Novel 1,3,4-oxadiazole compounds inhibit the tyrosinase and melanin level: Synthesis, in-vitro, and in-silico studies

Article abstract of DOI:10.1016/j.bmc.2021.116222

In this research work, we have designed and synthesized some biologically useful of 1,3,4-Oxadiazoles. The structural interpretation of the synthesized compounds has been validated by using FT-IR, LC-MS, HRMS, ¹H NMR and ¹³C NMR techniques. Moreover, the in-vitro mushroom tyrosinase inhibitory potential of the target compounds was assessed. The in-vitro study reveals that, all compounds demonstrate an excellent tyrosinase inhibitory activity. Especially, 2-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-phenylacetamide (IC₅₀ = 0.003 ± 0.00 μM) confirms much more significant potent inhibition activity compared with standard drug kojic acid (IC₅₀ = 16.83 ± 1.16 μM). Subsequently, the most potent five oxadiazole compounds were screened for cytotoxicity study against B16F10 melanoma cells using an MTT assay method. The survival rate for the most potent compound was more pleasant than other compounds. Furthermore, the western blot results proved that the most potent compound considerably decreased the expression level of tyrosinase at 50 μM (P < 0.05). The molecular docking investigation exposed that the utmost potent compound displayed the significant interactions pattern within the active region of the tyrosinase enzyme and which might be responsible for the decent inhibitory activity towards the enzyme. A molecular dynamic simulation experiment was presented to recognize the residual backbone stability of protein structure.

Full text of DOI:10.1016/j.bmc.2021.116222

Bioorg. Med. Chem. 41 (2021) 116222
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 1,3,4-oxadiazole compounds inhibit the tyrosinase and melanin
level: Synthesis, in-vitro, and in-silico studies
Balasaheb D. Vanjarea, Nam Gyu Choia, Prasad G. Mahajanb, Hussain Razac,
,
*
Mubashir Hassand, Yohan Hanc, Seon-Mi Yuc, Song Ja Kimc, Sung-Yum Seoc, Ki Hwan Leea
a Dept. of Chemistry, Kongju National University, Gongju, Chungnam 32588, Republic of Korea
b Vidya Pratishthan’s Arts, Science & Commerce College, Vidyanagari, Baramati, Maharashtra 413133, India
c Department of Biological Science, Kongju National University, Gongju, Chungnam 32588, Republic of Korea
d Institute of Molecular Biology and Biotechnology, The University of Lahore, 54590, Pakistan
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this research work, we have designed and synthesized some biologically useful of 1,3,4-Oxadiazoles. The
structural interpretation of the synthesized compounds has been validated by using FT-IR, LC-MS, HRMS, 1H
NMR and 13C NMR techniques. Moreover, the in-vitro mushroom tyrosinase inhibitory potential of the target
compounds was assessed. The in-vitro study reveals that, all compounds demonstrate an excellent tyrosinase
1,3,4-oxadiazole
Cytotoxicity
Dynamic simulation
Melanin content
Molecular docking
Tyrosinase inhibitor
inhibitory activity. Especially, 2-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-phenylacetamide (IC50
=
0.003 ± 0.00 µM) confirms much more significant potent inhibition activity compared with standard drug kojic
acid (IC50 = 16.83 ± 1.16 µM). Subsequently, the most potent five oxadiazole compounds were screened for
cytotoxicity study against B16F10 melanoma cells using an MTT assay method. The survival rate for the most
potent compound was more pleasant than other compounds. Furthermore, the western blot results proved that
the most potent compound considerably decreased the expression level of tyrosinase at 50 µM (P < 0.05). The
molecular docking investigation exposed that the utmost potent compound displayed the significant interactions
pattern within the active region of the tyrosinase enzyme and which might be responsible for the decent
inhibitory activity towards the enzyme. A molecular dynamic simulation experiment was presented to recognize
the residual backbone stability of protein structure.
1. Introduction
tyrosinase inhibitor which can regulate the melanin synthesis process.
Hence, it is imperative to synthesize and development of tyrosinase in-
Tyrosinase (EC 1.14.18.1) is a copper encompassing metalloenzyme
plays an influential part in the biosynthesis of melanin from tyrosine
with the help of melanogenesis practice.1 It is mostly found in animals,
microorganism territories and plants. The melanogenesis process was
carried out in two steps in which tyrosinase act as a catalyst.2 Initially,
the ortho diphenol (O-diphenol) conversion takes place from mono-
phenol by hydroxylation. Secondly, the ortho diphenol gets converted
into ortho quinone (O-quinone) by oxidation process and the established
quinone product is extremely reactive species for the synthesis of the
melanin pigments.3,4 The formation of melanin in skin symbolizes the
major protection action against exposure to UV radiation, excessive
melanin formation can affect other skin diseases including solar lentigo,
cancer, melasma, inflammatory melanoderma, flecks, age spots and
ephelis.5,6 To defeat this challenge, there is a necessity of a decent
hibitor that can regulate the synthesis of melanin in the human body.
