Microwave-assisted synthesis and biological evaluation of new thiazolylhydrazone derivatives as tyrosinase inhibitors and antioxidants

Article abstract of DOI:10.1002/jhet.3760

In this work, we have synthesized a series of 2-thiazolylhydrazone derivatives (1–27) and investigated their biological activities as tyrosinase inhibitors and antioxidants. Some compounds showed potent tyrosinase inhibitory activities and 4-(2-(2-(1-(4-Aminophenyl)ethylidene)-hydrazinyl)thiazol-4-yl) phenol (26) showed more potent inhibitory effect than the standard tyrosinase inhibitor kojic acid (IC₅₀: 9.8 μM vs. 23.6 μM). Compounds 2, 14, and 26 exhibited high antioxidant activities in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The structure–activity relationship (SAR) indicated that the substitutions of bromine, hydroxyl group, and amino groups cause great effect to the inhibition effect against tyrosinase. The mechanism and kinetic studies demonstrated that the inhibitory effect of compound 26 on the tyrosinase by acting as the reversible and uncompetitive inhibitor. Docking studies suggests that compound 26 interacts strongly with mushroom tyrosinase via hydrogen bonding.

Full text of DOI:10.1002/jhet.3760

Received: 11 December 2018
DOI: 10.1002/jhet.3760
Revised: 2 September 2019
Accepted: 12 September 2019
A R T I C L E
Microwave-assisted synthesis and biological evaluation of
new thiazolylhydrazone derivatives as tyrosinase inhibitors
and antioxidants
Yu Zhang | Xi Fu | Yangting Yan | Jinbing Liu
Department of Food and Chemical
Abstract
Engineering, Shaoyang University, Shao
Shui Xi Road, Shaoyang, 422100, People's
Republic of China
In this work, we have synthesized a series of 2-thiazolylhydrazone derivatives
(1–27) and investigated their biological activities as tyrosinase inhibitors and
antioxidants. Some compounds showed potent tyrosinase inhibitory activities
and 4-(2-(2-(1-(4-Aminophenyl)ethylidene)-hydrazinyl)thiazol-4-yl) phenol
(26) showed more potent inhibitory effect than the standard tyrosinase inhibi-
tor kojic acid (IC50: 9.8 μM vs. 23.6 μM). Compounds 2, 14, and 26 exhibited
high antioxidant activities in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-
azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The struc-
ture–activity relationship (SAR) indicated that the substitutions of bromine,
hydroxyl group, and amino groups cause great effect to the inhibition effect
against tyrosinase. The mechanism and kinetic studies demonstrated that the
inhibitory effect of compound 26 on the tyrosinase by acting as the reversible
and uncompetitive inhibitor. Docking studies suggests that compound 26 inter-
acts strongly with mushroom tyrosinase via hydrogen bonding.
Correspondence
Jinbing Liu, Department of Food and
Chemical Engineering, Shaoyang
University, Shao Shui Xi Road, Shaoyang
422100. People's Republic of China.
Email: syuliujb@163.com
1 | INTRODUCTION
tyrosinase is implicated in the pathogenesis of Parkinson's
disease and other neurodegenerative disease processes.[9,10]
Tyrosinase (monophenol or o-diphenol, oxygen oxidore-
ductase, EC 1.14.18.1, syn. Polyphenol oxidase) is a
multifunctional copper containing oxidase, which is widely
distributed in mammals, plants, bacteria, and fungi.[1,2]
Tyrosinase enables to catalyze the hydroxylation of mono-
phenols to o-diphenols (monophenolase activity) and the
oxidation of o-diphenols to o-quinone (diphenolase activ-
ity).[3,4] The dopaquinone could transform to melanin pig-
ment in melanin biosynthesis pathway,[5] which can block
ultraviolent light rays and protect skin cells from harmful
ultraviolent light radiation.[6,7] However, overproduction
and accumulation of melanin can cause a number of skin
diseases, such as melasma, freckles, ephelide, and solar
lentigines.[8] Tyrosinase has been identified to be able to
oxidize dopamine to form melanin in the brain. Excessive
production of dopaquinone by oxidation of dopamine
results in neuronal damage and cell death, and thus
The browning of fruits, vegetables, and beverages poses
serious threat to agro-economic countries. Tyrosinase is
one of the main factors for most fruits and vegetables qual-
ity loss during post-harvest handling and processing, lead-
ing to fast degradation and the shortening of storage
life.[11] Thus, how to prevent this unfavorable browning
reaction remains challenge in food science. Tyrosinase is
also associated with three biochemical processes of insects,
including sclerotization of cuticle, defensive encapsulation
and melanization of foreign organism and wound
healing.[12,13] These processes provide potential targets for
developing safe and effective tyrosinase inhibitors as insec-
ticides. These phenomena encourage researchers to
develop new tyrosinase inhibitors using in medicinal, agri-
cultural, food sciences, and cosmetic industries.[14,15] By
far, some natural and synthetic tyrosinase inhibitors have
been reported, for example, hydroquinone, ascorbic acid,
J Heterocyclic Chem. 2020;1–12.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

arbutin, kojic acid, aromatic aldehydes, aromatic acids, aro-
matic alcohols, tropolone, and polyphenols.[16–19] However,
only few compounds such as kojic acid, arbutin, tropolone,
and 1-phenyl-2-thiourea (PTU) have been used as cosmetic
products and therapeutic agents.[20,21] Moreover, it has
been reported that arbutin decomposes at room tempera-
ture (10% decomposition at 20ꢀC for 15 days).[22] Hydroqui-
none, a widely used skin-lightening agent, is still
controversial with respect to its biosafety owing to its cyto-
toxic and mitogenic properties.[23] Kojic acid as the com-
monly used tyrosinase inhibitor has been banned in many
countries due to its serious side effects and unstable prop-
erty.[23] Hence, there is still an urgent need for developing
novel tyrosinase inhibitors with high activities and low side
effects. Klabunde et al. reported that phenyl thioureas, thi-
azole ring, and alkyl thioureas exhibited weak to
moderate inhibition of tyrosinase activity.[24] Similarly,
thiosemicarbazide derivatives and curcumin showed
potential ability to coordinate the two copper ions in the
active site of tyrosinase.[25,26] Lately, our group described
that thiosemicarbazide derivatives exhibited potent inhibi-
tory activities against mushroom tyrosinase and the lead
compounds could potentially bind to the binuclear active
site of tyrosinase to inhibit its activity.[27,28] In addition,
we found that 1,3,4-Thiadiazole-2(3H)-thiones and
1,3,4-thiadiazole Schiff base derivatives performed potent
inhibitory activities against mushroom tyrosinase.[29,30]
Inspired by the literatures and our previous work, here we
rationally design and synthesize novel tyrosinase
inhibitors.
