Novel 1,2,4-triazole analogues as mushroom tyrosinase inhibitors: synthesis, kinetic mechanism, cytotoxicity and computational studies

Article abstract of DOI:10.1007/s11030-020-10102-5

We have created a novel series of mushroom tyrosinase inhibitors with 1,2,4-triazole as fundamental skeleton. The target compound 1,2,4-triazol-3-ylthio)-N-phenyl acetamide derivatives 9(a–l) were synthesized by the reaction of 4- and 5-substituted 1,2,4-triazole-3-thiol derivatives 6(a–c) with 2-chloro-N-sub/un-substituted phenyl acetamide derivatives 8(a–d) under basic condition. By using the analytical techniques for instance, FTIR, LC–MS, ¹H NMR and ¹³C NMR, the structural verification was evaluated. The novel series of the target compounds 9(a–l) has been scanned for biological activity (mushroom tyrosinase inhibition potential) which demonstrates adequate results. Interestingly, compound 9k (IC₅₀ = 0.0048 ± 0.0016?μM) exhibits 3500 times more activity compared with standard drug kojic acid (IC₅₀ = 16.8320 ± 1.1600?μM) against mushroom tyrosinase inhibitor. Furthermore, the cytotoxicity experiment was carried out for the highly effective target compounds (9d, 9i, 9j and 9k) by using MTT assay method for A375 human melanoma cells to define the nontoxic performance of the most effective compounds ranging from 1 to 25?μM. Furthermore, the molecular docking study delivers the thought concerning the interface of the ligand with an enzyme. Also, the dynamic simulation was accomplished for compound 9k to govern the plausible binding model.

Full text of DOI:10.1007/s11030-020-10102-5

Molecular Diversity
https://doi.org/10.1007/s11030-020-10102-5
ORIGINAL ARTICLE
Novel 1,2,4‑triazole analogues as mushroom tyrosinase inhibitors:
synthesis, kinetic mechanism, cytotoxicity and computational studies
Balasaheb D. Vanjare1 · Prasad G. Mahajan1 · Nilam C. Dige2 · Hussain Raza2 · Mubashir Hassan3 · Yohan Han2 ·
Song Ja Kim2 · Sung‑Yum Seo2 · Ki Hwan Lee1
Received: 31 January 2020 / Accepted: 29 April 2020
© Springer Nature Switzerland AG 2020
Abstract
We have created a novel series of mushroom tyrosinase inhibitors with 1,2,4-triazole as fundamental skeleton. The target
compound 1,2,4-triazol-3-ylthio)-N-phenyl acetamide derivatives 9(a–l) were synthesized by the reaction of 4- and 5-substi-
tuted 1,2,4-triazole-3-thiol derivatives 6(a–c) with 2-chloro-N-sub/un-substituted phenyl acetamide derivatives 8(a–d) under
basic condition. By using the analytical techniques for instance, FTIR, LC–MS, 1H NMR and 13C NMR, the structural veri-
fcation was evaluated. The novel series of the target compounds 9(a–l) has been scanned for biological activity (mushroom
tyrosinase inhibition potential) which demonstrates adequate results. Interestingly, compound 9k (IC50 =0.0048 0.0016 µM)
exhibits 3500 times more activity compared with standard drug kojic acid (IC50 =16.8320 1.1600 µM) against mushroom
tyrosinase inhibitor. Furthermore, the cytotoxicity experiment was carried out for the highly efective target compounds (9d,
9i, 9j and 9k) by using MTT assay method for A375 human melanoma cells to defne the nontoxic performance of the most
efective compounds ranging from 1 to 25 µM. Furthermore, the molecular docking study delivers the thought concerning
the interface of the ligand with an enzyme. Also, the dynamic simulation was accomplished for compound 9k to govern the
plausible binding model.
Keywords 1,2,4-Triazole · Tyrosinase activity · Cytotoxicity · Molecular docking · Dynamic simulation
Introduction
groups are presently working on the synthesis and biologi-
cal application of heterocyclic compounds. One of the vital
groups of heterocyclic compounds holding three nitrogen
atoms within the fve-membered ring is recognized as tria-
zole [11–13]. It covers two kinds of isomer with variation in
their virtual arrangement of the three nitrogen atoms within
the fve-membered ring, for instance, 1,2,3 and 1,2,4-tria-
zoles. Here, in this research work, we are focusing on the
synthesis of the 1,2,4-triazole skeleton.
Heterocyclic compounds are extensively dispersed in nature
as they perform a major part in the metabolic system which
is important for the living things [1–5]. In addition, owing
to their diferent assets, these compounds are observed in
many segments, including medicine, agriculture, polymer
and material science [5–10]. Thus, the maximum research
Nowadays, a lot of articles are ofered in the synthesis
of the Schif base, natural products and heterocyclic com-
pounds, exhibiting biological activity which belongs to this
class [14–17]. Besides, 1,2,4-triazole is one of the extremely
considerable scafolds that can be accountable in the feld
of the pharmaceutical chemistry which ofers a disparity of
the biological actions: for instance, anti-infammatory [18],
anticancer [19], antihypertensive [20], antimalarial [21],
antibiotic [22], anti-HIV [23, 24], and tyrosinase PDGF-
RTK inhibiting agents [25]. Additionally, there are numer-
ous drugs which are available in the market that have tria-
zole core, such as fuconazole used as an antifungal [26];
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10102-5) contains
supplementary material, which is available to authorized users.
* Ki Hwan Lee
khlee@kongju.ac.kr
1
Department of Chemistry, Kongju National University,
Gongju, Chungnam 32588, Republic of Korea
2
Department of Biological Sciences, Kongju National
University, Gongju, Chungnam 32588, Republic of Korea
3
Institute of Molecular Biology and Biotechnology, The
University of Lahore, Lahore 54590, Pakistan
Vol.:(0123456789)
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Molecular Diversity
ribavirin used as an antiviral [27]; anastrozole, letrozole
used as an antineoplastic [28]; trazodone used as an antide-
pressant [29]; and rilmazafon used as anxiolytic [30].
Tyrosinase is a copper-containing metalloenzyme that
plays a crucial title role in the biosynthesis of melanin from
tyrosine by the process known as melanogenesis. It is pri-
marily observed in the plant, microorganism and animal tis-
sue [31–35]. It is participating in the frst two steps of the
melanin biosynthesis: (1) conversion of monophenol into
ortho-diphenol by the ortho-hydroxylation and (2) oxida-
tion of the catechol to the ortho-quinone [36]. In brief, the
melanin and additional pigments are formed from the tyros-
ine by the oxidation process catalysed by tyrosinase under
various conditions, which is known as melanogenesis. Also,
all the processes are carried out in specifc organelles and
melanosomes in the melanocytes. The real skin colour is
determined by the melanin’s circulation pattern. Likewise,
melanin performs a critical part in absorbing the free radical
and in shielding skin from UV rays. Moreover, excessive
melanin formation generates various harmful impacts on the
skin, for example cancer, melasma, solar lentigo and post-
infammatory melanoderma [37–39]. Therefore, to regulate
the melanin synthesis and to cure dermatological-related
disease, it is very much valuable to synthesize or to create
such an inhibitor, which should not be injurious or not cause
further side efects to health.