Over recent decades, there have been findings of numerous tyrosinase
inhibitors, but a few display adequate strength and reliability in the use
in medicinal and food industries. In this perspective, heterocyclic com-
pounds for instance, triazole, triazolothiadiazole, thiazole, sulfonamides
and quinolines tender attracting class of tyrosinase inhibitors. But
among all tyrosinase inhibitors, azole framework has been described by
high potency.7–10
Oxadiazole is a group of the heterocyclic compound belongs to the
azole family. It holds two carbon, two nitrogen and one oxygen atom
associated together within a five-membered ring. It exists in four forms
(isomers) namely, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole
and 1,3,4-oxadiazole, respectively which is shown in Fig. 1.11 Among
all, 1,3,4-oxadiazole has developed a vital creation pattern for the
* Corresponding author.
E-mail address: khlee@kongju.ac.kr (K.H. Lee).
https://doi.org/10.1016/j.bmc.2021.116222
Received 18 November 2020; Received in revised form 12 May 2021; Accepted 13 May 2021
Available online 21 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
expansion of the novel drugs and have more ability to undergo a
different type of chemical reactions which made them more important
for the development of the new molecule.12 Therefore, it has been
spotted that different azoles and its derivatives have earned additional
appreciation for their bioactive nature.13,14 Fig. 2 illustrates several
leading clinical drugs which holds a 1,3,4-oxadiazole core structure.
Likewise, compounds comprising oxadiazole core demonstrating a
broad range of biological activity for instance, anti-inflammatory,
antifungal, antiviral, an alkaline phosphatase inhibitor, antihyperten-
sive, anticonvulsant, antidiabetic, anticancer, and tyrosinase inhibition
properties.15–18 Additionally, oxadiazole acquires significant compe-
tence as a tyrosinase inhibitor and several of them examined oxadiazole
correspondents have been proven to be more effective inhibitors when
an associated with standard drugs.19
In this research work, we report the multi-step synthesis of hybrid
molecules, spectroscopic characterization, mushroom tyrosinase inhi-
bition assay, molecular docking, and dynamic simulation studies of
novel 1,3,4-oxadiazole comprising scaffolds. Molecular modelling and
enzyme inhibition studies revealed a terrific association with the
experimental findings.
Fig. 2. Drugs with 1,3,4-oxadiazole core structure.
hydrazine hydrate in ethanol at reflux condition, the substituted hy-
drazide compounds 3(a-c) were afforded with 70–80% yield. Later, un/
substituted 5-phenyl-1,3,4-oxadiazole-2-thiol compounds 5(a-c) were
synthesized in two-step reaction. In which the hydrazide compounds 3
(a-c), treated with carbon disulphide (CS2) during the basic condition to
form its open ring type of corresponding hydrazide salt 4(a-c). After-
wards, the hydrazide salt compounds 4(a-c) was taken as such for cyc-
lisation process by treating with water in heating condition to form
corresponding cyclised un/substituted 5-phenyl-1,3,4-oxadiazole-2-
thiol compounds 5(a-c) with 65–75% yield. Additionally, in synthetic
route B, various 2-chloro-N-phenyl-halogen un/substituted acetamide
compounds 8(a-d) were synthesized by following our previously pub-
lished article.2 Finally, the route C signifies the coupling reaction among
the thiol and alpha-chloro-N-phenylacetamide functionality in presence
of weak base potassium carbonate (K2CO3) in DMF to form a corre-
2. Results and discussion
2.1. Chemistry
2.1.1. Design approach, synthesis, and characterization
In this research work, we have systematically synthesized twelve
novel 1,3,4-oxadiazole founded compounds by modifying the several
structural aspects of the previously described tyrosinase inhibitors.20,21
The innovative structural aspects constructed in our objective com-
pounds have been displayed in Fig. 3. From the literature survey, it has
been noticed that Lam et al., and co-workers successfully employed
numerous 3,5-substituted oxadiazole-2-thione compounds as a tyrosi-
nase inhibitor. From the results, it has been observed that the 3-position
nitrogen atom was substituted with diverse cyclic amino groups in a
1,3,4-oxadiazole-2(3H)-thiones which exhibit a good tyrosinase inhibi-
tory activity varies from 0.87 to 1.49 µM, compared with standard drug
kojic acid having IC50 value 9.18 ± 0.28 µM.20 In which, we have
replaced the 3-position substation to the second position, and 5-position
naphthyl group to un/substituted phenyl group in 1,3,4 oxadiazole
framework. Similarly, Bandar et al., and co-workers effectively synthe-
sized some N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-
benzamide/amide compounds as a tyrosinase inhibitor with IC50 value
sponding
un/substituted
N-phenyl-2-(5-phenyl-1,3,4-oxadiazol-2-
ylthio) acetamide compounds with 80–95% yield.