Nitrogen heterocyclic compounds are an important
class in medicinal chemistry. Thiazoles and their deriva-
tives have attracted continuing interest over the years
because of the compounds with thiazole structures
impose a broad spectrum of biological activities such as
anticonvulsant, antiinflammatory, analgesic, anti-
tuberculosis, antiviral, pesticidal, antimicrobial, antican-
cer, antitumor, and enzyme inhibition activities.[31,32]
This heterocyclic system has broad application in drug
development for the treatment of allergies, hypertension,
inflammation, schizophrenia, bacterial, HIV infections,
hypnotics, and pain.[33] Hydrazinylthiazoles are an
important class of compounds of profound interest to
medicinal and industrial chemists because compounds
bearing the hydrazinylthiazolyl moieties exhibit diverse
biological properties such as antioxidant, antitubercular,
adenosine receptor antagonist, xanthine oxidase
inhibitors, antimicrobial, anti-inflammatory, and
antiplasmodial activity.[34–36] To the best of our knowl-
edge, the tyrosinase inhibitors with hydrazinylthiazolyl
moiety have never been reported. In this work, we are
going to design and synthesize some hydrazone deriva-
tives bearing thiazole and phenyl moieties, and evaluate
their inhibitory activities against mushroom tyrosinase
and antioxidative performances. We further analyze the
structure–activity relationships (SARs) of as-synthesized
compounds and tyrosinase by enzymatic kinetic assays
and docking calculations.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
As shown in Scheme 1, a series of 2-thiazolylhydrazone
derivatives (1–27) have been synthesized using the gen-
eral procedure. A mixture of different aromatic aldehyde
or aromatic ketone with thiosemicarbazide in ethanol
was refluxed for 24 h catalyzed by acetic acid under
microwave irradiation, affording the corresponding
thiosemicarbazone compounds after recrystallization.
Under microwave irradiation, the 2-thiazolylhydrazone
derivatives were obtained using equimolar of the as-pre-
pared thiosemicarbazone and α-bromo acetophenones in
moderate to high yields. The microwave assisted organic
syntheses (MAOS) shows highly yielding and eco-
friendly. All the target compounds were characterized by
chemical and spectral methods.
2.2 | Tyrosinase inhibitory activity assay
According to the reported protocol,[30] the compounds 1–
27 were subjected to tyrosinase inhibition assay with L-
DOPA as substrate. The tyrosinase inhibitory activity of
kojic acid was selected as a reference substance. The IC50
values of the thiazolylhydrazone derivatives against
tyrosinase were summarized in Table 1.
Most of the 2-thiazolylhydrazone derivatives displayed
potent inhibition activities against mushroom tyrosinase
with IC50 values ranged from 9.80 to 98.16 μM. Compared
to the reference inhibitor kojic acid, compound 3 showed a
comparable tyrosinase inhibitory activity. Particularly,
compound 26 exhibited the most potent tyrosinase inhibi-
tory activity with an IC50 value of 9.80 μM, that performs
better than the reference inhibitor kojic acid. From
Table 1, the results suggested that the substitution pattern
of the benzene ring and substitution position on the ben-
zene ring of 2-thiazolylhydrazone derivatives cause signifi-
cant influence to the tyrosinase inhibitory activity.
Considering the tyrosinase inhibitory activities of com-
pounds 1–12 originating from aromatic aldehydes, com-
pound
3
exhibited better inhibitory activity than
compounds 1, 2, and 4. The similar phenomena were
observed for compound 7 versus compounds 5, 6, 8, and
compound 11 versus compounds 9, 10, 12. These results

ZHANG ET AL.
3
indicate that the type of substitution group (R3) at the ben-
zene ring might modulate the activities and bromine atom
(R3) might be an important active group to target the
enzyme active site or amino acid residues. Moreover, the
IC50 value of the compound 3 was 28.08 μM that performed
better inhibitory activity than compounds 7 and 11. How-
ever, compared to compounds 1–12 bearing R3 as hydroxyl
group caused decreased inhibitory activity. This result sug-
gests that hydroxyl group (R1) in 2-thiazolylhydrazone
derivatives cause great effect to the inhibitory activity.
Therefore, there were different effects on inhibitory activi-
ties when the hydroxyl groups were introduced in different
benzene ring. Hydroxyl group (R1) might be beneficial to
increasing the inhibitory activity and it could interact with
the enzyme active site or amino acid residues. However,
Hydroxyl group (R3) could not interact with the enzyme
active site or amino acid residues. For the compounds 13–
27 from aromatic ketones, the corresponding inhibitory
activities were certainly enhanced once the hydroxyl group
(R1) was replaced by bromine atom (R1). Among the com-
pounds 16, 20, 24, and 25, compound 25 (R1 = methyl
group) exhibited the lowest potent tyrosinase inhibitory
activity with IC50 value of more than 100 μM. These results
indicate that bromine atom positively affects the tyrosinase
inhibitory activity yet methyl group causes adverse influ-
ences to the tyrosinase inhibitory activities. Compound 26
(R1 is amino group) showed more potent inhibitory activity
than compounds 14, 18, and 22. This data suggest that
amino group might play an important role to the inhibitory
owing the lone pair electrons of amino group. From
Table 1, we also can found that tyrosinase inhibitory activ-
ity of compound synthesized from aromatic aldehyde is
better than the tyrosinase inhibitory activity of
corresponding compound synthesized by aromatic ketone.
group could enhance the radical scavenging activity.
However, the compound bearing NO2 group on phenyl
ring (4, 8, 12, 16, 20, 24, 25) always showed lower radical
scavenging ability. Concerning the effect of the R2 group,
compounds 13–24 showed less activity compare with the
corresponding compounds 1–12, indicating that the
R2 = methyl group might exert negative effect to the anti-
oxidant activity. Additionally, compounds with halogen
atoms in phenyl ring possessed moderate radical scaveng-
ing ability compared with BHT.
2.4 | Inhibitory mechanism on
tyrosinase
The inhibition mechanism of compound 26 on mushroom
tyrosinase for the oxidation of L-DOPA was explored.
Figure 1 shows the relationship between compound 26 at
different concentrations and the corresponding enzymatic
activities. With compound 26 at any concentration, the
plots showed linear profiles. With increasing the concen-
tration of compound 26, the decreased slope was obtained,
indicating that the inhibition of compound 26 on mush-
room tyrosinase was reversible.
To further get insight into the inhibition mechanism,
the inhibitory type of the compound 26 on mushroom
tyrosinase for the oxidation of L-DOPA was fitted to the
Line Weaver–Burk double reciprocal plots. Figure 2
shows the double-reciprocal plots of the enzymatic kinet-
ics of mushroom tyrosinase using compound 26. The
result displayed that the plots of 1/V versus 1/[S] gave a
family of parallel straight lines with the same slopes.
Meanwhile, As the concentration of compound increas-
ing, the values of both Km and Vm decreased, but the
ratio of Km/Vm remained unchanged. This result indi-
cated that compound 26 was the uncompetitive tyrosi-
nase inhibitor.