Results and discussion
Chemistry
Design approach
In this investigation, we have manufactured 12 novel
1,2,4-triazole derivatives by modifying several structural
aspects of the previously defned tyrosinase inhibitors
[40–42]. The advanced structural features designed in our
target compounds are shown in Fig. 1. From the literature
survey, we come to know that Xie et al. synthesized sev-
eral novel kojic acid correspondents’ derivatives which are
screened for the tyrosinase inhibition study. The results
expose that derivatives in which the 4-position is substi-
tuted with diferent benzyl amino and benzylidene amino
groups in a 1,2,4-triazole core successfully inhibit the
tyrosinase activity [41]. Further, in the potential tyrosinase
inhibitors, the N-(3-(3-(9-methyl-9H-carbazol-3-yl) acry-
loyl) phenyl)-substituted amide functionality was replaced
with diferent substituted phenyl acetamides [40]. After
all, established on the bioisosterism auxiliary hypothesis,
we have incorporated two distinct scafolds to originate
new scafold, which was exploited for the tyrosinase inhi-
bition study. The fragment-founded synthetic strategy in
the present studies is shown in Fig. 1.
To conclude, we have developed and produced a series of
12 new 1,2,4-triazole founded derivatives with good yield.
All the synthesized derivatives were examined for inhibitory
activity of mushroom tyrosinase. From the in vitro report,
it has been noticed that all the compounds demonstrate the
decent result for the tyrosinase inhibition ranging from
(IC50 =0.0048 0.0016 µM) to (IC50 =0.6545 0.0988 µM).
Besides, compound 9k (IC50 = 0.0048 0.0016 µM) dis-
played an outstanding outcome for the inhibition compared
with other synthesized derivatives as well as with standard
drug kojic acid (IC50 =16.8320 1.1600 µM). In addition, it
is very important to clarify the toxic infuence of the potent
compounds. Therefore, we have screened the most potent
target compounds (9d, 9i, 9j and 9k) for the cytotoxicity
experiment by using MTT assay for A375 human melanoma
cells. The results reveal nontoxic performance of 9d, 9i, 9j
and 9k triazole derivatives as compared with nontreated
cells even at high concentration ranging from 1 to 25 µM. In
addition, the molecular docking reports provide an insight
to the enzyme–inhibitors interaction. Also, the molecular
dynamic simulation supplied supportive data for complex
inhibition of compound 9k by recognizing binding residues
in the vicinity of the binding pocket.
Synthesis
The synthesis path towards the synthesis of the intermedi-
ates 2(a–b), 3(a–b), 5(a–c), 6(a–c), halogen-substituted/
unsubstituted 2-chloro-N-phenyl acetamide derivatives
8(a–d) and the target compounds of 4-, 5-substituted
1,2,4-triazol-3-ylthio)-halogen sub/unsubstituted N-phe-
nyl acetamide 9(a–l) is outlined in Scheme 1. The 3, 4
and 5-substituted 1,2,4-triazole-3-thiol derivatives 6(a–c)
were synthesized by using benzoic acid and 2-fuoroben-
zoic acid as one of the beginning raw materials. Firstly,
sub/unsubstituted benzoic acid derivatives 1(a–b) are con-
verted into their corresponding ester derivatives 2(a–b)
at refux temperature under acidic condition. Further, the
nucleophilic substitution reaction with hydrazine hydrate
in ethanol at refux state was carried out for the synthesis
of the corresponding hydrazide derivative 3(a–b). After-
wards, the hydrazide derivatives were condensed with
distinct isothiocyanate functionality to form an open-ring
intermediate 5(a–c) under inert atmosphere with accept-
able yield which was taken as such for further reaction.
The open-ring intermediate 5(a–c) was cyclised, when
refuxed under basic condition to form one of the prereq-
uisite key intermediates termed as a 1,2,4-triazole-3-thiol
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Molecular Diversity
Fig. 1 Fragment-founded strategy of proposed tyrosinase inhibitors
unsubstituted aniline derivatives 7(a–d) and 2-chloroa-
cetyl chloride (2CAC) in the presence of triethyl amine
as a base to form corresponding 2-chloro-N-halogen sub/
unsubstituted phenyl acetamide derivatives 8(a–d). Lastly,
the 1,2,4-triazole-3-thiol derivatives 6(a–c) were coupled
with electrophile (benzamide derivatives) 8(a–d) in exist-
ence of a base (K2CO3) using DMF as solvent to form a
corresponding target compound 9(a–l) with decent yield.
The general representative structure of the target com-
pounds appeared is formulated in Table 1. Furthermore,
the progress of the chemical reactions was authenticated
by TLC methodology and the manufactured compounds
were purifed by using column chromatography method.
The structural interpretation of the target compounds
1
9(a–l) was done by using FTIR, LC–MS, H NMR and
13C NMR analytical skills.
In vitro tyrosinase activity and structural activity
relationship (SAR)
The novel synthesized triazole-based heterocyclic target
compounds 9(a–l) were screened in contradiction to mush-
room tyrosinase enzyme. The gained result reveals that
these molecules’ IC50 value ranges from 0.0048 0.0016
to 0.6545 0.0988 µM relative to standard inhibitor kojic
acid having IC50 value 16.8320 1.1600 µM as presented
in Table 1. In practice, the achieved inhibitory activity in a
molecule is due to the involvement of the entire molecule
but a partial SAR study was streamlined by the presence of
the distinct substituents in respective target compounds, in
Scheme 1 Synthesis route, reagents and conditions: (i) EtOH, conc.
H2SO4; (ii) hydrazine hydrate, EtOH, refux; (iii) ethanol, 12 h, RT;
(iv) DM water, 2 N NaOH, 70–80 °C, (v) triethyl amine, MDC; (vi)
potassium carbonate, DMF, RT
6(a–c). In order to complete the synthesis of the target
compound, another key intermediate has been synthe-
sized by the simple coupling reaction of the diferent sub/
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Molecular Diversity
Table 1 Diferent substituents
present in target compounds and
IC50 value for tyrosinase activity
Tyrosinase activity
IC50 SEM (µM)
Sr. No Compound
-R1
-R2
-X
1
2
3
4
9a
9b
9c
9d
-F
-F
-F
-F
-H
-F
0.5425 0.0715
0.1691 0.0086
0.1537 0.0024
0.0605 0.0096
-Br
-I
5
6
7
8
9
9e
9f
9g
9h
9i
-H
-H
-H
-H
-H
-H
-F
0.2144 0.0368
0.3696 0.2293
0.3431 0.0749
0.2179 0.0478
0.0238 0.0077
-Br
-I
-H
10
11
12
13
9j
9k
9l
-H
-H
-H
-F
-Br
-I
0.0802 0.0032
0.0048 0.0016
0.6545 0.0988
16.8320 1.1600
Kojic Acid (Standard)
SEM=standard error of the mean; values are expressed in mean SEM
which it has more prospects to fuctuate the inhibitory activ-
ity of the corresponding target compound because of the
presence of the distinct interaction pattern with the enzyme,
which was clearly distinguished by SAR study. Furthermore,
all the integrated compounds 9(a–l) possess good activity,
but among all of them, compound 9k exhibits the most
efective inhibitory activity against mushroom tyrosinase
enzyme.