The characterization of the synthesized target compounds 9(a-l) was
accomplished by using 1H NMR, 13C NMR, LC-MS, HRMS, and FT-IR
techniques. The structural elucidation of the compound 9e is disused
in details. The compound 9e was acquired a white solid in 85% yield,
possessing melting point around 138–142 ◦C. The molecular mass of the
synthesized compound was identified by its molecular ion peak at 342.2
(M+1) in its LC-MS spectra. Besides, the number of proton and type of
proton has been evaluated using 1H NMR spectroscopy method. In
which, the highly deshielded peak was observed for an amide proton at δ
10.44 (s, 1H). Also, the aromatic benzoyl group present in compound 9e
was appeared by its different signals at 7.62–7.57 (m, 2H), 7.34 (dd, J =
10.8, 5.1 Hz, 2H) and 7.09 (br.t, J = 7.4 Hz, 1H). Likewise, the numerous
signals of 2-methoxy phenyl unit substituted at 5-position in 1,3,4-oxa-
diazole core were appeared at 7.55 (dt, J = 7.7, 1.3 Hz, 1H), 7.50 (t, J =
7.9 Hz, 1H), 7.45 (dd, J = 2.5, 1.5 Hz, 1H) and 7.20 (ddd, J = 8.0, 2.6,
1.2 Hz, 1H). Apart from the aromatic region, the compound 9e acquire
two signals for the aliphatic region comprising for methoxy and meth-
ylene groups appeared at 3.83 (s, 3H) and 4.36 (s, 2H) respectively. Also,
the carbon skeleton of the synthesized compound 9e was systematically
validated using the 13C NMR spectrum. Compound 9e comprises amide
functionality in which the carbonyl carbon signal has been detected at δ
ranges from 14.01 to > 100 µM when equated with kojic acid (IC50
=
16.22 ± 1.74 µM).21 Later, we have founded on the Bioisosterism
auxiliary assumption, we have unified two distinct scaffolds to establish
another platform which was further utilized for tyrosinase inhibition
study.
The essential 1,3,4-oxadiazole compounds 9(a-l) were synthesized
by following synthetic routes A/B/C as presented in Scheme 1. Firstly, in
synthetic route A, different substituted and unsubstituted benzoic acid
compounds 1(a-c) were converted into its corresponding ester com-
pounds 2(a-c) by the esterification reaction. In which the acid com-
pounds were treated with ethyl alcohol and in presence of concentrated
sulphuric acid. Secondly, the converted phenyl ester compounds 2(a-c)
were converted into hydrazide compounds 3(a-c), by the reaction of
Fig. 1. Four different isomers of oxadiazole.
2

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
Fig. 3. Fragment-originated approach of proposed tyrosinase inhibitors.
Scheme 1. Synthetic routes (A/ B & C), reagents and conditions; i) Ethanol, conc. H2SO4, reflux; ii) hydrazine hydrate, ethanol, reflux; iii) carbon disulphide (CS2),
ethanol, reflux 8 h; iv) water, reflux 4–5 h; v) triethyl amine, dichloromethane, 0–5 ◦C. vi) potassium carbonate, dimethyl formamide, ambient temperature.
165.5 (C-14). Furthermore, the formation of the 4,5-substituted 1,3,4-
oxadiazole framework has been approved by the formation of two
quaternary carbon which is observed at δ 165.3 (C-9) and 163.9 (C-7),
respectively. Similarly, the compound involves two aromatic benzene
rings also contains three quaternary carbon and are their signal
appeared at δ 160.1 (C-4), 118.5 (C-5), and 139.1 (C-17) and the
remaining aromatic signals appeared δ 131.2 (C-2), 129.3 (C-21 & C-
19), 124.6 (C-20), 124.17 (C-1), 119.6 (C-18 & C-22), 119.1 (C-6), 111.6
(C-3) correspondingly. Moreover, the two aliphatic signals for methoxy
and methylene group appeared at 55.9 (C-24) and 37.3 (C-13) present in
respective compound 9e. To validate different functional characteris-
tics, vibrational studies of the molecules were analysed through FT-IR
spectroscopy. The significant bands appeared in IR spectrum at υ 3268
2
3
–
–
(Ar-NHCO-), 3057 (aromatic SP C H), 2927 (SP C H), 1660 (Ar-NH-
3
–
–
CO-), 1601, 1547 (aromatic C C Bending), 1443 (SP C H, bending),
–
1176 (Ar-O-CH3); 704.