2.3 | Antioxidant activity
DPPH assays and ABTS assays were employed to exam-
ine the antioxidant activities of the 2-thiazolylhydrazone
derivatives. As shown in Table 2, most of the
2-thiazolylhydrazone derivatives showed promising anti-
oxidant activities. In DPPH assay, compared to BHT with
IC50 of 55.35 μM, some compounds (1, 2, 3, 6, 10, 14, 15,
26) exhibited higher antioxidant activities and compound
2 showed the best performance of antioxidant activity
with IC50 of 12.53 μM. In ABTS assay, compounds 2, 14,
and 26 exhibited potent activity with IC50 values of
1.06, 2.35, and 1.82 μM, respectively, that showed
better performances than that of standard of BHT
(IC50 = 3.12 μM). Notably, all of the compounds with
potent antioxidant activities contain phenolic hydroxyl
groups in phenyl ring, indicating that phenolic hydroxyl
2.5 | In silico docking between
tyrosinase and the compound 26
The binding affinities of the inhibitor to the receptor
tyrosinase were calculated using computational docking
studies. The binding energy between compound 26 and
tyrosinase was −5.06 kcal/mol, suggesting that com-
pound 26 was bound tightly to tyrosinase. As shown in
Figure 3, amino group of compound 26 formed hydrogen
bonds with Ala246 (1.87 Å) and Glu322 (2.91 Å). Nitro-
gen atom of thiazole ring formed hydrogen bond with
Asn260 with a distance of 3.01 Å, and the nitrogen atom
of hydrazone formed hydrogen bond with His244 with a
distance of 2.43 Å. Thiazole ring exhibited a Pi–S

4
ZHANG ET AL.
SCHEME 1 Synthesis routes of
2-thiazolylhydrazone derivatives. Reagents and
conditions: (i) thiosemicarbazide, ethanol, reflux, or
microwave; (ii) substituted α-bromo acetophenone,
2-propanol, r.t, or microwave
1. R1 = 4-OH, R2 = H, R3 = Cl;
3. R1 = 4-OH, R2 = H, R3 = Br;
5. R1 = 3-Cl, R2 = H, R3 = Cl;
2. R1 = 4-OH, R2 = H, R3 = OH;
4. R1 = 4-OH, R2 = H, R3 = NO2;
6. R1 = 3-Cl, R2 = H, R3 = OH;
8. R1 = 3-Cl, R2 = H, R3 = NO2;
10. R1 = 4-Br, R2 = H, R3 = OH;
12. R1 = 4-Br, R2 = H, R3 = NO2;
14. R1 = 4-OH, R2 = CH3, R3 = OH;
16. R1 = 4-OH, R2 = CH3, R3 = NO2;
18. R1 = 3-Cl, R2 = CH3, R3 = OH;
20. R1 = 3-Cl, R2 = CH3, R3 = NO2;
22. R1 = 4-Br, R2 = CH3, R3 = OH;
24. R1 = 4-Br, R2 = CH3, R3 = NO2;
26. R1 = 4-NH2, R2 = CH3, R3 = OH;
7. R1 = 3-Cl, R2 = H, R3 = Br;
9. R1 = 4-Br, R2 = H, R3 = Cl;
11. R1 = 4-Br, R2 = H, R3 = Br;
13. R1 = 4-OH, R2 = CH3, R3 = Cl;
15. R1 = 4-OH, R2 = CH3, R3 = Br;
17. R1 = 3-Cl, R2 = CH3, R3 = Cl;
19. R1 = 3-Cl, R2 = CH3, R3 = Br;
21. R1 = 4-Br, R2 = CH3, R3 = Cl;
23. R1 = 4-Br, R2 = CH3, R3 = Br;
25. R1 = 4-CH3, R2 = CH3, R3 = NO2;
27. R1 = 4-NH2, R2 = CH3, R3 = NO2
interaction with Phe264 with a distance of 5.37 Å. Ben-
zene ring involved in Pi–Alkyl with Val283 (5.37 Å),
His85 (5.49 Å), and His263 (4.93 Å), respectively. From
the docking results, no interaction between inhibitor and
the copper atom active center of tyrosinase was observed,
indicating that a distinct inhibition type exists between
tyrosinase and compound 26.
hydroxyl groups were introduced into different benzene
ring; (4) amino group (R1) might play an important
role in the increase of inhibitory activity. Moreover,
the inhibition mechanism and kinetic studies reveal
that the compound 26 acts as the reversible and
uncompetitive inhibitor to tyrosinase by acting as the
reversible and uncompetitive inhibitor. Docking studies
suggest that compound 26 interact strongly with mush-
room tyrosinase where amino group forms hydrogen
bonds with Ala246 and Glu322 and the thiazole ring
and amino acid residues interact with each other.
3 | CONCLUSIONS
In summary, we have synthesized
a
series of
2-thiazolylhydrazone derivatives (1–27) for biological
investigations. Some compounds show potent tyrosi-
nase inhibitory activities and the lead compound 26 is
the best tyrosinase inhibitor with an IC50 value of
9.80 μM. Most of the compounds exhibit promising
antioxidant activities in DPPH and ABTS assays. Com-
pound 2 is found to be the most active antioxidative
compound with IC50 values of 12.53 μM and 1.06 μM
in DPPH and ABTS assays, respectively. Structure–
activity relationship (SAR) analysis indicates that
(1) bromine atom (R3) might be an important active
group of this kind of compounds synthesized by aro-
matic aldehyde; (2) hydroxyl group (R1) may be benefi-
cial to increasing the inhibitory activity; (3) There are
different effects on inhibitory activities when the
4 | EXPERIMENTAL
Melting points were measured on SGW-X4 melting point
1
apparatus and are uncorrected. H NMR and 13C NMR
spectra were recorded on a Bruker 300 at 25ꢀC in DMSO-
d6 using tetramethyl silane (TMS) as an internal
standard. Infrared (IR) spectra were obtained on a Nico-
let is 5 spectrophotometer (KBr disks). Mass (MS) spectra
were reported in m/z using the LCMS-2010A. Tyrosinase,
L-3,4-dhydroxyphenylalanine (L-DOPA), and kojic acid
were purchased from Sigma-Aldrich Chemical Co.
(Shanghai, China). Other chemicals were obtained from
commercial suppliers and used without further
purification.

ZHANG ET AL.