in Fig. 2. In fact, compound 9a (IC50 =0.5425 0.0715 µM)
does not contain any halogen substituent or contains hydro-
gen atom at para position in acetamide functional group,
while the compounds 9b (IC50 = 0.1691 0.0086 µM),
9 c ( I C 5 0 = 0 . 1 5 3 7
0 . 0 0 2 4 µ M ) a n d 9 d
(IC50 =0.0605 0.0096 µM) have fuoro, bromo, and iodo
substituents at para-position, respectively. As a result, the
compound which holds an iodo group possesses a good
inhibitory activity. From the in vitro study, we observed that
the compounds (9c, 9d and 9d) which cover halogen atoms
(–F, –Br and –I) at para-position in acetamide functionality
display better activity than compound (9a) which does not
contain halogen substituent at para-position. It means that
as the size of the halogen atoms increases at the para-posi-
tion in the acetamide functional group, the interaction with
As displayed in Fig. 2, All the four target compounds
9a, 9b, 9c and 9d consist of same scafold (i.e.1,2,4-tria-
zol-3-ylthio)-N-phenyl acetamide) but only the change in
para-substituents of acetamide group. At this point, the 4-
and 5-positions in 1,2,4-triazole scafold possess an ortho-
methoxy phenyl group and an ortho-fuoro phenyl group,
which were unchanged for these four derivatives as indicated
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Molecular Diversity
Fig. 2 Structural activity rela-
tionship of 9a, 9b, 9c and 9d
the enzyme becomes more and more powerful, resulting in
increased activity trend in the respective triazole deriva-
tives. The isomer can be organized as per the inhibitory
activity: 9d>9c>9b>9a. Therefore, from the SAR study,
compound 9d confrms a good inhibitory activity counter to
mushroom tyrosinase enzyme since it contains more heavier
halogen (-I) atom at the para-position in the acetamide func-
tionality of the triazole derivatives.
four derivatives 9(e–h) but only the substituents (X = H,
F, Br or I) are changed at the para-position in aceta-
mide functionality as shown in Fig. 3. Hereby, the com-
pound 9e (IC50 = 0.2144 0.0368 µM) bearing hydrogen,
similarly compounds 9f (IC50 = 0.3696 0.2293 µM),
9 g ( I C5 0 = 0 . 3 4 3 1
0 . 0 7 4 9 µ M ) a n d 9 h
(IC50 =0.2179 0.0478 µM) contain fuoro, bromo and iodo
substituents at the para-position in the acetamide functional
group. In the case of compounds 9e and 9h, carrying hydro-
gen (-H) and iodo (-I) substituents at the para-position in
acetamide functionality demonstrate almost the same inter-
action with enzyme and exhibit auspicious inhibitory activ-
ity against tyrosinase. But in the case of fuoro and bromo
Furthermore, the scafold was changed at 4 and 5 posi-
tions in 1,2,4-triazol-3-ylthio)-N-phenyl acetamide. In
compounds 9e, 9f, 9g and 9h, the 4- and 5-positions
have been replaced by aliphatic n-butyl and aromatic
phenyl groups, respectively, which was ordinary for
Fig. 3 Structural activity rela-
tionship of 9e, 9f, 9g and 9 h
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Molecular Diversity
substituents (9f and 9g), the interaction with the enzyme was
not much more efective. Additionally, after evaluating the
in vitro outcomes of the target derivatives 9(a–d) with the
respective compounds 9(e–h), the result indicates that com-
pounds 9(b-d) confrm superior interaction than the respec-
tive compounds 9(f–h) with exception of nonhalogenated
compounds 9a and 9e, respectively (Figs. 2, 3).
acetamide functionality. But this trend was proved up to
bromo substitution; afterward, the irregular behaviour of
the iodo compound appears. The synthesized target com-
pounds 9(a–l) are organized corresponding to their efec-
tive interaction pattern achieved as per in vitro report, for
instance 9k > 9i > 9d > 9j > 9c > 9b > 9e > 9h > 9g > 9f >
9a > 9l (IC50 values are mentioned in Table 1 and Fig. 2,
3, 4).
Also, the compounds 9(a–d) have been slightly modifed
by replacing the ortho/fuoro phenyl group to phenyl moi-
ety at 5-position in the 1,2,4-triazole scafold to ofer four
new compounds 9i, 9j, 9k and 9l, respectively, as shown in
Fig. 4. Compounds 9i (IC50 =0.0238 0.0077 µM) and 9j
(IC50 = 0.0802 0.0032 µM) display capable activity, and
they contain a hydrogen and fuorine atom at para-position
concerning acetamide functional group. But in the case of
bromo substitution, it shows an excellent activity among the
series as well as compared with standard drug kojic acid
(IC50 =16.8320 1.1600 µM) but in case of iodo-substituted
compounds, i.e., 9l (IC50 =0.6545 0.0988 µM), it exhibits
a reverse trend. Additionally, after comparing the IC50 val-
ues of the 9(i-l) with the respective compounds 9(a–d) and
9(e–h), it was observed that the target compounds 9d, 9i,
9j and 9k confrm the most efective interaction among the
series, but in the case of iodo-substituted derivatives, they
exhibit anomalous comportment.
In vitro analysis
Kinetic analysis
Based on our results, we select the most potent compound
9k to determine inhibition type and inhibition constant of
tyrosinase. The potential of these compounds to inhibit
free enzyme and enzyme–substrate complex was deter-
mined in terms of EI and ESI constants, respectively. The
kinetic studies of the enzyme by the Lineweaver–Burk plot
of 1/V versus 1/[S] in the presence of diferent compounds
concentrations gave a series of straight lines (Fig. 5A).
The results of compound 9k showed that compound inter-
sected within the second quadrant. The analysis showed
that Vmax decreased to new increasing doses of inhibitors
and on the other hand Km remains the same. This behav-
iour indicates that compound 9k inhibits the tyrosinase
noncompetitively to form the enzyme–inhibitor complex.
The secondary plot of slope against the concentration of
inhibitors showed enzyme inhibitor dissociation constant
(Ki) (Fig. 5B). The kinetic results are presented in Table 2
(kinetic parameter table).
Finally, from the structural activity relationship (SAR)
assessment, it was noticed that the compounds which pos-
sess aromatic substituent at 4-position in 1,2,4-triazol-
3-ylthio)-N-(phenyl) acetamide scafold demonstrate good
inhibition outcome than aliphatic-substituted compound
(Figs. 2, 3, 4). Also, the trend was acceptable when the
halogens are substituted at para-position with respect to
Fig. 4 Structural activity rela-
tionship of 9i, 9j, 9k and 9l
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Molecular Diversity
Fig. 5 Lineweaver–Burk plots for inhibition of tyrosinase in the pres-
ence of compound 9k. (A) Concentrations of 9k were 0.00, 0.0024,
0.0048 and 0.0096 µM, respectively. Substrate L-DOPA concentra-
tions were 0.0625, 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B)
The insets represent the plot of the slope versus inhibitor 9k concen-
trations to determine inhibition constant. The lines were drawn using
linear least squares ft
Table 2 Kinetic parameters of the mushroom tyrosinase for L-DOPA
activity in the presence of various concentrations of 9k
all the compounds demonstrate cell viability of approxi-
mately 80%, against nontreated cells. Thus, all the potent
compounds are not toxic to the A375 human melanoma
cells in the concentration scale between 0 and 25 μg/ml.