The different substituents attached to the synthesized 1,3,4-oxadia-
zole compounds 9(a-l) are listed in Table 1.
2.1.2. In-Vitro tyrosinase activity and structural activity relationship (SAR)
Herein, the synthesized novel heterocyclic compounds 9(a-l) were
screened for their mushroom tyrosinase inhibitory capability. All these
compounds 9(a-l) displayed exceptionally good tyrosinase inhibitory
activity ranging from 0.003 ± 0.00 to 0.396 ± 0.01 µM, when
3

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
µM), 9k (IC50 = 0.145 ± 0.09 µM) and 9l (IC50 0.088 ± 0.10 µM)
Table 1
=
Structures of the synthesized 1,3,4-oxadiazole compounds 9(a-l).
compared with 9e-9h (Table 2). It has been observed that, the activity
trend was almost similar for compound 9(e-h) and 9(i-l), but it was
irregular in 9(a-h). It suggests that ortho-substituted strong electron-
donating unit found to be more effective to enhance the inhibition
rate than meta-substituted or unsubstituted compounds at 5-position
substituted phenyl group in an 1,3,4-oxadaizole core. Additionally,
however, oxadiazole compounds 9i, 9j, 9k and 9l holds same func-
tionality expect the substitution at para position with regards to N-
phenyl acetamide functional group. In which 9i possess no substituents
at para position relating to acetamide functionality while 9j, 9k and 9l
comprises fluoro, bromo and iodo substituents. The results suggest that,
in absence of halogen substituent in the oxadiazole compounds presents
excellent interaction pattern with the enzyme but it is practically
opposite in case para substituted halogen atoms.
Sr. No
Compound
-R1
-R2
-X
1
9a
9b
9c
9d
9e
9f
-H
-H
-H
-F
2
-H
-H
3
-H
-H
-Br
-I
4
-H
-H
5
–OCH3
–OCH3
–OCH3
–OCH3
-H
-H
-H
-F
6
-H
7
9g
9h
9i
-H
-Br
-I
8
-H
9
–OCH3
–OCH3
–OCH3
–OCH3
-H
-F
In conclusion, from the structural activity relationship assessment, it
was spotted that ortho and meta-methoxy phenyl group when
substituted at 5-position in 1,3,4-oxadiazole compounds reveals com-
parable activity trend in presence or absence of halogen groups
regarding amide functionality. While, in case of only aromatic benzene
substituted group in 1,3,4-oxadiazole core reveals varying activity style.
Also, it can be asserted from the docking study that (Fig. 8), methoxy
phenyl containing compounds 9(e-l) exhibit good binding energy
pattern than un-substituted methoxy compounds 9(a-d) at 5-position in
1,3,4-oxadiazole core. The activity movement for synthesized 1,3,4-oxa-
diazole compounds 9(a-l) are arranged as per their interaction rela-
tionship with enzyme explored through in-vitro evaluation analysis, for
example 9e>9f>9i>9h>9j>9c>9g>9d>9l>9k>9a>9b (IC50 values
are arranged in Table. 2).
10
11
12
9j
-H
9k
9l
-H
-Br
-I
-H
differentiated with the standard drug kojic acid (IC50 = 16.832 ± 1.16
µM). However, certain compounds involving; 9f, 9h, 9i & 9j, demon-
strate good inhibitory activity. But among the series 9(a-l), compound
9e believed to be an extremely active tyrosinase inhibitor than standard
drug kojic acid. The accomplished inhibitory activity in a molecule is
caused by the participation of the entire molecule, yet a limited SAR
study was simplified by the existence of the different substituents in
corresponding 1,3,4-oxadiazole compounds.
