E.M. of three to five independent experiments
[b] Standard inhibitor tyrosinase
Kinetic studies of enzyme inhibition
The mechanism of tyrosinase inhibition was investigated by enzyme kinetics, following the similar
procedure of the tyrosinase inhibition assay.[18] Lineweaver-Burk graphics were used to estimate
the type of inhibition.[23-25] Enzyme kinetics was analyzed by recording substrate-velocity curves
in the absence and presence of the most potent compound 3d. Lineweaver–Burk plot introduces
the inhibition type as mixed-type, competitive or non-competitive. The graphical analysis of
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10.1002/cbdv.202000285
Chemistry & Biodiversity
steady-state inhibition data for compound 3d is shown in Fig. 2. Therefore, the pattern indicates
the inhibition mechanism 3d is mix-competitive.
140
0 μM
0.01 μM
0.025 μM
0.05 μM
0.1 μM
120
100
80
60
0.2 μM
40
20
0
-2
0
2
4
6
8
10
12
1/[S] (mM)-1
-20
Fig. 2. Lineweaver–Burk plot for the inhibition of mushroom tyrosinase-catalyzed L-dopa
oxidation by 3d at 0.010, 0.025, 0.050, 0.100 and 0.200 μM
DPPH antioxidant assay
In order to evaluate the antioxidant profile of compounds 3e and 3f as the most potent
compounds, the DPPH• scavenging capacity was applied.[26-28] The IC50 values, that correspond
to the concentration of compound that reduces 50% of 2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical. Compound 3e and 3f displayed a modest dose-dependent antioxidant activity with IC50
of 106.9 and 133.6 µM, respectively. Comparison of this value with data from other studies
based on the DPPH assays, it was understood that the presence of the nitro group through their
lone pairs of electrons could serve as binding sites in the reaction media to inactivate reactive
oxygen and nitrogen species (RONS). Besides, the OH group may giving out active hydrogen to
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10.1002/cbdv.202000285
Chemistry & Biodiversity
the free radical to obtain antioxidant properties.[29, 30] Although tested compounds exhibited weak
antioxidant activity compare to that of quercetin as positive control
Table 4. Antioxidant properties of 3e and 3f compounds using DPPH assay
IC50 µM [a]
Compounds
% Inhibition
at 100 µM [a]
% Inhibition
at 250 µM [a]
% Inhibition
at 500 µM [a]
47.63±2.2
28.95±0.96
-
73.59±2.6
83.81±2.9
-
86.11±1.32
93.15±0.35
-
3e
3f
106.9±1.8
133.6±0.04
4.62±2.26
Quercetin [b]
[a] Data presented here are the mean ± S.E.M. of three to five independent experiments
[b] Positive control
Docking analyses
All scoring functions available in Gold software were investigated in terms of root-mean-square
deviation (RMSD) of the redocking process. The only scoring function capable of the generation
of an acceptable RMSD was ChemScore. Thus, the docking interaction analysis of all compounds
against the tyrosinase enzyme has been done using Gold software using the ChemScore fitness
function. The validation process resulted in RMSD 2.0 Å. The ChemScore fitness value of 3d, (as
the most potent compound) and 3k (the least active compound) plus their interactions with amino
acid residues in the tyrosinase active site were tabulated in Table 5. Compound 3d as the most
potent compound in the list, IC50= 0.05 µM, generated the ChemScore value of 32.42. Compound
3k with IC50 17.77 µM generated a low score value 21.87 which shows that the scoring function
could nearly categorize compounds based on their experimental activities.
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Table 5. Docking scores and interactions of compounds against Tyrosinase
enzyme
Compound
ChemScore
32.42
Interactions
Hydrogen bonds: Val283
Pi-pi interactions: His263
Hydrogen bonds: Asn260
3d
3k
21.87
Metal acceptor interactions: one Cu2+
3D interaction pattern of compounds 3d, the best tyrosinase inhibitors in terms of IC50 values, have
been shown in Fig. 3a. Compound 3d showed a pi-pi stacked interaction with His263 via its
Nitrobenzene and donated a hydrogen bond in thiourea to residue Val283. The least active
compound in the set is compound 3k with IC50 value 17.77 µM and Chemscore value of 21.87
which is of a low value. 3D interaction pattern of compound 3k shows two hydrogen bonds with
Asn260 from NHs in Thiourea but made the ligand to create a bump with His244 that lowered the
binding ability. Although this compound shows a metal acceptor interaction with one of Cu2+ ions,
it missed pi-pi interaction with His263 as was shown critical in compounds 3d (Fig 3b.).