Nevertheless, after comparison among the series, the most
potent compounds 9d, 9i, 9j and 9k are proved to have the
more acceptable result in cell cytotoxicity experimenta-
tion. Hence, the target compound 9k could be employed
as excellent tyrosinase inhibitor in the feld of medicinal
chemistry for the development of the new drug.
Concentra- Vmax (ΔA/S)
Km (mM) Inhibition type
Ki (µM)
tion (µM)
0.00
0.00013
0.28
0.28
Noncompetitive 0.007
0.0024
0.0048
0.0096
8.236×10−5
4.8551×10−5 0.28
3.2204×10−5 0.28
Vmax is the reaction velocity, Km is the Michaelis–Menten constant, Ki
is the EI dissociation constant
In silico analysis
Cytotoxicity study
Binding energy evaluation of the synthesized derivatives
There are many skin-lighting compounds available in the
market, namely kojic acid, PTU, hydroquinone arbutin,
etc., which are being broadly exploited as a skin-whiting
agent [43, 44]. But to full fll, the criteria of the potent skin
whiting agent should be reliable, without objectionable
cytotoxic side efect. Herein, we have synthesized 12 novel
triazole founded tyrosinase inhibitors and all proved to
have decent activity against mushroom tyrosinase inhibi-
tor compared with standard kojic acid. But among all, we
have preferred four extremely potent inhibitors (9d, 9i,
9j and 9k) for cytotoxicity experiment. The cell toxicity
experiment was carried out by using the MTT assay for
A375 human melanoma cells. The cells were cultivated
for 24 h at various concentrations (1, 5, 10, 15, 20, and
25 μg/ml) of the most potent compounds 9d, 9i, 9j and
9k, respectively, and the results on cell viability assay are
specifed in Fig. 6. From the cell viability experiment,
To predict the best-ftted conformational position of syn-
thesized ligands 9(a–l) within the active region of the tar-
get protein, the generated docked complexes were observed
based on glide docking energy values (kcal/mol) and bond-
ing interaction (hydrogen/hydrophobic) pattern. The low-
est binding energy value depicts the best conformational
position of the ligand within the active region of the target
protein. The docking results showed that all the synthesized
ligands 9(a–l) were bound within the active site of the tar-
get protein with diferent conformational poses and energy
values, respectively (Fig. 7A, B). The comparative results
showed that compounds exhibited good docking energy val-
ues. The basic skeleton of all the synthesized compounds
was similar; therefore, no considerable variation was noticed
in docking energy of all synthesized ligands.
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Molecular Diversity
Fig. 6 Cell viability measure-
ment using the MTT assay.
Cells were untreated (control)
or treated with most potent
compounds 9d, 9i, 9j and 9k at
diferent concentrations (1, 5,
10, 15 and 25 μg/ml) in DMSO
for 24 h
Fig. 7 (A) Docking complex of 9(a–l) within the active region of target protein. (B) Graphical presentation of docking energy values of com-
plexes 9(a–l)
Ligand‑binding analysis of tyrosinase docked complexes
The binding pocket analysis depicted that 9k showed good
binding conformation within binding pocket residues. The
2D graphical depiction of the docking complex is done in
Fig. 8B. Literature data also ensured the importance of these
residues in bonding with other tyrosinase inhibitors which
strengthen our docking results [45–48].
Based on in vitro results, the 9k docking complex was
selected to check the binding conformational analysis. In
detailed docking analysis, couple of hydrogen bonds and
three π–π interactions were observed in the best potent
ligand docking complex. The carbonyl oxygen atom of
9k formed two hydrogen bonds with Arg268 with a bond-
ing distance of 2.20 Å (Fig. 8A). Moreover, phenyl ring
of 4-bromobenzene and another phenyl ring substituted
at 3-position in 1,2,4-triazole core of 9k formed two π–π
interactions with Phe264. Similarly, the 1,2,4-triazole ring
also interacted with the same residue Phe264 through π–π
interaction. The hydrogen and π–π bonds gave more sta-
bility to 9k within the active region of the target protein.
Molecular dynamic simulations (MDS)
Root‑mean‑square deviation and fuctuation (RMSD/
RMSF) To evaluate the residual fexibility of receptor
through MD simulation, RMSD and RMSF graphs were
evaluated to determine the protein structural behaviour. The
RMSD is used to measure the average change in displace-
ment of a selection of atoms for a frame with respect to a
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Molecular Diversity
Fig. 8 (A) 3D docking depiction of 9k complex against tyrosinase. (B) 2D docking depiction of 9k complex against tyrosinase
reference frame. The RMSD graph result of 9k interprets
the protein residual deviation in 100 ns simulation time
frame. Initially, the graph lines showed an increasing trend
from 0 to 20 ns having RMSD value range from 1.2 to 2.4
Å for Cα, backbone and side chains, respectively. From 20
to 60 ns, Cα and backbone graph lines remain stable and
no high fuctuations were observed and RMSD values
remained the same. Moreover, after 60 ns, a little increasing
fuctuation was observed and RMSD values for Cα, back-
bone and side chains rose to 1.6 and 2.8 Å, respectively. Fur-
thermore, from 60 to 100 ns, all graph lines remained steady
stable and no high fuctuations were observed. The overall
RMSD analysis showed that fuctuations of all graphs in the
whole simulation are within the standard range of RMSD
1–3 Å (Fig. 9). The RMSF is useful for characterizing local
changes along with the protein structure. The generated plot
indicated protein behaviour fuctuations during the simu-
lation in Cα residues. The overall results showed that the
tails N-and C-terminals little fuctuated expect the residues
around 250 amino acids. Secondary structure elements such
as alpha helices and beta strands are usually more rigid than
the unstructured part of the protein and thus fuctuated less
than the loop regions (Fig. 10).
Protein interactions with the ligand can be monitored
throughout the simulation. It has been obtained that pro-
tein–ligand interactions are categorized into four types:
(1) hydrogen bonds, (2) hydrophobic, (3) ionic and (4)
water bridges. The generated stacked bar charts showed
that a couple of residues Phe264 and Trp227 have inter-
acted with the ligand. The interaction fractions for both
residues were higher than 0.7, which suggested that 70%
of the simulation time is specifcally maintained in interac-
tion behaviour (Fig. 11).
Fig. 9 RMSD graph of 9k dock-
ing complex at 100 ns
1 3

Molecular Diversity
Fig. 10 RMSF graph of 9k
docking complex at 100 ns
Fig. 11 Protein–ligand contacts
of 9k docking complex
(9d, 9i, 9j, 9k) has been calculated at various concentra-
tions ranging from 0 to 25 μg/ml. The results reveal that
all the potent compounds confrm nontoxic performance
against A375 human melanoma cells even treated at high
concentrations. Furthermore, the docking assessment was
conducted to analyse the binding contact of the ligand with
an enzyme. Besides, the simulation experiment gives us
an idea about the binding pattern of the ligand with an
enzyme, which shows that residues Phe264 and Trp227
more efectively interacted with a ligand. Overall, it can be
assumed from the experiment that the synthesized target
compound 9k could be used as a good candidate of the
drug in a pharmaceutical feld for the development of the
new drug.