5
TABLE 1 Tyrosinase inhibitory activities of the synthesized
TABLE 2 Antioxidant activities of the synthesized compounds
compounds
Compounds
DPPH IC50 (μM)
52.31
ABTS IC50 (μM)
6.87
Compounds
CLog Pa
5.645
4.461
5.795
4.686
6.244
5.065
6.394
5.281
6.394
5.215
6.544
5.431
4.742
3.562
4.892
3.779
6.122
4.942
6.272
5.159
6.272
5.092
6.422
5.309
4.945
3.002
3.219
—
IC50 (μM)b
40.53
55.15
28.08
60.83
98.16
>100
71.72
79.39
>100
92.73
76.56
>100
>100
>100
46.26
73.13
>100
>100
82.27
95.28
43.13
88.18
82.72
62.57
>100
9.80
1
1
2
12.53
1.06
2
3
45.34
6.32
3
4
67.89
26.91
78.36
62.18
58.21
55.28
89.15
53.10
46.37
88.49
16.26
2.35
4
5
91.68
5
6
38.16
6
7
97.15
7
8
98.65
8
9
94.45
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
BHT
41.78
10
95.17
11
117.25
58.06
12
13
19.17
14
50.19
34.67
63.14
128.08
51.33
99.45
94.75
95.36
55.76
82.77
152.68
>200
1.82
15
96.55
16
126.15
74.52
17
18
123.07
148.65
138.26
69.13
19
20
21
22
>200
23
>200
24
>200
25
18.93
26
58.16
64.25
3.02
27
>100
23.6
55.15
Kojic acid
aValue of CLog P was obtained by ChemBioDraw Ultra 12.0.
temperature. The appearing precipitate was filtered and
recrystallized from 95% alcohol to obtain the
corresponding thiosemicarbazone compounds.
bValues were determined from logarithmic concentration–inhibition curves
(at least eight points) and are given as means of three experiments.
Equimolar of the as-prepared thiosemicarbazone
(10 mmol) and substituted α-bromo acetophenone
(10 mmol) were dissolved in 2-propanol (20 mL). The
mixture was stirred at room temperature until the
thiosemicarbazone was disappeared on TLC. The precipi-
tate was filtered and dried to give the compounds 1–27 in
moderate to high yields.
4.1 | General procedure for the synthesis
of 2-thiazolylhydrazone derivatives 1–27
4.1.1 | Method 1
The procedure was adapted from the literature
reported.[37] To a solution of appropriate aldehyde or
ketone (10 mmol) in anhydrous ethanol (20 mL),
thiosemicarbazide (10 mmol), and acetic acid (0.5 mL)
were added. The reaction mixture was refluxed for 24 h.
The reaction progress was monitored by thin layer
chromatography (TLC). After the reaction was
completed, the reaction mixture was cooled to room
4.1.2 | Method 2
To a solution of substituted acetophenone (1.0 mmol) or
benzaldehyde (1.0 mmol) in anhydrous ethanol (10 mL),
thiosemicarbazide (1.0 mmol), and acetic acid (catalytic

6
ZHANG ET AL.
15 min. After the reaction was finished, the reaction
mixture was cooled to room temperature. The resulted
precipitate was filtered and washed with ethanol. The
crude product was recrystallized from 95% ethanol to
afford the compounds 1–27.
4.2 | 4-((2-(4-(4-chlorophenyl)thiazol-
2-yl)hydrazono)methyl)phenol (1)
White solid; Yield: 74.49%; mp: >280ꢀC; IR (KBr, ν/cm−1
)
1623, 1516, 1215, 1159, 1096. 1H NMR (DMSO-d6,
300 MHz) δ 8.25 (s, 1H, =CH), 7.87 (d, J = 8.6 Hz, 2H,
ph-H), 7.49–7.42 (m, 4H, ph-H), 7.36 (s, 1H, thiazol-H),
6.35 (d, J = 8.6 Hz, 2H, ph-H); 13C NMR (DMSO-d6,
FIGURE 1 Determination of the inhibitory effect of
compound 26 on mushroom tyrosinase for the oxidation of L-
DOPA. The concentrations of compound 26 for curves 1–3 were
12 μM, 8 μM, and 0 μM, respectively [Color figure can be viewed at
wileyonlinelibrary.com]
amount) were added. The reaction mixture was put in a
round-bottomed flask and placed in a microwave reactor
under irradiation at 800 W for 5–10 min. After the reac-
tion was completed, the reaction mixture was cooled to
room temperature. The appearing precipitate was filtered
and recrystallized from 95% alcohol to obtain the
corresponding thiosemicarbazone compounds.
A mixture of thiosemicarbazone (1.0 mmol) and
substituted α-bromo acetophenone (1.0 mmol) was added
in 2-propanol (10 mL). The reaction mixture was placed
in a microwave reactor and irradiated at 200 W for 8–
FIGURE 3 A, the simulated docking model of the interactions
of compound 26 with the active site of mushroom tyrosinase by
AutoDock4.2. B, the schematic representation of the interactions of
compound 26 in the binding pocket of mushroom tyrosinase
derived from the docking model. Dashed lines represent bond
distances between interacting functionalities of the ligand and
receptor. The legends inset represent the type of interaction
between the ligand atoms and the amino acid residues of the
protein [Color figure can be viewed at wileyonlinelibrary.com]
FIGURE 2 Kinetic inhibition assay of compound 26 against
the oxidation of L-DOPA by tyrosinase. The mode of inhibition type
exhibited through Lineweaver–Burk plot with different
concentrations of compound 26. The concentrations of compound
26 for curves 1–3 were 0 μM, 8 μM, and 12 μM, respectively [Color
figure can be viewed at wileyonlinelibrary.com]

ZHANG ET AL.
7
75 MHz) δ 168.0, 160.1, 157.7, 143.6, 132.0, 128.6,
128.2, 127.3, 111.4, 107.9, 103.8, 102.4; ESI-MS,
m/z = 329 (M-H)−.
136.5, 133.6, 132.0, 130.7, 129.5, 128.6, 127.2, 125.5, 124.9,
104.8; ESI-MS, m/z = 348 (M + H)+.
4.7 | 3-((2-(4-(4-hydroxyphenyl)thiazol-
2-yl)hydrazono)methyl)phenol (6)
4.3 | 4-(2-(2-(4-hydroxybenzylidene)
hydrazinyl)thiazol-4-yl)phenol (2)
Yellow solid; Yield: 27.58%; mp: 143.9–146.8ꢀC; IR (KBr,
ν/cm−1) 3213, 1624, 1513, 1362, 1276, 1215. 1H NMR
(DMSO-d6, 300 MHz) δ 8.02 (s, 1H, =CH), 7.70–7.61 (m,
4H, ph-H), 7.46 (d, J = 7.5 Hz, 2H, ph-H), 7.08 (s, 1H,
thiazol-H), 7.68 (d, J = 8.7 Hz, 2H, ph-H); 13C NMR
(DMSO-d6, 75 MHz) δ 168.1, 160.2, 157.1, 148.2, 143.0,
133.6, 131.2, 130.7, 128.6, 126.9, 125.4, 124.9, 115.3, 100.9;
ESI-MS, m/z = 329 (M-H)−.