Overall, it was shown that pi-pi interaction with phenyl ring is critical and potent compounds
made it to His263 mostly.
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Fig. 3. 3D interaction pattern of compounds 3d (a) and 3k (b) in the active site of TYR enzyme
Pharmacophore modeling
A set of pharmacophore models has been developed and the best constructed model 1 generated a
score value of 33.68. All the input compounds could map all the features, as these compounds
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10.1002/cbdv.202000285
Chemistry & Biodiversity
share a common substructure. Model 1 contained 4 features including 2 hydrogen bond donor, one
acceptor and one aromatic features, see Fig. 4. One aromatic feature overlapped aromatic ring of
benzoic acid in compound 3d and one hydrogen bond acceptor feature covered sulfur in thiourea.
Docking interaction analyses has shown that compounds 3d, 3b, 3c, 3l, 3d, 3e and 3f accepted
hydrogen bonds via sulfur in thiourea from Val283, or Arg268. Aromatic ring of phenyl
contributed in important interaction of pi-pi with His263 and was seen in all compounds except
for 3k and in compound 3l was seen with Ser282. Both two hydrogen bond donor features,
overlapped two NH groups in thiourea. Amino groups in thiourea donated hydrogen bonds to
Asn260 in compounds 3f, 3i and to Gly281 in compounds 3a, 3g, and 3h.
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10.1002/cbdv.202000285
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Fig. 4. Pharmacophore model 1 aligned on compound 3d. (a) AR: Aromatic feature, DON: Hydrogen
bond donor feature, ACC: hydrogen bond acceptor feature. (b) All compounds except 3k aligned on
model 1 (b).
Conclusion
Following our expertise in the rational design of tyrosinase inhibitor, herein, we designed,
synthesized and evaluated highly potent tyrosinase inhibitors from the thiosemicarbazide family.
Most compounds exhibited pronounced anti-tyrosinase activity compare the reference compound
kojic acid, with IC50 values in the range of 0.05–17.77ꢀµM for L-dopa as substrate and 0.027–
2.26ꢀµM in the presence of L-tyrosine. It is important to note that all of these compounds, due to
the presence of hydrazinecarboxamide on the phenyl ring, have a high affinity for tyrosine enzyme.
Molecular docking study of 3d into TYR revealed that this molecule completely embedded into
the TYR pocket and showed a pi-pi stacked interaction with His263 via its benzyl pendant. Besides
the additional hydrogen binding interactions with Val283 were also observed. Pharmacological
results proved that the aryl thiosemicarbazone is a privileged scaffold or even fragment for the
design and discovery of novel anti-tyrosinase.
Material and method
Chemistry
All reagents were reagent grade quality and obtained from Sigma-Aldrich (Prague, Czech
Republic). The reaction process was monitored using thin-layer chromatography on the glass-
backed silica gel sheets (Silica Gel 60 GF254) and visualized under UV light (254 nm). Column
1
chromatography was performed on silica gel (90–150ꢀmm; Merck Chemical Inc.). H and 13C
NMR spectra were determined by a Bruker FT-300 MHz spectrometer in DMSO-d6. All the
chemical shifts were reported as (δ) values (ppm). Mass spectra were obtained on the Agilent
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10.1002/cbdv.202000285
Chemistry & Biodiversity
7890A spectrometer at 70 eV. The infrared (IR) spectra were run as KBr disk on Perki-Elmer
Spectrum RXI FTIR.