Conclusion
In summary, we have defned the successful synthesis
of the 1,2,4-triazole derivatives via a multistep pathway
under simplistic conditions and these 12 novel target com-
pounds were achieved with good yields. The synthesized
target derivatives were well substantiated through spectral
data analysis of FTIR, LCMC, 1H & 13C NMR; and inves-
tigated for their in vitro study, counter to mushroom tyrosi-
nase inhibition. From the in vitro study, it was observed
that all the synthesized target compounds 9(a–l) exhibit
decent activity against mushroom tyrosinase enzyme com-
pared with reference kojic acid. Besides, among all the
derivatives, compounds 9k (IC50 = 0.0048 0.0016 µM)
are almost 3500-fold more active with the aggres-
sive mechanism of action than standard drug kojic acid
(IC50 = 16.8320 1.1600 µM). Also, to validate the non-
toxic actions of the most potent compounds, has been
proven by using the cytotoxicity experiment, which was
carried out using MTT assay method for A375 human mel-
anoma cells. Cell viability of the most potent compounds
Materials and methods
Chemistry
The chemicals required for the synthesis of the target
compounds were bought from Sigma-Aldrich (Munich,
1 3

Molecular Diversity
Germany) and Samchun Chemicals (Daejeon, South Korea)
General synthetic procedure synthesis for the key
intermediate 8(a–d)
1
and used without purification. 13C NMR and H NMR
spectra were taken in DMSO-d6 using a Bruker Avance
(Germany) NMR spectrophotometer at 101 and 400 MHz,
respectively. The mass analysis (LC–MS) was performed on
2795/ZQ2000 (waters) spectrometer. The IR spectra were
flmed on Frontier IR spectrophotometer (Perkin Elmer,
USA). The improvement in the reactions was monitored by
a thin-layer chromatography (TLC) technique. The physi-
cal parameters (such as melting points) of the compounds
(9a–9l) were determined by using Fisher Scientifc (USA)
melting point tool and are uncorrected. The coupling con-
stant and chemical shift values are mentioned in ppm and
Hz, respectively.
As stated in Scheme 1, the respective aniline compound
7(a–d) (1 mmol) was taken in dichloromethane (MDC) and
chilled to 0 °C. Subsequently, triethyl amine (1.2 mmol)
was added to the precooled reaction mass and stirred for
15–20 min. Meanwhile, in another flask, a solution of
2-Chloroacetyl chloride (2-CAC) (1 mmol) was taken in
MDC under atmospheric nitrogen condition and cooled to
0 °C. The above precooled reaction mass of respective ani-
line and triethyl amine was gradually added to the 2-CAC
reaction mass at 0 °C (evolution of HCl gas). After com-
pletion of the addition, the reaction mixture was continued
to be stirred for another 2 h, and then the temperature was
steadily raised to RT and retained for an additional 1–2 h.
The reaction improvement was noticed by TLC. After the
achievement of the reaction, distilled water was added to the
reaction mixture, which was then quenched with water and
extracted with MDC. The MDC layer was concentrated, and
crude residue was purifed by using column chromatography
technique eluting with hexane/ethyl acetate (1–10%) as a
solvent to aford a pure compound 8(a–d), which was used
for the synthesis of the objective compounds 9(a–l).
General synthetic procedure for the key intermediates
(2a–2g) and (3a–3b)
The intermediates 2(a–b) and 3(a–b) were prepared by fol-
lowing previously reported method [49, 50].
General synthetic procedure for the key intermediate
5(a–c)
The respective hydrazide intermediate compound, i.e.,
3(a–b) (1 mmol) in ethanol (2 mL), was added to a RBF
at room temperature under atmospheric nitrogen and it was
continued to be stirred for an additional 10–15 min, and then
corresponding isothiocyanate compound 4(a–b) (1 mmol)
was added under nitrogen environment. The reaction was
continued till the accomplishment of the reaction (10–12 h).
The formation of the product was verifed by TLC system.
The precipitated solid was fltered and washed with cold
ethanol and then dried to aford an open-ring intermediate
5(a–c) with good yield and utilized as such for the next step.
General procedure for the synthesis of the target
compounds 9(a–l)
As displayed in Scheme 1, an intermediate compound 6(a–c)
(1 mmol) and potassium carbonate were taken in dimethyl-
formamide (DMF) and stirred at RT for 15–20 min. Sub-
sequently, intermediate compound 8(a–d) (1 mmol) was
added and continued to be stirred for 4–5 h, until the com-
pletion of the reaction (reaction enhancement supported
via TLC approach). The reaction accomplishment was ana-
lysed by TLC. Later, distilled coldwater was added to the
reaction mass and stirred for 20 min (solid was obtained).
The acquired solid was fltered, washed several times with
distilled cold water and suck dried. Further, the solid was
dried and purifed by using column chromatography tech-
nique eluting with the hexane and ethyl acetate as a solvent
to aford a pure 1,2,4-triazole derivatives 9(a–l) with good
yield.
General synthetic procedure for the key intermediate 6
(a–c)
As shown in Scheme 1, the diferent intermediate com-
pounds 5(a–c) (1 mmol) were taken in distilled water and
heated to 35–40 °C for 10 min and then 2 N NaOH (pH of
reaction mixture>10) solution was added to it, and then the
mixture was heated for 4–5 h at 70–80 °C. Product confrma-
tion was checked by TLC technique. Afterward, the reaction
mass was slowly cooled down to RT (room temperature) and
distilled water was added and then it was cooled to 0–5 °C.
The pH (5–6) of the reaction mass was adjusted by adding
2 N HCl solution (Conc. HCl was used wherever necessary).
After maintaining the pH, the reaction mass was continued
to be stirred for 1–2 h at 10–15 °C, and then the solid was
fltered and dried to aford a compound 6(a–c), which was
taken as it is for the next step.
2‑(5‑(2‑Fluorophenyl)‑4‑(2‑methoxyphenyl)‑4H‑1,2,4‑tria‑
zol‑3‑ylthio)‑N‑phenylacetamide (9a) White solid; isolated
1 3

Molecular Diversity
yield: 80%; M.P: 154 °C; (ESI, Fig. S1): 1H NMR data: δ H
(400 MHz, DMSO-d6) 10.37 (1 H, s), 7.58 (2 H, d, J=7.6),
7.52–7.44 (2 H, m), 7.41 (1 H, dd, J=7.7, 1.8), 7.37–7.30 (3
H, m), 7.26–7.18 (2 H, m), 7.16 (1 H, dd, J=8.4, 0.9), 7.08
(1 H, t, J=7.4), 7.05–6.99 (1 H, m), 4.24 (1 H, d, J=15.0),
4.20 (1 H, d, J = 15.0), 3.62 (3 H, s); (ESI, Fig. S2): 13C
NMR (101 MHz, DMSO-d6) δ 165.98, 154.41, 132.27,
131.90, 129.29, 129.08, 124.01, 121.28, 119.59, 116.44,
113.35, 56.22, 37.39; (ESI, Fig. S3): IR (KBr): 3250, 3193,
3131, 3093, 2933, 2845, 1678, 1622, 1177, 1094, 820 and
687 cm−1; (ESI, Fig. S4): LC–MS:435.2 (m/z).
2845.47,1678.69, 1622.18, 1177.60, 1163.22, 1021.20,
1001.91, 750.88 and 987.54 cm−1; (ESI, Fig. S12): LC–
MS: 515.1 (m/z).