Yellow solid; Yield: 87.76%; mp: 267.5–270.3ꢀC; IR (KBr,
1
ν/cm−1) 3329, 1628, 1509, 1310, 1243. H NMR (DMSO-
d6, 300 MHz) δ 8.30 (s, 1H, =CH), 7.65 (d, J = 8.7 Hz, 2H,
ph-H), 7.46 (d, J = 8.7 Hz, 2H, ph-H), 7.03 (s, 1H, thiazol-
H), 6.83 (d, J = 8.7 Hz, 2H, ph-H), 6.36 (d, J = 8.7 Hz,
2H, ph-H); 13C NMR (DMSO-d6, 75 MHz) δ 167.7, 158.0,
157.5, 143.2, 128.4, 127.2, 115.3, 111.3, 107.9, 102.4, 100.1,
99.5; ESI-MS, m/z = 328 (M + NH4)+.
4.8 | 3-((2-(4-(4-bromophenyl)thiazol-
2-yl)hydrazono)methyl)phenol (7)
4.4 | 4-((2-(4-(4-bromophenyl)thiazol-
2-yl)hydrazono)methyl)phenol (3)[38]
White solid; Yield: 66.67%; mp: 262.8–265.5ꢀC; IR
(KBr, ν/cm−1) 1623, 1484, 1108, 1071, 1005. 1H NMR
(DMSO-d6, 300 MHz) δ 8.03 (s, 1H, =CH), 7.83 (d,
J = 8.1 Hz, 2H, ph-H), 7.69–7.59(m, 4H, ph-H, thiazol-
H), 7.47–7.42(m, 3H, ph-H); 13C NMR (DMSO-d6,
75 MHz) δ 168.1, 149.2, 140.9, 139.7, 136.5, 133.6,
131.5, 130.7, 128.9, 127.5, 125.5, 124.9, 120.6, 104.9;
ESI-MS, m/z = 392 (M-H)−.
Yellow solid; Yield: 89.23%; mp: 253.5–255.9ꢀC; IR (KBr,
ν/cm−1) 1619, 1495, 1312, 1214. 1H NMR (DMSO-d6,
300 MHz) δ 8.23 (s, 1H, =CH), 7.81 (d, J = 8.7 Hz, 2H,
ph-H), 7.62 (d, J = 8.7 Hz, 2H, ph-H), 7.44 (d, J = 9.0 Hz,
2H, ph-H), 7.37 (s, 1H, thiazol-H), 6.36 (d, J = 8.7 Hz,
2H, ph-H); 13C NMR (DMSO-d6, 75 MHz) δ 167.7, 160.1,
157.7, 141.6, 133.5, 131.5, 128.2, 127.6, 120.6, 111.5, 107.8,
103.7; ESI-MS, m/z = 390 (M + K)+.
4.9 | 3-((2-(4-(4-nitrophenyl)thiazol-2-yl)
hydrazono)methyl)phenol (8)
4.5 | 4-((2-(4-(4-nitrophenyl)thiazol-2-yl)
hydrazono)methyl)phenol (4)
White solid; Yield: 93.59%; mp: 265.5–266.7ꢀC; IR (KBr, ν/
1
Yellow solid; Yield: 96.07%; mp: >280ꢀC; IR (KBr, ν/
cm−1) 1631, 1502, 1343, 1108. 1H NMR (DMSO-d6,
300 MHz) δ 8.29 (s, 1H, =CH), 8.26 (d, J = 7.8 Hz, 2H,
ph-H), 8.11 (d, J = 7.8 Hz, 2H, ph-H), 7.67 (s, 1H, thiazol-
H), 7.45 (d, J = 8.1 Hz, 1H, ph-H), 6.36 (d, J = 8.7 Hz,
2H, ph-H); 13C NMR (DMSO-d6, 75 MHz) δ 168.4, 160.1,
157.6, 148.5, 146.1, 141.3, 128.1, 126.3, 124.1, 111.5, 107.9,
102.4; ESI-MS, m/z = 357 (M + NH4)+.
cm−1) 1641, 1513, 1341, 1109, 1056. H NMR (DMSO-d6,
300 MHz) δ 8.30 (d, J = 9.0 Hz, 2H, ph-H), 8.13 (d,
J = 9.0 Hz, 2H, ph-H), 8.05 (s, 1H, =CH), 7.76 (s, 1H, ph-
H), 7.71 (s, 1H, thiazol-H), 7.65–7.62(m, 1H, ph-H), 7.47–
7.45(m, 1H, ph-H); 13C NMR (DMSO-d6, 75 MHz) δ 168.4,
148.5, 146.2, 140.5, 140.0, 136.5, 133.6, 130.7, 129.0, 126.3,
125.5, 125.0, 124.1, 108.9; ESI-MS, m/z = 359 (M + H)+.
4.10 | 2-(2-(4-bromobenzylidene)
hydrazinyl)-4-(4-chlorophenyl)thiazole
(9)[39]
4.6 | 3-((2-(4-(4-chlorophenyl)thiazol-
2-yl)hydrazono)methyl)phenol (5)
Yellow solid; Yield: 97.99%; mp: 191.5–194.5ꢀC; IR (KBr,
ν/cm−1) 1620, 1487, 1323, 1089. 1H NMR (DMSO-d6,
300 MHz) δ 8.03 (s, 1H, =CH), 7.91–7.86 (m, 2H, ph-H),
7.70 (s, 1H, thiazol-H), 7.64–7.61 (m, 1H, ph-H), 7.56 (d,
J = 9.0 Hz, 1H, ph-H), 7.49–7.44 (m, 4H, ph-H); 13C
NMR (DMSO-d6, 75 MHz) δ 168.1, 149.2, 140.8, 139.7,
Yellow solid; Yield: 63.15%; mp: 197.8–201.2ꢀC; IR (KBr,
ν/cm−1) 1617, 1558, 1491, 1093, 1010, 830. 1H NMR
(DMSO-d6, 300 MHz) δ 8.22 (s, 1H, =CH), 7.82 (d,
J = 8.4 Hz, 2H, ph-H), 7.71 (d, J = 8.4 Hz, 2H, ph-H),
7.55–7.52 (m, 4H, ph-H), 7.31 (s, 1H, thiazol-H); 13C
NMR (DMSO-d6, 75 MHz) δ 170.1, 153.6, 143.6, 134.8,

8
ZHANG ET AL.
132.4, 131.1, 129.8, 128.9, 128.2, 127.3, 125.6, 103.4; ESI-
158.3, 147.2, 143.1, 134.5, 131.9, 129.3, 128.6, 128.3, 127.2,
MS, m/z = 394 (M + H)+.
115.3, 104.6, 14.0; ESI-MS, m/z = 342 (M-H)−.