Procedure for the synthesis of thiosemicarbazones derivatives (3a-l)
All substituted thiosemicarbazones were prepared by treatment of thiosemicarbazid with various
benzaldehydes. Briefly, 1mmol thiosemicarbazide was added to 1mmol respective substituted
benzaldehyde. The reaction mixture was stirred at room temperature for 2ꢀh followed by refluxing
for 6ꢀh. The progress of reaction is monitored by TLC. After cooling, the solvent was evaporated
under reduced pressure and the formed precipitate was recrystallized from methanol.
Synthesis of (E)-1-benzylidenethiosemicarbazide (3a)
White powder; yield: 53.4%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.46 (s, 1H, NH), 8.22
(s, 1H, N=CH), 8.05 (brs, 1H, NH2), 8.01 (brs, 1H, NH2), 7.77-7.79 (m, 2H, phenyl-C2,6-H), 7.39-
13
7.40 (m, 3H, phenyl C3,4,5-H); C NMR (DMSO-d6, 125 MHz): δc (ppm): 178.4 (C=S), 142.7
(HC=N), 134.6 (C1), 130.2 (C4), 129.1 (C3,C5), 127.7 (C2,C6); MS (EI), m/z (%): 179.1 (M+, 100),
162 (10), 119.1 (55.5), 104.1 (63.8), 93 (27.7), 76 (63.3), 60 (50), 43 (77.7); IR (KBr) v (cm-1):
3468.9 (NH2), 3394.08 (NH), 3145.40 (=CH strech), 1582.9 , 1601.13 (C=C aromatic), 1326(C-
N), 1159.50 (C=S strech).
Synthesis of (E)-1-(4-chlorobenzylidene)thiosemicarbazide (3b)
White powder; yield: 48.6%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.50 (s, 1H, NH), 8.26
(s, 1H, HC=N), 8.09 (brs, 1H, NH2), 8.02 (brs, 1H, NH2), 7.44-7.46 (m, 2H, phenyl-C2,6-H), 7.82-
7.86 (m, 2H, phenyl-C3,5-H); 13C NMR (DMSO-d6, 125 MHz): δc (ppm): 178.5 (C=S), 141.2
(HC=N), 133.9 (C4), 132 (C1), 129.6 (C3,5), 123.4 (C2,6); MS (EI),m/z (%): 213.1 (M+, 84.6), 196
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Chemistry & Biodiversity
(11.5), 153.1 (34.6), 138 (57.6), 111 (38.4), 89 (42.3), 76 (80.7), 60 (57.6), 43 (100); IR(KBr) v
(cm-1): 3165.3, 3280.57 (NH2), 3437.56 (NH), 1525, 1600.7 (C=C), 1282.8 (C-N), 1090.5 (aryl
chloride), 1016.9 (C=S).
Synthesis of (E)-1-(4-bromobenzylidene)thiosemicarbazide (3c)
White powder; yield: 42.62%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.50 (s, 1H, NH), 8.26
(s, 1H, HC=N), 8.09 (brs, 1H, NH2), 8.00 (brs, 1H, NH2), 7.75-7.78 (m, 2H, phenyl-C3,5-H), 7.57-
7.6 (m, 2H, phenyl-C2,6-H); 13C NMR (DMSO-d6, 125 MHz): δc (ppm): 178.5 (C=S), 141.3
(HC=N), 133.9 (C4), 132.0 (C1), 129.6 (C3,5), 123.4 (C2,6); MS (EI), m/z (%): 259 ((M+2)+, 87.5),
257 (M+, 87), 199 (18.7), 184 (40.6), 157 (21.8), 118 (50), 102 (28.1), 76 (90.6), 60 (84.3), 43
(100); IR (KBr) v (cm-1): 3165.9, 3287 (NH2), 3436.2 (NH), 1523.5, 1600.8 (C=C), 1286 (C-N),
1089 (C=S).