2‑(5‑(2‑fluorophenyl)‑4‑(2‑methoxyphenyl)‑4H‑1,2,4‑tri‑
N‑(4‑fuorophenyl)‑2‑(5‑(2‑fuorophenyl)‑4‑(2‑methoxyph
azol‑3‑ylthio)‑N‑(4‑iodophenyl) acetamide (9d) Brown
solid; isolated yield: 85%; M.P: 220 °C; (ESI, Fig. S13):
1H NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 7.69–7.65
(m, 2H), 7.52–7.38 (m, 5H), 7.34 (dd, J=7.8, 1.5 Hz, 1H),
7.28–7.11 (m, 3H), 7.03 (dd, J=7.6, 1.1 Hz, 1H), 4.23 (d,
J = 15.1 Hz, 1H), 4.18 (d, J = 15.1 Hz, 1H), 3.62 (s, 3H);
(ESI, Fig. S14): 13C NMR (101 MHz, DMSO-d6) δ 166.20,
139.10, 137.95, 132.23, 131.90, 129.07, 121.78, 121.28,
116.44, 116.23, 113.36, 56.22 and 37.37; (ESI, Fig. S15):
IR (KBr): 3250.82, 3192.87, 3133.61, 3071.26, 3038.34,
2923.51, 2849.77, 1678.72, 1622.14, 1177.65, 1024.75,
750.92 and 687.70 cm−1; (ESI, Fig. S16): LC–MS: 561.1
(m/z).
enyl)‑4H‑1,2,4‑triazol‑3‑ylthio) acetamide (9b) Light-grey
solid; isolated yield: 82%; M.P: 105 °C; (ESI, Fig. S5):1H
NMR (400 MHz, DMSO-d6) δ 10.48 (s, 1H), 7.96 (s, 1H),
7.67–7.55 (m, 2H), 7.52–7.37 (m, 3H), 7.34 (dd, J = 7.8,
1.5 Hz, 1H), 7.28–7.10 (m, 4H), 7.03 (ddd, J=7.7, 1.0 Hz,
1H), 4.20 (s, 2H), 3.62 (s, 3H); (ESI, Fig. S6): 13C NMR
(101 MHz, DMSO-d6) δ 162.78, 132.27, 131.89, 129.08,
121.42, 121.34, 121.27, 115.97, 115.74, 113.35, 56.22,
52.62, 36.25; (ESI, Fig. S7): IR (KBr): 3251, 3192, 3132,
3038, 2933, 2845, 1679, 1622, 1177, 1163, 1094, 751 and
687 cm−1; (ESI, Fig. S8): LC–MS: 453.2 (m/z).
2‑(4‑butyl‑5‑phenyl‑4H‑1,2,4‑triazol‑3‑ylthio)‑N‑phenyl
N‑(4‑bromophenyl)‑2‑(5‑(2‑fuorophenyl)‑4‑(2‑methoxyph
acetamide (9e) White solid; isolated yield: 82%; M.P:
103 °C; (ESI, Fig. S17): 1H NMR (400 MHz, DMSO-d6) δ
10.40 (s, 1H), 7.65–7.61 (m, 2H), 7.61–7.54 (m,5H), 7.36–
7.28 (m, 2H), 4.21 (s, 1H), 4.02 (t, 2H), 1.54 (qui, 2H), 1.13
(sxt, 2H), 0.74 (t, J=7.4 Hz, 3H); (ESI, Fig. S18): 13C NMR
(101 MHz, DMSO-d6) δ 166.16, 155.56, 139.29, 130.52,
129.46, 129.28, 128.91, 127.83, 124.01, 119.59, 52.62, 44.45,
38.15, 29.51, 29.16, 22.55, 19.43, 14.42; (ESI, Fig. S19):
IR (KBr): 3249.97, 3192.64, 3132.27, 3018.51, 2921.02,
2850.87, 1679.01, 1622.07, 1177.86, 1094.37, 751.52 and
687.81 cm−1; (ESI, Fig. S20): LC–MS: 367.2 (m/z).
enyl)‑4H‑1,2,4‑triazol‑3‑ylthio) acetamide (9c) Dark-grey
solid; isolated yield: 82.5%; M.P: 112 °C; (ESI, Fig. S9): 1H
NMR δ H (400 MHz, DMSO-d6) 10.54 (1 H, s), 7.60–7.54
(2 H, m), 7.50 (3 H, ddd, J=8.0, 7.3, 4.1), 7.47–7.44 (1 H,
m), 7.41 (1 H, td, J=7.6, 1.8), 7.34 (1 H, dd, J=7.8, 1.5),
7.22 (2 H, ddd, J=8.7, 6.8, 3.3), 7.18–7.13 (1 H, m), 7.03
(1 H, td, J = 7.7, 1.0), 4.26–4.16 (2 H, s), 3.62 (3 H, s);
(ESI, Fig. S10): 13C NMR (101 MHz, DMSO-d6) δ 132.28,
132.12, 131.89, 129.07, 127.20, 124.99, 121.53, 121.28,
115.59, 113.36, 56.23, 52.63, 37.34; (ESI, Fig. S11): IR
(KBr): 3250, 3193, 31,32, 3071.85, 3038.88, 2933.32,
2 ‑ ( 4 ‑ b u t y l ‑ 5 ‑ p h e n y l ‑ 4 H ‑ 1 , 2 , 4 ‑ t r i a ‑
z o l ‑ 3 ‑ y l t h i o ) ‑ N ‑ ( 4 ‑ f l u o r o p h e n y l ) a c e t a m i d e
1 3

Molecular Diversity
2H), 1.53 (qui, 2H), 1.13 (Sxt, 2H), 0.74 (t, J=7.4 Hz, 3H);
(ESI, Fig. S30): 13C NMR (101 MHz, DMSO) δ 166.37,
155.57, 150.47, 139.11, 137.96, 130.54, 129.46, 128.92,
127.79, 121.76, 87.54, 44.45, 38.09, 31.63, 19.43, 13.60.
(ESI, Fig. S31): IR (KBr):3250.76, 3192.30, 3039.34,
2922.09, 2851.49, 2249.80, 1679.29, 1622.49, 1550.55,
1177.99, 1094.61, 820.53, 752.45 and 688.24 cm−1; (ESI,
Fig. S32): LC–MS: 493.1 (m/z).