All the target compounds 9(a-l) consist of 2-(1,3,4-oxadiazol-2-
ylthio)-N-phenylacetamide as a fundamental framework. In which the 5-
postion of the 1,3,4-oxadiazole were substituted with various aromatic
phenyl groups for instance, phenyl group in 9(a-d), 2-methoxy phenyl in
9(e-h) and 3-methoxy phenyl in 9(i-l), correspondingly. Also, the 4-po-
sition of the N-phenyl acetamide were unsubstituted or substituted with
different halogens (Such as, -F, -Br and -I) which is shown in Scheme 1.
Furthermore, the target compound 9a (IC50 = 0.231 ± 0.09) acquire no
substituent, while 9b (IC50 = 0.396 ± 0.09), 9c (IC50 = 0.039 ± 0.01)
and 9d (IC50 = 0.085 ± 0.08) hold fluoro, bromo and iodo halogen
atoms as shown in Table 1. All these four compounds 9(a-d) exhibit
virtuous inhibitory activity as matched to the standard drug kojic acid
(IC50 = 16.83 ± 1.16). Moreover, the tyrosinase inhibitory activity for
un-substituted 9a and fluoro substituted derivative 9b was not that
much effective as compared with the compounds bearing bromo 9c and
iodo 9d group at para position concerning to N-phenyl acetamide
functionality. Since, as the size of the halogen atom increases at para-
position, the interaction with enzyme improves as a result pointing
decent inhibitory activity.
2.2. Biology analysis
2.2.1. Kinetic mechanism
Kinetic analysis was accomplished to check the mechanism of most
potent compound 9e, the compound was nominated based on IC50
outcomes. The inhibition kinetics of 9e on the mushroom tyrosinase was
analysed using the Lineweaver-Burk plot. The outcomes are presented in
Fig. 4A, showed that the plots of 1/V versus 1/[S] gave a series of
straight lines which intersected within the second quadrant. These data
showed 9e inhibit the tyrosinase in a non-competitive manner. The Ki
value was obtained from the (Fig. 4B) of slope against the concentrations
of inhibitor. The kinetic results are presented in Table 3. (Kinetic
parameter table).
2.2.2. Effect of compound 9e on the expression of tyrosinase
In order to explore the impact of the compound 9e over tyrosinase
expression, we have organized a western-blot experimental study. In
which the most potent compound 9e were treated with the B16F10 cells
at various concentrations including, 10, 20, 30 and 50 µM indepen-
Also, as presented in Table 1, the 5-position of the 1,3,4-oxadiazole
heterocyclic ring was substituted with the ortho-methoxy phenyl unit
and the N-phenyl acetamide functional group holds no substituent,
fluoro, bromo or iodo groups at para position in respective target com-
pounds 9(e-f). Herein, all the compounds present extremely great in-
hibition rate as equated with compounds 9(a-d). It suggests that the
ortho-substituted methoxy phenyl group present in the respective target
compounds create a good interaction pattern with an enzyme which
causes to exhibit good inhibition rate. Nevertheless, among the ortho-
methoxy substituted compounds (9e, 9d, 9e & 9f), the compound 9e
(IC50 = 0.003 ± 0.00 µM) demonstrate excellent activity because it does
not hold any substituent concerning to the N-phenyl acetamide func-
tionality. Whereas other compounds (9f, 9g and 9h) acquires halogen
unit at para-position indicates reduced inhibitory activity. The result
indicates that, as the halogen size increases the interaction with enzyme
becomes lower, however these findings are opposite in accordance with
the compounds 9(a-d).
dently. Herein,
α
-MSH was employed as a melanogenic inducers.22 The
results are shown in Fig. 5A, designates that α-MSH significantly induces
Table 2
Various substituents present to target compounds 9(a-l) and its IC50 value for
tyrosinase activity. (SEM = Standard error of the mean; values are expressed in
mean ± SEM).
Compound
Tyrosinase activity
Compound
Tyrosinase activity
IC50 SEM (µM)
IC50 SEM (µM)
9a
9b
9c
9d
9e
9f
0.231 ± 0.09
0.396 ± 0.09
0.039 ± 0.01
0.085 ± 0.08
0.003 0.00
0.018 ± 0.08
9g
0.046 ± 0.88
0.023 ± 0.89
0.019 ± 0.00
0.027 ± 0.10
0.145 ± 0.09
0.088 ± 0.10
9h
9i
9j
9k
9l
Similarly, when the inhibitory potential of meta-substituted methoxy
phenyl compounds, 9i (IC50 = 0.019 ± 0.00 µM), 9j (IC50 = 0.027 ± 0.10
Kojic Acid (Standard)
16.832 1.16µM
SEM = Standard error of the mean; values are expressed in mean ± SEM.