Synthesis of (E)-1-(4-nitrobenzylidene)thiosemicarbazide (3d)
Yellow powder; yield: 71 %; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.72 (s, 1H, NH), 8.42
(s, 1H, HC=N), 8.26 (brs, 1H, NH2), 8.18 (brs, 1H, NH2), 8.06-8.08 (m, 4H, phenyl-C2,3,5,6-H);13C
NMR (DMSO-d6, 125 MHz): δc (ppm): 178.9 (C=S), 147.9 (HC=N), 141.1 (C4), 139.9 (C1), 128.5
(C3,5), 124.2 (C2,6); MS (EI), m/z (%): 224 (M+, 65.6), 165 (11.25), 149 (12.5), 135 (15.6), 118
(25), 102 (18.7), 89 (21.8), 76 (81.2), 60 (62.5), 43 (100); IR (KBr) v (cm-1): 3092.7, 3142.1 (NH2),
3491.2 (NH), 1588.7, 1578.1 (C=C), 1338.6, 1525 (NO2), 1288.1 (C=N), 1097.8 (C=S).
Synthesis of (E)-1-(4-nitrobenzylidene)thiosemicarbazide (3e)
1
Pale yellow powder; yield: 71.62%; H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.195 (s, 1H,
NH), 8.01 (s, 1H, HC=N), 7.93 (brs, 1H, NH2), 7.77 (brs, 1H, NH2), 7.56-7.59 (m, 2H, phenyl-
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Chemistry & Biodiversity
C3,5-H), 6.67-6.70 (m, 2H, phenyl-C2,6-H), 2.95 (s, 6H, 2CH3); 13C NMR (DMSO-d6, 125 MHz):
δc (ppm)= 177.4 (C=S), 151.8 (HC=N), 143.8 (C1), 129.0 (C4), 121.8 (C2,6), 112.1 (C3,5), 40.6
(2CH3); MS (EI), m/z (%): 222 (M+, 90), 205 (87.5), 188 (50) , 162 (27.5), 147 (100), 118 (25),
91 (30), 60 (28.7), 43 (30); IR (KBr) v (cm-1): 3249, 3147.9 (NH2), 3404 (NH), 1553, 1589 (C=C),
1358.4 (C-N aromatic tertiary amine), 1056.2 (C-N), 1184.2 (C=S).
Synthesis of (E)-1-(2,3-dihydroxybenzylidene)thiosemicarbazide (3f)
Gray powder, yield: 90.09%; MS (EI), m/z (%):1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.40
(s, 1H, NH), 9.69 (s, 1H, HC=N), 8.96 (brs, 1H, NH2), 8.37 (brs, 1H, NH2), 6.61-7.3 (m, 3H,
phenyl-C4,5,6-H), 7.36 (s, 1H, OH-C2), 7.87 (s, 1H, OH-C3); 13C NMR (DMSO-d6, 125 MHz): δc
(ppm)= 177.9 (C=S), 145.6 (HC=N), 141.16 (C2), 141.12 (C3), 121.2 (C1), 119.6 (C4), 117.6 (C6),
116.0 (C5); 211 (M+, 90), 162 (18.1), 120 (100), 92 (22.7), 65 (16.3), 43 (9); IR (KBr) v (cm-1):
3357.6 (NH2), 3461 (NH), 3254.1 (OH), 1234.4 (C-O), 1614, 1697 (C=C), 1221 (C=S).
Synthesis of (E)-1-(2-chloro-6-fluorobenzylidene)thiosemicarbazide (3g)
White powder; yield: 43.85%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.79 (s, 1H, NH), 8.48
(brs, 1H, NH2), 8.36 (brs, 1H, NH2), 7.37-7.44 (m, 3H, phenyl-C3,4,5-H), 7.34 (s, 1H, HC=N); 13C
NMR (DMSO-d6, 125 MHz): δc (ppm)= 178.8 (C=S), 135.9 (HC=N), 126.7 (C6), 126.6 (C2), 120.8
(C5), 120.6 (C3), 116 (C1), 115.7 (C4); MS (EI), m/z (%): 231 (M+, 56.2), 156 (37.5), 137 (40.6),
107 (50), 76 (72.8), 60 (62.5), 43 (100); IR(KBr) v (cm-1): 3146.5 (NH2), 3408 (NH), 1597, 1538
(C=C), 1239 (C=S), 1286 (C-N), 1167 (Ar-F), 1094 (Ar-Cl).