4.11 | 4-(2-(2-(4-bromobenzylidene)
4.15 | 4-(2-(2-(1-(4-hydroxyphenyl)
ethylidene)hydrazinyl)thiazol-4-yl)phenol
(14)
hydrazinyl)thiazol-4-yl)phenol (10)[40]
Yellow solid; Yield: 55.89%; mp: 224.6–227.1ꢀC; IR (KBr,
ν/cm−1) 3326, 1609, 1514, 1275, 1233, 1121, 832. 1H NMR
(DMSO-d6, 300 MHz) δ 7.85 (d, J = 8.7 Hz, 2H, ph-H),
7.71 (d, J = 8.7 Hz, 2H, ph-H), 7.58 (d, J = 8.7 Hz, 2H,
ph-H), 7.46 (d, J = 8.7 Hz, 2H, ph-H), 7.36 (s, 1H, thiazol-
H); 13C NMR (DMSO-d6, 75 MHz) δ 164.7, 158.1, 154.7,
134.2, 133.3, 131.8, 129.7, 128.6, 128.2, 127.2, 122.7, 105.3;
ESI-MS, m/z = 373 (M-H)−.
White solid; Yield 71.38%; mp: 239.5–240.7ꢀC; IR (KBr, ν/
cm−1) 3327, 3128, 1621, 1523, 1269, 1242. 1H NMR
(DMSO-d6, 300 MHz) δ 7.84 (d, J = 8.4 Hz, 2H, ph-H),
7.66 (d, J = 7.2 Hz, 2H, ph-H), 7.60 (d, J = 8.4 Hz, 2H,
ph-H), 7.39 (s, 1H, thiazol-H), 6.83 (d, J = 7.2 Hz, 2H, ph-
H), 2.28 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ
170.1, 158.8, 147.2, 142.0, 132.7, 131.0, 129.2, 127.9, 127.4,
117.8, 115.4, 106.4, 14.1; ESI-MS, m/z = 324 (M-H)−.
4.12 | 2-(2-(4-bromobenzylidene)
hydrazinyl)-4-(4-bromophenyl)thiazole
(11)[39]
4.16 | 4-(1-(2-(4-(4-bromophenyl)thiazol-
2-yl)hydrazono)ethyl)phenol (15)
Yellow solid; Yield: 63.82%; mp: 227.6–229.1ꢀC; IR (KBr,
ν/cm−1) 1603, 1508, 1488, 1207, 1096, 1007, 829. 1H NMR
(DMSO-d6, 300 MHz) δ 8.23 (s, 1H, =CH), 7.81 (d,
J = 8.7 Hz, 2H, ph-H), 7.72 (d, J = 8.7 Hz, 2H, ph-H), 7.64
(d, J = 8.7 Hz, 2H, ph-H), 7.52 (d, J = 8.7 Hz, 2H, ph-H),
7.37 (s, 1H, thiazol-H); 13C NMR (DMSO-d6, 75 MHz) δ
167.7, 160.1, 157.7, 141.6, 133.5, 131.5, 128.2, 127.6, 120.6,
111.5, 107.8, 103.7; ESI-MS, m/z = 438 (M + H)+.
White solid; Yield 96.13%; mp: 283.2–285.6ꢀC; IR (KBr, ν/
cm−1) 1610, 1571, 1279, 1169. 1H NMR (DMSO-d6,
300 MHz) δ 7.84 (d, J = 8.4 Hz, 2H, ph-H), 7.70 (d,
J = 7.2 Hz, 2H, ph-H), 7.51 (d, J = 8.4 Hz, 2H, ph-H),
7.08 (s, 1H, thiazol-H), 6.85 (d, J = 7.2 Hz, 2H, ph-H),
2.28 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 170.2,
158.5, 147.6, 143.8,133.4, 131.3, 129.3, 128.4, 128.3, 127.2,
115.1, 111.3, 14.0; ESI-MS, m/z = 387 (M-H)−.
4.13 | 2-(2-(4-bromobenzylidene)
hydrazinyl)-4-(4-nitrophenyl)thiazole (12)
4.17 | 4-(1-(2-(4-(4-nitrophenyl)thiazol-
2-yl)hydrazono)ethyl)phenol (16)
Yellow solid; Yield: 73.91%; mp: 261.5–263.2ꢀC; IR (KBr,
Yellow solid; Yield 88.71%; mp: 263.4–265.8ꢀC; IR (KBr,
1
1
ν/cm−1) 1624, 1594, 1507, 1339, 1139, 1070, 1049, 829. H
ν/cm−1) 2987, 1617, 1541, 1345, 1190. H NMR (DMSO-
NMR (DMSO-d6, 300 MHz) δ 8.32 (s, 1H, =CH), 8.15 (d,
J = 7.8 Hz, 2H, ph-H), 8.06 (d, J = 8.1 Hz, 2H, ph-H), 7.72
(d, J = 8.1 Hz, 2H, ph-H),7.67 (s, 1H, thiazol-H), 7.58 (d,
J = 8.1 Hz, 2H, ph-H); 13C NMR (DMSO-d6, 75 MHz) δ
169.8, 161.1, 156.6, 143.3, 139.1, 132.7, 131.7, 128.5, 126.2,
125.4, 124.4, 105.1; ESI-MS, m/z = 421 (M + NH4)+.
d6, 300 MHz) δ 8.30 (d, J = 9.0 Hz, 2H, ph-H), 8.15
(d, J = 9.0 Hz, 2H, ph-H), 7.71 (s, 1H, thiazol-H), 7.65 (d,
J = 8.7 Hz, 2H, ph-H), 6.83 (d, J = 8.7 Hz, 2H, ph-H),
2.28 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 170.3,
158.3, 147.5, 146.1,140.8, 131.3, 128.6, 127.2, 126.3, 124.1,
115.2, 108.7, 14.0; ESI-MS, m/z = 355 (M + H)+.
4.14 | 4-(1-(2-(4-(4-chlorophenyl)thiazol-
4.18 | 4-(4-chlorophenyl)-
2-(2-(1-(3-chlorophenyl)ethylidene)
hydrazinyl)thiazole (17)
2-yl)hydrazono)ethyl)phenol (13)[41]
White solid; Yield: 86.08%; mp: 263.4–265.2ꢀC; IR (KBr,
ν/cm−1) 3386, 1617, 1490, 1196, 1171.1H NMR (DMSO-d6,
300 MHz) δ 7.90 (d, J = 8.4 Hz, 2H, ph-H), 7.64 (d,
J = 7.2 Hz, 2H, ph-H), 7.48 (d, J = 8.4 Hz, 2H, ph-H),
7.37 (s, 1H, thiazol-H), 6.81 (d, J = 7.2 Hz, 2H, ph-H),
2.26 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 170.0,
Yellow solid; Yield 72.65%; mp: 193.1–194.9ꢀC; IR (KBr, ν/
cm−1) 1607, 1488, 1195, 1124, 1090, 828. 1H NMR (DMSO-
d6, 300 MHz) δ 7.97 (d, J = 7.5 Hz, 2H, ph-H), 7.91 (s, 1H,
ph-H), 7.36–7.69 (m, 5H, ph-H), 7.05 (s, 1H, thiazol-H),
2.60 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 163.9,

ZHANG ET AL.