4

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
Fig. 4. Lineweaver–Burk plots for the inhibition of tyrosinase in the presence of compound 9e. (A) Concentrations of 9e were 0.00, 0.0017, 0.0033 and 0.0066 µM,
respectively. Substrate L-DOPA concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B) Slope against 9e concentrations. The lines were drawn using
linear least squares fit.
mammalian skin and for protection from UV radiation. Also, it is very
Table 3
well known that α-melanocyte stimulating hormone (α-MSH) primarily
Kinetic parameters of 9e on mushroom tyrosinase inhibition for L-DOPA activity.
utilized for the activation of the melanogenesis process.23–25 Hence, we
Concentration
Vmax (ΔA
Km
Inhibition Type
Ki (µM)
have utilized α-MSH as a melanogenic inducer. Furthermore, to recog-
(µM)
/Sec)
(mM)
nize the content of the melanin in B16F10 cells, initially, the cells were
preserved with -MSH followed by a co-treatment with an 1,3,4-oxadia-
0.00
5.818 × 10-6
4.970 × 10-6
3.636 × 10-6
2.273 × 10-6
0.25
0.25
0.25
0.25
Non-
0.0031
α
0.0017
0.0033
0.0066
Competitive
zole compound 9e at distinct concentrations comprising 10, 20, 30, and
50 µM, respectively. The prepared samples were incubated with a period
of 24 h. Afterwards, the absorbance (optical density) was measured for
each sample at 470 nm, to determine the melanin formation inhibitory
impact of the compound 9e. As presented in Fig. 6, it exposes that
compound 9e, passionately and effectively diminishes the content of the
melanin as equated with the control. The oxadiazole derivative 9e de-
creases the melanin contents to 16%, 62%, 77% and 78% at 10, 20, 30
Vmax is the reaction velocity, Km is the Michaelis-Menten constant, Ki is the EI
dissociation constant.
the tyrosinase expression as equated with the control. Also, when the
compound 9e incubated with α-MSH at 10 µM, the tyrosinase expression
was slightly decreased. Nevertheless, a drastic shift was detected after
the concentration was raised between 20 and 30 µM and then relatively
stable at 50 µM as shown in Fig. 5B. These results exhibit that 1,3,4-oxa-
and 50 µM concentrations relating to the α-MSH. Hence, it suggests that
compound 9e sharply reduces the melanin content present in α-MSH
stimulated in B16F10 cells in a concentration dependant approach.
diazole compound 9e drastically diminished α-MSH stimulated melanin
synthesis.
2.2.4. Cell viability
To develop a skin lighting agent or compound, it must have to
accomplish the criteria that, it should be harmless to consume without
any adverse effects. At present, there are several previously registered
2.2.3. Melanin contents assay of compound 9e
Melanin plays an important role in determining the colour of the
Fig. 5. The effects of compound 9e on the
α-MSH induced tyrosinase expression in B16F10 cells. Cells were exposed with or without α-MSH in the presence of
different concentration of compound 9e (10, 20, 30 & 50 µM) for 24 h. Tyrosinase expressions were determined by western-blot analysis using GAPDH as loading
control (A) α-MSH increased the expression of tyrosinase in comparison to normal control (B) Compound 9e reduced the α-MSH induced expression of tyrosinase. The
asterisks signify the considerable variation among the columns: *P < 0.05; and #P < 0.001.
5

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
could be treated as outstanding tyrosinase inhibitor in the vicinity of the
pharmaceutical chemistry for the improvement of the new drug.
2.3. In-Silico analysis
2.3.1. Mushroom tyrosinase structural assessment
Mushroom tyrosinase, a copper-containing enzyme comprises 391
residues.27 The detailed structure analysis target protein showed that it
consists of 39%
α-helices, 14% β-sheets and 46% coils. The X-ray
diffraction study confirmed its resolution 2.78 Å, R-value 0.238 and unit
cell crystal dimensions such as coordinates length and angles. The
Ramachandran plots and values indicated that 95.90% of protein resi-
dues were present in the favoured region and 100.0% residues were lie
in the allowed region showed in Fig. S73. The Ramachandran graph
values showed the good accuracy of phi (φ) and psi (ψ) angles among the
coordinates of receptor and most of the residues were plunged in
acceptable region.