Synthesis of (E)-1-(3,4-dimethoxybenzylidene)thiosemicarbazide (3h)
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10.1002/cbdv.202000285
Chemistry & Biodiversity
White powder; yield: 57.94%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.34 (s, 1H, NH), 8.18
(s, 1H, HC=N), 8.04 (brs, 1H, NH2), 7.97 (brs, 1H, NH2), 7.52 (s, 1H, phenyl-C2-H), 7.13 (d, J=
8.4 Hz, 1H, phenyl-C6-H), 6.94 (d, J= 8.4 Hz, 1H, phenyl-C5-H), 3.81 (s, 3H, OCH3), 3.77 (s, 3H,
13
OCH3); C NMR (DMSO-d6, 125 MHz): δc (ppm)= 177.9 (C=S), 151.0 (HC=N), 149.5 (C3),
143.0 (C4), 127.3 (C2), 122.6 (C5), 111.6 (C1), 108.9 (C6), 55.9 (CH3), 56.1 (CH3); MS (EI), m/z
(%): 239 (M+, 88.4), 222 (88.4), 168 (100), 137 (42.3), 119 (44.2), 92 (57.6), 77 (61.5), 60 (50),
43 (76.9); IR (KBr) v(cm-1): 3117.1, 3182 (NH2), 3351 (NH), 1536, 1618 (C=C), 1146 (Ar-O-C),
1018 (C-N).
Synthesis of (E)-1-(2-nitrobenzylidene)thiosemicarbazide (3i)
Yellow powder; yield: 91.29%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.75 (s, 1H, NH), 8.46
(s, 1H, HC=N), 8.44 (brs, 1H, NH2), 8.42 (brs, 1H, NH2), 8.14 (d, J= 8.4 Hz, 1H, phenyl-C6-H ),
8.02 (d, J= 8.4 Hz, 1H, phenyl-C3-H ), 7.73 (t, J= 7.8 Hz, 1H, phenyl-C5-H), 7.61 (t, J= 7.8 Hz,
1H, phenyl-C4-H); 13C NMR (DMSO-d6, 125 MHz): δc (ppm)= 178.8 (C=S), 143.7 (HC=N), 137.6
(C2), 133.7 (C1), 130.7 (C3), 128.8 (C4), 128.7 (C6), 124.9 (C5); MS (EI), m/z (%): 224 (M+, 28.5),
207 (53.5), 188 (89.2), 167 (10.7), 149 (28.5), 120 (21.4), 91 (42.8), 76 (71), 60 (71.4), 43 (100);
IR (KBr) v (cm-1): 3114 (NH2), 3365 (NH), 1432, 1606 (C=C), 1231 (C=S), 1101 (C-N), 1333,
1542 (NO2).
Synthesis of (E)-1-(3-nitrobenzylidene)thiosemicarbazide (3j)
White powder; yield: 76.65%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.64 (s, 1H, NH), 8.65
(s, 1H, phenyl-C2-H), 8.35 (brs, 1H, NH2), 8.33 (brs, 1H, NH2), 8.22-8.26 (m, phenyl-C6-H ), 8.16-
13
8.21 (m, 1H, phenyl-C4-H ), 8.13 (brs, 1H, HC=N), 7.67 (t, J= 8.0 Hz, 1H, phenyl-C5-H); C
NMR (DMSO-d6, 125 MHz): δc (ppm)= 178.7 (C=S), 148.8 (HC=N), 140.3 (C3), 136.6 (C2), 134.0
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10.1002/cbdv.202000285
Chemistry & Biodiversity
(C4), 130.5 (C1), 124.3 (C5), 121.8 (C6); MS (EI), m/z (%): 224 (M+, 25), 207 (57.1), 188 (92.8),
149 (28.5), 120 (20), 105 (21.4), 76 (71.4), 60 (71.4), 43 (100); IR (KBr) v (cm-1): 3114 (NH2),
3365 (NH), 1432, 1606 (C=C), 1231 (C=S), 1101 (C-N), 1333, 1542 (NO2).