9
154.2, 149.6, 138.5, 133.6, 133.3, 132.8, 130.7, 129.1, 127.7,
7.07 (s, 1H, thiazol-H), 6.73 (d, J = 8.7 Hz, 2H, ph-H),
2.28 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 170.2,
157.2, 146.8, 136.8, 136.5, 130.6, 128.0, 127.2, 124.9, 122.1,
115.1, 106.6, 13.9; ESI-MS, m/z = 387 (M-H)−.
126.8, 125.9, 116.4, 115.3, 18.5; ESI-MS, m/z = 361 (M-H)−.
4.19 | 4-(2-(2-(1-(3-chlorophenyl)
ethylidene)hydrazinyl)thiazol-4-yl)phenol
(18)
4.23 | 4-(2-(2-(1-(4-Bromophenyl)
ethylidene)hydrazinyl)thiazol-4-yl)phenol
(22)
Yellow solid; Yield76.76%; mp: 194.6–195.9ꢀC; IR (KBr, ν/
cm−1) 3116, 1591, 1514, 1397, 1275, 1042. 1H NMR
(DMSO-d6, 300 MHz) δ 7.90 (s, 1H, ph-H), 7.83 (d,
J = 8.1 Hz, 1H, ph-H), 7.60 (d, J = 8.1 Hz, 1H, ph-H), 7.46
(d, J = 9.3 Hz, 2H, ph-H), 7.29 (s, 1H, thiazol-H), 7.25 (d,
J = 9.3 Hz, 2H, ph-H), 6.82–6.77 (m, 1H, ph-H), 2.30 (s,
3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 169.7, 149.0,
145.7, 137.5, 136.2, 133.7, 132.8, 131.5, 130.2, 128.9, 127.5,
127.0, 123.2, 120.3, 18.5; ESI-MS, m/z = 342 (M-H)−.
Yellow solid; Yield 61.59%; mp: 282.3–283.1ꢀC; IR (KBr,
ν/cm−1) 3126, 1606, 1509, 1397, 1271, 1080. 1H NMR
(DMSO-d6, 300 MHz) δ 7.91 (d, J = 8.7 Hz, 2H, ph-H),
7.74 (d, J = 8.7 Hz, 2H, ph-H), 7.64 (d, J = 8.7 Hz, 2H,
ph-H), 7.49 (d, J = 8.7 Hz, 2H, ph-H), 7.42 (s, 1H, thiazol-
H),, 2.30 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ
169.7, 168.5, 145.5, 137.0, 133.5, 131.9, 129.5, 128.6, 128.4,
127.7, 122.1, 105.0, 13.8; ESI-MS, m/z = 405 (M-H)−.
4.20 | 4-(4-bromophenyl)-
2-(2-(1-(3-chlorophenyl)ethylidene)
hydrazinyl)thiazole (19)
4.24 | 4-(4-Bromophenyl)-
2-(2-(1-(4-bromophenyl)ethylidene)
hydrazinyl)thiazole (23)[43]
Yellow solid; Yield 79.61%; mp: 180.7–182.0ꢀC; IR (KBr, ν/
cm−1) 1606, 1482, 1366, 1110, 1071, 835. 1H NMR (DMSO-
d6, 300 MHz) δ 7.99 (s, 1H, ph-H), 7.83 (d, J = 8.1 Hz, 1H,
ph-H), 7.76–7.21 (m, 7H, ph-H, thiazol-H), 2.60 (s, 3H,
CH3); 13C NMR (DMSO-d6, 75 MHz) δ 167.0, 165.4, 140.0,
138.5, 133.3, 132.8, 132.2, 130.6, 129.9, 129.2, 128.3, 127.7,
126.8, 120.8, 16.3; ESI-MS, m/z = 407 (M + H)+.
Yellow solid; Yield 85.14%; mp: 247.7–249.3ꢀC; IR (KBr, ν/
1
cm−1) 1623, 1487, 1398, 1110, 1073. H NMR (DMSO-d6,
300 MHz) δ 7.86 (d, J = 8.7 Hz, 2H, ph-H), 7.74(d,
J = 8.7 Hz, 2H, ph-H), 7.64–7.60 (m, 4H, ph-H), 7.43 (s,
1H, thiazol-H), 2.30 (s, 3H, CH3); 13C NMR (DMSO-d6,
75 MHz) δ 169.7, 168.5, 145.5, 137.0, 133.8, 131.3, 129.8,
127.6, 122.1, 121.4, 120.5, 105.1, 14.0; ESI-MS, m/z = 450
(M-H)−.
4.21 | 2-(2-(1-(3-chlorophenyl)
ethylidene)hydrazinyl)-4-(4-nitrophenyl)
thiazole (20)
4.25 | 2-(2-(1-(4-Bromophenyl)
ethylidene)hydrazinyl)-4-(4-nitrophenyl)
thiazole (24)[42]
Yellow solid; Yield 77.74%; mp: 245.7–247.9ꢀC; IR (KBr,
1
ν/cm−1) 1609, 1598, 1521, 1341, 1112. H NMR (DMSO-
d6, 300 MHz) δ 7.99 (s, 1H, ph-H), 7.88 (d, J = 8.1 Hz,
1H, ph-H), 7.83–7.34 (m, 7H, ph-H, thiazol-H), 2.61 (s,
3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ 176.2, 165.3,
151.2, 145.1, 132.8, 132.4, 131.3, 130.7, 130.2, 129.4, 128.1,
127.7, 126.8, 125.5, 16.3; ESI-MS, m/z = 373 (M + H)+.
Yellow solid; Yield 93.05%; mp: 245.1–247.7ꢀC; IR (KBr, ν/
1
cm−1) 1617, 1600, 1514, 1343, 1081. H NMR (DMSO-d6,
300 MHz) δ 7.70–7.61 (m, 4H, ph-H), 7.46–7.43 (m, 2H,
ph-H), 7.08 (s, 1H, thiazol-H), 6.81 (d, J = 8.7 Hz, 2H, ph-
H), 2.30 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ
169.9, 168.6, 157.1, 137.0, 133.6, 130.7, 129.8, 126.9, 125.4,
124.9, 115.3, 100.9, 13.8; ESI-MS, m/z = 418 (M + H)+.
4.22 | 2-(2-(1-(4-bromophenyl)
ethylidene)hydrazinyl)-4-(4-chlorophenyl)
thiazole (21)[42]
4.26 | 4-(4-Nitrophenyl)-2-(2-(1-(p-tolyl)
ethylidene)hydrazinyl)thiazole (25)[44]
Yellow solid; Yield 76.66%; mp: 247.3–248.5ꢀC; IR (KBr,
1
ν/cm−1) 1612, 1490, 1370, 1113, 1093. H NMR (DMSO-
Yellow solid; Yield 96.59%; mp: 267.6–269.7ꢀC; IR (KBr,
1
d6, 300 MHz) δ 7.73 (d, J = 7.8 Hz, 2H, ph-H), 7.66 (d,
J = 9.0 Hz, 2H, ph-H), 7.65 (d, J = 8.7 Hz, 2H, ph-H),
ν/cm−1) 3340, 1654, 1507, 1344, 1137, 842, 717; H NMR
(DMSO-d6, 300 MHz) δ 8.31 (d, J = 9.0 Hz, 2H, ph-H),

10
ZHANG ET AL.