(9f) Of-white solid; isolated yield: 81%; M.P: 108 °C;
1
(ESI, Fig. S21): H NMR (400 MHz, DMSO-d6) δ 10.45
2‑(4‑(2‑methox yphenyl)‑5‑phenyl‑4H‑1,2,4‑tria‑
(s, 1H), 7.66–7.53 (m, 7H), 7.21–7.13 (m, 2H), 4.20 (s,
2H), 4.06–3.97 (m, 2H), 1.54 (qui, 2H), 1.13 (Sxt, J=14.6,
7.3 Hz, 3H), 0.74 (t, J=7.4 Hz, 3H); (ESI, Fig. S22): 13
C
NMR (101 MHz, DMSO) δ 166.09, 155.56, 150.49, 135.69,
130.53, 129.46, 128.91, 127.82, 121.41, 121.33, 115.98,
115.76, 52.63, 44.45, 38.01, 31.75, 31.64, 29.51, 19.43,
13.60; (ESI, Fig. S23): IR (KBr): 3252, 3194, 3023, 2922,
2851, 1679, 1601, 1178, 1095, 752 and 688 cm−1; Fig. (ESI,
Fig. S24): LC–MS: 385.2 (m/z).
zol‑3‑ylthio)‑N‑phenylacetamide (9i) White solid; iso-
1
lated yield: 82%; M.P: 173 °C; (ESI, Fig. S33): H NMR
(400 MHz, DMSO-d6) δ 10.37 (s, 1H), 7.56 (dd, J =13.3,
4.8 Hz, 3H), 7.46–7.41 (m, 1H), 7.39–7.30 (m, 7H), 7.27
(d, J=8.3 Hz, 1H), 7.09 (dt, J=12.6, 7.5 Hz, 2H), 4.21 (d,
J = 15.0 Hz, 1H), 4.17 (d, J = 15.0 Hz, 1H), 3.66 (s, 3H);
(ESI, Fig. S34): 13C NMR (101 MHz, DMSO-d6) δ 166.01,
155.15, 154.84, 139.27, 132.57, 130.23, 129.61, 127.55,
127.35, 124.00, 122.54, 121.70, 119.58, 113.71, 56.43,
37.34; (ESI, Fig. S35): IR (KBr): 3250, 3192, 3132, 2923,
2851, 2294, 1678, 1622, 1177 and 820 cm−1; (ESI, Fig.
S36): LC–MS: 417.2 (m/z).
N‑(4‑bromophenyl)‑2‑(4‑butyl‑5‑phenyl‑4H‑1,2,4‑tria‑
zol‑3‑ylthio) acetamide (9g) Grey solid; isolated yield: 79%;
M.P: 104 °C; (ESI, Fig. S25): 1H NMR (400 MHz, DMSO-
d6) δ 10.53 (s, 1H), 7.62 (dd, J=10.4, 3.3 Hz, 2H), 7.59–7.55
(m, 5H), 7.53–7.49 (m, 2H), 4.21 (s, 2H), 4.04–3.98 (m, 2H),
1.53 (qui, J=14.9, 7.5 Hz, 2H), 1.12 (Sxt, J=14.6, 7.4 Hz,
2H), 0.74 (t, J= 7.4 Hz, 3H); (ESI, Fig. S26): 13C NMR
(101 MHz, DMSO) δ 166.38, 155.57, 150.45, 138.66, 132.13,
130.53, 129.46, 128.91, 127.80, 121.51, 115.61, 52.63,
44.45, 38.06, 31.63, 29.51, 19.43, 13.60; (ESI, Fig. S27): IR
(KBr):3249.99, 3192.92, 3038.00, 2919.51, 2850.81, 1679.87,
1622.02, 1177.82, 1094.36, 752.19 and 687.89 cm−1; (ESI,
Fig. S28): LC–MS: 447.1 (m/z).
N‑(4‑fluorophenyl)‑2‑(4‑(2‑methoxyphenyl)‑5‑phe‑
nyl‑4H‑1,2,4‑triazol‑3‑ylthio) acetamide (9j) Off-white
solid; isolated yield: 82%; M.P: 88 °C; (ESI, Fig. S37): 1H
NMR (400 MHz, DMSO-d6) δ 10.43 (s, 1H), 7.64–7.54
(m, 3H), 7.43 (dd, J = 7.8, 1.6 Hz, 1H), 7.40–7.31 (m,
5H), 7.26 (d, J=7.7 Hz, 1H), 7.21–7.14 (m, 2H), 7.11 (td,
J = 7.7, 1.0 Hz, 1H), 4.20 (d, J = 15.0 Hz, 1H), 4.15 (d,
J=15.0 Hz, 1H), 3.65 (s, 3H); (ESI, Fig. S38): 13C NMR
(101 MHz, DMSO-d6) δ 165.95, 155.17, 154.84, 152.36,
135.67, 132.57, 130.24, 129.60, 127.55, 127.35, 122.53,
121.70, 115.98, 113.71, 56.43, 37.22; (ESI, Fig. S39):
IR (KBr): 3250.25, 3132.75, 3038.62, 2922.42, 2851.51,
1678.71, 1678.71, 1622.19,1177.81, 1094.38, 752.53 and
688.17 cm−1; (ESI, Fig. S40): LC–MS: 435.1 (m/z).
2‑(4‑butyl‑5‑phenyl‑4H‑1,2,4‑triazol‑3‑ylthio)‑N‑(4‑iodophe‑
nyl) acetamide (9h) Dark-brown solid; isolated yield: 78%;
M.P: 90 °C; (ESI, Fig. S29): 1H NMR (400 MHz, DMSO-
d6) δ 10.48 (s, 1H), 7.70–7.64 (m, 2H), 7.64–7.59 (m, 2H),
7.59–7.54 (m, 3H), 7.46–7.40 (m, 2H), 4.20 (s, 2H), 4.01 (t,
1 3

Molecular Diversity
N‑(4‑bromophenyl)‑2‑(4‑(2‑methoxyphenyl)‑5‑phenyl‑4H
mushroom tyrosinase (30 U/mL) and 20 µL of the inhibitor
solution were placed in the wells of a 96-well microplate.
After pre-incubation for 10 min at room temperature, 20 µL
of L-DOPA (3,4-dihydroxyphenylalanine, Sigma Chemical,
USA) (0.85 mM) was added and the assay plate was further
incubated at 25 °C for 20 min. After the incubation time, the
absorbance was measured at 475 nm and the inhibition per-
centage was calculated concerning the control. Phosphate
bufer and kojic acid were tested under the same condi-
tions as the negative and positive controls, respectively. The
amount of inhibition by the test compounds was expressed
as the percentage of concentration necessary to achieve 50%
inhibition (IC50). Each concentration was analysed in three
independent experiments. IC50 values were calculated by
nonlinear regression using GraphPad Prism 5.0.
‑1,2,4‑triazol‑3‑ylthio) acetamide (9k) Light-grey solid;
isolated yield: 84%; M.P: 98 °C; (ESI, Fig. S41): 1H NMR
(400 MHz, DMSO-d6) δ 10.51 (s, 1H), 7.59–7.54 (m,
3H), 7.53–7.50 (m, 2H), 7.43 (dd, J = 7.8, 1.7 Hz, 1H),
7.41–7.34 (m, 5H), 7.26 (dd, J = 8.4, 1.0 Hz, 1H), 7.10
(td, J=7.6, 1.1 Hz, 1H), 4.20 (d, J=15.1 Hz, 1H), 4.16 (d,
J=15.1 Hz, 1H), 3.65 (s, 3H); (ESI, Fig. S42): 13C NMR
(101 MHz, DMSO-d6) δ 166.24, 155.17, 154.82, 138.64,
132.58, 132.12, 130.24, 129.60, 129.07, 127.55, 122.45,
121.51, 113.72, 56.43, 37.28; (ESI, Fig. S43): IR (KBr):
3193.01, 3038.63, 2925.19, 2852.90, 2300.62, 2050.74,
1678.54, 1601.77, 1178.78, 1163.21, 1021.71, 764.19 and
688.97 cm−1; (ESI, Fig. S44): LC–MS: 495.1 (m/z).