Synthesis of (E)- 1-((2-nitrothiophen-3-yl)methylene)thiosemicarbazide (3k)
Orange powder; yield: 60.17%; 1H NMR (DMSO-d6, 300 MHz) δH (ppm): 11.82 (s, 1H, NH), 8.63
13
(brs, 1H, NH2), 8.31 (brs, 1H, NH2), 8.04-8.31 (m, 2H, C=C), 7.50 (s, 1H, HC=N); C NMR
(DMSO-d6, 125 MHz): δc (ppm)= 178.6 (C=S), 151.1 (HC=N), 147.1 (C2), 135.7 (C3), 130.8 (C1),
129.6 (C4); m/z (%): 147 (10), 120 (100), 92 (36.6), 65 (30), 43 (16.6); IR (KBr) v (cm-1): 3025,
2985 (NH2), 3395 (NH), 1525, 1603 (C=C), 1106 (C=S), 1348 (C-N), 844 (thiophen ring).
Synthesis of (E)-1-((naphthalen-2-yl)methylene)thiosemicarbazide (3l)
White powder; yield: 68.88 %; 11.56 (s, 1H, NH), 8.28 (brs, 1H, NH2), 8.22 (s, 1H, HC=N), 8.13
(brs, 1H, NH2), 8.17 (d, J= 8.4 Hz, 1H, phenyl-C4-H), 8.11 (s, 1H, phenyl-C2-H ), 7.89-7.93 (m,
3H, phenyl-C5,6,7-H), 7.52-7.55 (m, 2H, phenyl-C9,10-H); 13C NMR (DMSO-d6, 125 MHz): δc
(ppm)= 178.4 (C=S), 142.7 (HC=N), 134 (C1), 133.3 (C2), 132.4 (C10), 129.4 (C9), 129.3 (C3),
128.6 (C8), 128.1 (C4), 127.4 (C7), 127.1 (C5), 123.5 (C6); MS (EI), m/z (%): 229 (M+, 77.7), 158
(100), 139 (38.8), 127 (50), 115 (38.8), 76 (22.2), 60 (27.7), 43 (27.7); IR (KBr) v (cm-1): 3245
(NH2), 3380 (NH), 1599, 1540 (C=C).
Tyrosinase inhibition assay
The tyrosinase activity was performed by assay of monophenolase and diphenolase activity. L-
dopa is a substrate for diphenolase assay activity and L-Tyr is a substrate for monophenolase assay
activity, At first 160 µL phosphate buffer (50 mM, pH= 6.8) was added to each well except blank
(190 µL) and then 10 µL tyrosinase (5771 units/mg solid) (0.5 mg/mL) was added to all wells and
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10.1002/cbdv.202000285
Chemistry & Biodiversity
afterwards inhibitors were added accordance to protocol and finally 10 µL kojic acid as negative
control and 10 µL DMSO as positive control were added. In second step the plate was incubated
for 20 min. In third step 20 µL L-dopa and L-tyr considering to related test was added. In L-dopa
test the absorbance immediately was read for 1min in 475 nm. But in L-Tyr test after adding
substrate the plate was incubated for 10min and then the absorbance was read. The IC50 was
calculated by CurveExpert v1.3, The IC50 values are presented in table 2 and 3.