2.3.2. Molecular docking analyses
The best way to study the binding conformation of ligands against
target protein is through molecular docking analysis and which was
conducted by following our previously reported articles with little
modifications.2,6,24,28–31 The docked complexes of the synthesized
compounds 9(a-l) against tyrosinase were analysed based on lowest
binding energy values (Kcal/mol) and hydrogen/hydrophobic interac-
tion pattern. Results showed that all the ligands 9(a-l), exhibited good
docking energy values and showed their interaction within active region
of target protein (Fig. 8). The docking energy values of all the docking
Fig. 6. The inhibitory effect of 9e in B16F10 cells. The melanin content of the
human B16F10 cells treated with different concentrations of 9e (10, 20, 30 &
#
50 µM). Data are presented as mean ± SD; n = 3; * P < 0.05; P < 0.001;
compared with the control groups.
skin whitening agents (tyrosinase inhibitors) are accessible in the mar-
ket including, hydroquinone, PTU, arbutin and kojic acid.2,26 Even
though, most of the pharmaceutical companies and research institutes
are engaged to synthesize and develop various tyrosinase inhibitors.
Herein, we have synthesized and developed twelve novel tyrosinase
inhibitors which show decent inhibitory activity. However, amongst all,
we have selected the first five potent compounds (9e, 9f, 9h, 9i & 9j) for
the cell viability analysis. The cell viability study was conducted by
following our previously published article. In which the cytotoxicity
study was conducted using a MTT assay method on B16F10 cells.
Initially, the B16F10 cells were cultivated in most five potent com-
pounds (9e, 9f, 9h, 9i & 9j) for 24 h (hrs) at various concentrations
including 1, 5, 10, 20, 30, 40 and 50 µg/mL using DMSO as a solvent and
the cell viability was determined. From cell viability results (Fig. 7)
demonstrate compounds 9e, 9f & 9h shows cell viability more than 80%
even at high concentration 50 µg/mL. But compound 9i & 9j show
nontoxic behaviour up to 10 µg/mL but afterwards it acted as toxic to the
B16F10 cells. Therefore, among all compounds compound 9e demon-
strates most promising potent inhibitory activity as well as exhibit good
survival rate against B16F10 cells. Therefore, in future compound 9e
Fig. 8. Energy graph of synthesized compounds.
Fig. 7. Cell viability measurement employing MTT assay method. Cells were untreated (control) or treated with most potent compounds 9e, 9f, 9h, 9i & 9j at
various concentrations (1, 5, 10, 20, 30, 40, 50 µg/mL) in DMSO for 24 h. (*P < 0.05).
6

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
complexes was calculated by using equation (1).
region residues of mushroom tyrosinase. The docking results showed
that 9e builds single hydrogen bond with His244 and one hydrophobic
interaction with Met280, respectively. The carbonyl oxygen atom from a
single hydrogen bond with His244 having good binding distance 1.88 Å.
Similarly, methyl group was involved with Met280 by hydrophobic
interaction with bond length 3.32 Å. Our incorporated functional moiety
directly indulges with functional residues of target protein and have
strong correlation with in-vitro results. Literature study also justified that
these binding pocket residues are significant in downstream signaling
pathways.4,27,33 The graphical depiction of 9e docking complex is
mentioned in Fig. 10 and all other complexes in Supplementary data
Figs. S61–S72.
ΔG binding = ΔG gauss + ΔG repulsion + ΔG hbond + ΔG hydrophobic
+ ΔG tors
(1)
Here, ΔG gauss: attractive term for dispersion of two gaussian
functions, ΔGrepulsion: square of the distance if closer than a threshold
value, ΔGhbond: ramp function also used for interactions with metal
ions, ΔGhydrophobic: ramp function, ΔGtors: proportional to the
number of rotatable bonds. In docking energy results compounds
selected as best having more than 2.5 kcal/mol energy value different
compared to other compounds. The standard error for Auto dock is re-
ported as 2.5 kcal/mol.32 Present docking results justified that the en-
ergy value difference among all docking complexes were lower than
standard error value. Therefore, based on both in-vitro and in-silico
docking energy results, 9e was ranked as best ligand which showed good
inhibitory potential against targeted enzyme as compared with other
compounds. Also, the binding affinity of the standard drug kojic acid
(-5.7 kcal/mol) was equated with the binding affinities of all the syn-
thesized compounds, in which it has been noticed that all the target
compounds exhibit respectable binding energy than kojic acid.24
Although, the basic nucleus of all the synthesized compounds were the
same, therefore most of the ligands possess good efficient energy values
and have no big energy fluctuations difference.
2.3.5. Root mean square deviation and fluctuation analysis
The RMSD results showed that protein backbone deviation and
fluctuations behaviour in the simulation time frame from 0 to 5000 ps.