8.15 (d, J = 9.0 Hz, 2H, ph-H), 7.96 (s, 1H, -NH), 7.72 (s,
1H, thiazol-H), 7.70 (d, J = 8.1 Hz, 2H, ph-H), 7.25 (d,
J = 8.1 Hz, 2H, ph-H), 2.90 (s, 3H, CH3), 2.30 (s, 3H,
CH3); 13C NMR (DMSO-d6, 75 MHz) δ 170.3, 162.3,
148.6, 147.0, 146.1, 140.8, 138.3, 135.0, 129.0, 126.3, 124.1,
108.9, 20.8, 14.0; ESI-MS, m/z = 353 (M + H)+.
before further calculation. IC50 values were determined
by interpolation of the dose–response curves and the
results are listed in Table 1.
4.30 | Kinetic assay of the inhibition of
tyrosinase
4.27 | 4-(2-(2-(1-(4-Aminophenyl)
ethylidene)hydrazinyl)thiazol-4-yl)phenol
(26)
The assay of inhibition mechanism and inhibitory type
of selected compound 26 on mushroom tyrosinase was
carried out according to the protocol as previously
reported.[30] Compound 26 (0, 8, 12 μM) and L-DOPA
(60–150 μM) were used in the kinetic studies. Pre-
incubation and measurement time were the same as
discussed in mushroom tyrosinase inhibition assay pro-
tocol. The inhibition type on the enzyme was evalu-
ated by Lineweaver–Burk plots of inverse of velocities
White solid; Yield 97.53%; mp: 260.6–262.2ꢀC; IR (KBr, ν/
1
cm−1) 3316, 1621, 1513, 1271, 1184, 844, 738; H NMR
(DMSO-d6, 300 MHz) δ 7.89 (d, J = 8.4 Hz, 2H, ph-H),
8.15 (d, J = 8.7 Hz, 2H, ph-H), 7.38 (d, J = 8.7 Hz, 2H,
ph-H), 7.08 (s, 1H, thiazol-H), 6.85 (d, J = 8.4 Hz, 2H, ph-
H), 2.34 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ
169.4, 157.1, 149.9, 145.9, 136.2, 133.8, 130.4, 126.9, 122.1,
115.3, 113.6, 101.1, 14.0; ESI-MS, m/z = 324 (M + H)−.
(1/V) versus inverse of substrate concentration
−1
1/[S] μM
.
4.31 | In silico docking simulation of
tyrosinase with compound 26
4.28 | 4-(1-(2-(4-(4-Nitrophenyl)thiazol-
2-yl)hydrazono)ethyl)aniline (27)
For docking simulations of tyrosinase to the compound
26, the AutoDock 4.2 program was employed according
to the reported procedure.[30] Crystal structure of
tyrosinase (PDB code: 2Y9X) was obtained from the
Protein DataBank (http://www.rcsb.org, Rutgers, and
UCSD/SDSC). To prepare the compound for the dock-
ing simulation, the following steps were performed:
(1) conversion of 2D structures into 3D structures,
(2) calculation of charges, and (3) addition of hydrogen
atoms using the ChemOffice program (http://www.
cambridgeoft.com).
Yellow solid; Yield: 88.38%; mp: >280ꢀC; IR (KBr, ν/
cm−1) 3319, 1617, 1571, 1335, 1142, 859, 730; H NMR
1
(DMSO-d6, 300 MHz) δ 8.32 (d, J = 8.4 Hz, 2H, ph-H),
8.16 (d, J = 8.7 Hz, 2H, ph-H), 7.90 (d, J = 8.1 Hz, 2H,
ph-H), 7.77 (s, 1H, thiazol-H), 7.41 (d, J = 8.4 Hz, 2H, ph-
H), 2.36 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) δ
170.1, 148.6, 146.1, 140.7, 136.5, 133.4, 130.4, 127.0, 126.3,
124.1, 122.4, 109.1, 14.1; ESI-MS, m/z = 355 (M + H)−.
4.29 | Tyrosinase activity assay
Tyrosinase inhibition assays were performed from our
reported procedure.[30] Briefly, thiazolylhydrazone deriv-
atives were screened for diphenolase inhibitory activity of
tyrosinase using L-DOPA as substrate. The compounds
1–27 and kojic acid (used as control) were respectively
dissolved in DMSO to make their final concentration at
2.0%. Phosphate buffer (50 mM, pH 6.8) was used to
dilute the DMSO stock solution of tested compounds.
Thirty units of mushroom tyrosinase (0.5 mg/mL) were
firstly pre-incubated with the compounds in phosphate
buffer (50 mM, pH 6.8), for 10 min at 25ꢀC. Then, the L-
DOPA (0.5 mM) was added to the reaction mixture and
the enzyme reaction was continuously monitored by
measuring the UV absorbance at 475 nm of formation of
the DOPA chrome for 1 min. The measurement was per-
formed in triplicate for each concentration and averaged
4.32 | Free radical scavenging activity
assay of DPPH
The antioxidation effect on 2,2-diphenyl-1-picrylhydrazyl
(DPPH) was determined adapting from the reported
method.[45] Reaction mixtures consisted of 100 μL of
DPPH (150 μM), 20 μL of increasing concentration of test
compounds, and the volume was adjusted to 200 μL in
each well with DMSO. The above solution was incubated
at room temperature (25ꢀC) for 30 min in the dark. After
completion of the incubation, the UV absorbance at
517 nm was measured using a micro plate reader. All of
the experiments were triply conducted using DMSO as
control and BHT as antioxidant standard. The DPPH rad-
ical scavenging activity was calculated followed by the
formula:

ZHANG ET AL.
11
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formed according to the literature.[45] Test solution of
ABTS decolorization assay was prepared by mixing
2.45 mM potassium persulfate and 7 mM ABTS at 8:12 vol-
ume/volume ratio. The working solution was kept in the
dark at room temperature for 16 to 18 h. Before ABTS radi-
cal scavenging test, the ABTS test solution was diluted with
DMSO to an absorbance of 0.7 at 734 nm. One milliliter of
diluted ABTS test solution was added to 10 μL of DMSO or
tested sample solutions. After the working solution was
kept in the dark at room temperature for 4 min, the UV
absorbance was measured at 734 nm. The measurement
was repeated triply with DMSO as a control and BHT as an
antioxidant standard. The absorbance of test samples was
calculated by following formula.
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ACKNOWLEDGMENTS
This work was financially supported by the Foundation
of Education Department of Hunan Province, China
(15A172).
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ORCID
Jinbing Liu https://orcid.org/0000-0002-2998-9110
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How to cite this article: Zhang Y, Fu X, Yan Y,
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as tyrosinase inhibitors and antioxidants.
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