Kinetic assay
The kinetic study was determined by Lineweaver-Burk plot analysis (Fig. 2.) and it was carried
out on several concentrations of 3d compound and L-dopa as the substrate. Pre-incubation and
measurement time was the same as discussed in the mushroom tyrosinase inhibition assay
protocol. Considering to relation of 1/V=1/[S] Km/Vm+ 1/Vm and line equation, the Michaelis
constant (Km) and the maximal velocity (Vmax) of the tyrosinase activity were determined at
various concentrations of L-dopa (0.1, 0.25, 0.5, 0.75 and 1.5 mM) as a substrate. The inhibition
type of the enzyme was assayed by Line weaver Burk plots of the inverse of velocities (1/V) versus
the inverse of substrate concentrations 1/[S] mM.[31-33]
Free radical scavenging activity
The antioxidant activity of purified compounds was measured by the DPPH method.[34-36] 20 µL
of different concentration of drugs (500, 250, 100 µM) were added to 180 µL of 110 μM of
metanolic solution of DPPH in 96 well plate. The mixture was shaken vigorously for 20 min at
room temperature under sodium light. The absorbance was determined at 517 nm and the
percentage of activity was calculated. Quercetin was used as positive control and methanol as the
negative control. The DPPH scavenging effect was calculated as follows:
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10.1002/cbdv.202000285
Chemistry & Biodiversity
(
) × 100
퐴0−퐴1
Scavenging effect (%) =
퐴0
Where A0 is the absorbance of the control, and A1 is the absorbance in the presence of the sample.
All determinations were performed in triplicate. All tests were undertaken on three replicates and
the results were averaged and compared with the quercetin as standard.[30]
Molecular docking
All compounds were sketched using MarvinSketch 18.20.0 and energy minimized by Open Babel
2.4.0 using the steepest descent algorithm. GOLD docking program was used for doing docking
analyses. X-ray crystal structure of Agaricus bisporus tyrosinase including tropolone as the native
ligand in the binding site with PDB code of 2Y9X was applied for doing docking analyses. The
binding site of the tyrosinase comprises Cu2+ metal ions which all were regarded for docking.
Enzyme structure was prepared using Discovery studio client in such a way that all hydrogens
were added and all ligands were removed. The binding site of the enzyme for the docking was
defined automatically using the coordinates of the native ligand tropolone in such a way that 8 Å
around the ligand was defined as the binding site. In order to run docking analyses for all
compounds, we tried all available scoring functions in GOLD namely CHEMPLP, ChemScore,
ASP, and GoldScore. To choose and validate the docking algorithm, tropolone inside the 2Y9X
was redocked using all mentioned scoring functions.[37-39]
Pharmacophore model building
All structures except for 3k with IC50 value 17.77 µM were collected as a multi mol file and fed
to PharmaGist server for pharmacophore model building. PharmaGist is a free web server for
detection of pharmacophores which are the spatial arrangement of features that enable a molecule
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10.1002/cbdv.202000285
Chemistry & Biodiversity
to interact with a specific target receptor. The highest-scoring pharmacophores were defined by
multiple flexible alignments of the input inhibitors.[40]
The list of abbreviations
L-Tyr: L-tyrosinase, L-dopa: L-dihydroxyphenylalanine, Val: Valine, Gly: Glycine, Glu: Glutamic acid,
Ala: Alanine, Asn: Asparagine, Arg: Arginine, His: Histidine, LGA: Lamarckian genetic algorithm,
DMSO-d6: deuterated dimethyl sulfoxide, 1H NMR: proton nuclear magnetic resonance; 13C NMR: carbon-
13 nuclear magnetic resonance, TYR: Tyrosinase, CNS: Central nervous system, DPPH: 2,2-diphenyl-1-
picrylhydrazyl, RMSD: Root-mean-square deviation.
Declarations
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on
reasonable request. We have presented all data in the form of tables and figures.
Competing interests
The authors declare that they have no competing interests.
Funding
The authors wish to thank the support of the Vice-Chancellor for Research of Shiraz University of Medical
Sciences. This study was part of the Ph.D. thesis of Hona Hosseinpour. This agency was not involved in
the design of the study and collection, analysis, and interpretation of data as well as in writing the
manuscript.
Authors' contributions
Me.K conceived the research idea. AI, Ma.K and H.H conducted the experiments, performed synthesize
and biological tests, analyzed the data in addition to preparing the first draft. N.E, Me.K and RM analyzed
and interpreted the data and provided technical guidance. S.P performed the docking study. H.H, AI, RM,
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10.1002/cbdv.202000285
Chemistry & Biodiversity
N.E and Me.K critically read and revised the paper. All authors read and approved the final manuscript.
Me.K supervised all phases of the study.
Corresponding author
Correspondence to Mehdi Khoshneviszadeh.
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