The % Inhibition of tyrosinase was calculated as follows:
[
]
Inhibition (%) = (B − S)∕B × 100
Here, the B and S are the absorbances for the blank and
samples.
N‑(4‑iodophenyl)‑2‑(4‑(2‑methoxyphenyl)‑5‑phenyl‑4H
In vitro kinetic analysis Based on IC50 value, we select the
most potent compound 9k for kinetic study. A series of
experiments were performed to determine the inhibition
kinetics of 9k by following the already reported methods
[43, 54, 55]. The inhibitor concentrations for 9k were 0.00,
0.0024, 0.0048 and 0.0096 µM. The substrate (L-DOPA)
concentrations were between 0.0625 to 2 mM in all kinetic
studies. Pre-incubation and measurement time were the
same as discussed in the mushroom tyrosinase inhibition
assay protocol. Maximal initial velocity was determined
from the initial linear portion of absorbance up to 5 min
after the addition of enzyme at 30-s interval. The inhibi-
tion type of the enzyme was assayed by Lineweaver–Burk
plots of the inverse of velocities (1/V) versus the inverse of
substrate concentration 1/[L-DOPA] mM−1. The EI disso-
ciation constant Ki was determined by the secondary plot of
1/V versus inhibitor concentrations.
‑1,2,4‑triazol‑3‑ylthio) acetamide (9l) Dark grey solid;
1
isolated yield: 87%; M.P: 203 °C; (ESI, Fig. S45): H
NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 7.70–7.64
(m, 2H), 7.61–7.52 (m, 1H), 7.46–7.33 (m, 8H), 7.26 (dd,
J=8.4, 1.0 Hz, 1H), 7.10 (td, J=7.6, 1.1 Hz, 1H), 4.20 (d,
J = 15.1 Hz, 1H), 4.15 (d, J = 15.1 Hz, 1H), 3.65 (s, 3H);
(ESI, Fig. S46): 13C NMR (101 MHz, DMSO-d6) δ 166.24,
154.83, 139.10, 137.95, 132.58, 130.24, 129.60, 129.07,
127.55, 122.50, 121.77, 113.71, 56.43, 37.31; (ESI, Fig.
S47): IR (KBr): 3193.15, 3038.30, 2923.53, 2852.20,
2300.28, 1678.70, 1601.50, 1244.19, 1178.52, 1021.54,
757.85 and 688.96 cm−1; (ESI, Fig. S48): LC–MS: 543.1
(m/z).
Cytotoxicity
Cell culture and treatment of 9d, 9i, 9j and 9k Human malig-
nant melanoma A375 cells were procured from Korean Cell
Line Bank (KCLB no.80003, Seoul, Korea). The cells were
developed in Dulbecco’s altered Eagle’s medium (DMEM,
Gibco/Invitrogen, Carlsbad, CA, USA) containing 10% foe-
tal bovine serum (FBS, Gibco/Invitrogen) in an incubator
with 37 °C, 5% CO2 and 95% air.
Biology
In vitro methodology
In vitro tyrosinase assay The inhibition of mushroom tyrosi-
nase was determined by a modifcation of the dopachrome
method using L-DOPA as a substrate [51–53]. In a detail,
140 µL of phosphate bufer (20 mM, pH 6.8), 20 µL of
Cell proliferation assay Cells were seeded into 96-well
plates with 0.4 × 105 cells/well in 100 μL medium and cul-
1 3

Molecular Diversity
tured for 24 h. Following an overnight incubation, the cell
medium was removed, and the cells were treated with 9d,
9i, 9j and 9k concentration gradients (1, 5, 10, 15, 20, and
25 μg/ml) for 24 h. Each treatment included three duplicate
wells. The cells with DMSO were used as negative control.
The medium was removed, and 0.5 mg/ml 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
added to each well and incubated for 6 h at 37 °C in an
incubator. The medium was removed, and 100 μL solubi-
lization bufer (10% SDS, 0.01 N HCl) was added to each
well to terminate the MTT reaction. The plate was covered
with foil and agitated on orbital shaker for 1 h to dissolve
formazan crystals for the determination of formazan crystal
amount, which was measured by the absorbance at 595 nm
using a microplate reader (Molecular Devices, Sunnyvale,
CA, USA).
residues were performed by Glide/SP/XP and induced ft
docking (IFD) approaches [61].
Molecular dynamics
The molecular dynamics simulation experiment was carried
out using the Desmond simulation package of Schrödinger
[62]. In all runs, the NPT (isothermal–isobaric) ensemble
was applied with a temperature of 300k and pressure of
1 bar. The simulation length was 100 ns, with relaxation
time 1 ps. The force feld parameters for each simulation
were according to OPLS_2005 [63]. The long-range elec-
trostatic interactions were calculated using the particle mesh
Ewald (PME) method [64]. The cutof radius in Coulomb
interactions was 9.0 Å. The water molecules were described
using a simple point charge model (SPC) [65]. The Mar-
tyna–Tuckerman–Klein chain coupling scheme [66] with
a coupling constant of 2.0 ps was used for pressure con-
trol and the Nose–Hoover chain coupling scheme for tem-
perature control. Nonbonded forces were calculated using
an r-RESPA integrator, where the short-range forces were
updated every step and the long-range forces were updated
every three steps. The trajectories were saved at 4.8 ps inter-
vals for analysis. To analyse the behaviour and interactions
between the ligands and protein, we used the Simulation
Interactions Diagram tool implemented in the Desmond
molecular dynamics package.
Statistical analysis Values are presented as a mean of three
diferent experiments standard deviation (SD). Diferences
between the calculated means of each individual group were
evaluated using a one-way ANOVA.
Docking
Retrieval of mushroom tyrosinase structure from PDB
The three-dimensional (3D) crystal structure of mush-
room tyrosinase (Agaricus bisporus) (PDBID: 2Y9X) was
accessed from the Protein Data Bank (PDB) (http://www.
rcsb.org). The selected protein was energy minimized by
employing conjugate gradient algorithm and amber force
feld in UCSF Chimera 1.10.1 [56].
Acknowledgements This research was supported by Basic Sci-
ence Research Program through the National Research Foun-
dation of Korea (NRF) funded by the Ministry of Education
(NRF-2019R1I1A3A01059089).
Compliance with ethical standards
Grid generation and molecular docking
Conflict of interest The authors declare that they have no confict of
Prior to molecular docking, the optimized tyrosinase struc-
ture was prepared using the “Protein Preparation Wizard” by
Maestro interface in Schrödinger Suite. Bond orders were
assigned, and hydrogen atoms were added to the protein.
The structure was then minimized to reach the converged
root-mean-square deviation (RMSD) of 0.30 Å with the
OPLS_2005 force feld. The active site of the enzyme is
defned from the co-crystallized ligands from Protein Data
Bank and literature data [57–59]. The synthesized ligands
9(a–l) were sketched in s2D sketcher in Schrödinger Suite
and saved in Maestro for docking experiment. Furthermore,
the docking experiment was performed against all synthe-
sized ligands 9(a–l) and target protein by using the Glide
docking protocol [60]. The predicted binding energies (dock-
ing scores) and conformational positions of ligands within
the active region of protein were also performed using Glide
experiment. Throughout the docking simulations, both
partial fexibility and full fexibility around the active site
interest.
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