The 9e docked complex (blue) graph line depicted good stable and
steady behaviour throughout the simulation period (Fig. 11). Initially,
graph line showed increasing fluctuations to attain stability with RMSD
value range form 0.1–0.27 nm from simulation time 0–500 ps. From
time 500–1000 ps, 9e graph line remained static having stable RMSD
value 0.15 nm. From 1000 to 5000 ps docked complexes showed similar
stable behaviour with minute fluctuations pattern. The observed results
showed that our complex showed good stable behaviour after binding
with newly synthesized ligands.
Also, Fig. 12 showed the stability of 9e docking structure dynami-
cally fluctuated from residues N to C terminus. The fluctuation peaks
within loops region were observed at both terminus N and C-terminal
regions.
2.3.3. Binding pocket and ligands binding conformations
The binding pocket analysis showed that 9e were confined in the
active region of target protein nearby copper metal. All the docked
structures were superimposed to check the binding configuration of all
ligands in the active region of mushroom tyrosinase. Results showed that
the synthesized ligands were bind in the binding pocket having at least
similar conformational pattern. All ligands showed little deviation
around their axis in configuration shape. The aromatic benzene ring and
an oxygen atom of the carbonyl functionality appear in target compound
9e proved their binding pattern in the opening gate of binding pocket.
Whereas the presence of different incorporated functional moiety
showed their attachment inside the binding pocket near the copper
metal (Fig. 9A, B). The 9e-docking complex showed that incorporated
functional group showed its penetration inside the binding pocket and
may have potential to binds with active site residues of target protein.
This incorporation may result in suitable configuration and conforma-
tion to ligands to be fitted in the binding pocket of mushroom tyrosinase.
2.3.6. Radius of gyration and solvent accessible surface area analyses
The structural compactness in protein docking complex was
observed by radius of gyration (Rg). The stably folded proteins show the
relatively steady value of Rg in equilibrium state whereas, in static phase
it remains stable with little fluctuations and Rg valued also remained
stable throughout the simulation time. The generated results depicted
that Rg graph showed downward trend from 0 to 1000 ps with Rg value
ranged from 2.0 to 2.04 nm. After that Rg graph line remained stable
from 1000 to 4000 ps with constant Rg value 2 nm. From 4000 to 5000
ps again little fluctuation was observed, however, the Rg value remained
stable in the simulation period (Fig. 13). The solvent-accessible surface
areas (SASA) were also observed and shown in (Fig. 14). Results showed
that the SASA values of 9e docking complex were centered on 205–210
nm in the simulation time 0–5000 ps.
2.3.4. Hydrogen and hydrophobic binding interaction between 9e and
target protein
The binding interaction showed that 9e directly binds with active
Fig. 9. A, B. Binding of ligands within active region of target protein. The receptor molecule is highlighted in grey colour in surface format. However, ligand is
justified in purple colour.
7

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
Fig. 10. Molecular docking interaction of 9e with receptor molecule. A) The protein structure is represented in khaki and red colours in ribbon format while ligand is
highlighted purple colour while their functional groups such as oxygen, amino and sulfur are showed in red, blue, and yellow colours, respectively. B) The interactive
residues are justified in green colour whereas, amino acids involve in hydrogen bonds and hydrophobic interactions are labelled in red and dark blue, respectively.
The dotted lines represent bond distances in angstrom (Å). Two copper ions are represented in light cyan circles.
Fig. 11. RMSD graph of compound 9e.
Fig. 13. Rg graph of compound 9e.
Fig. 12. RMSF graph of compound 9e.
composites, which are converted into un/substituted 5-phenyl-1,3,4-
oxadiazole-2-thiol molecule by employing multistep chemical re-
actions and ultimately it coupled with 2-chloro-N-phenyl-halogen un/
substituted acetamide compounds to offer a target compounds 9(a-l)
with good yields. The structural elucidation of the target compounds
was carried out via FT-IR, LC-MS, HRMS, 1H NMR and 13C NMR
3. Conclusion
To conclude, we have designed and synthesized twelve novel 1,3,4-
oxadiazole compounds through multistep reaction pathway. The syn-
thesis of the target compounds was initiated with different aromatic acid
8

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
Fig. 14. SASA graph of compound 9e.
practices. Furthermore, all the 1,3,4-oxadiazole compounds were eval-
uated against mushroom tyrosinase activity. The results showed that, all
compounds 9(a-l) present good inhibitory activity against mushroom
tyrosinase enzyme than standard drug kojic acid. Mainly, compound 9e
seems to be extremely effective throughout the series. The inhibition
kinetics for most potent compound 9e was analysed by using
Lineweaver-Burk plots and is found to be non-competitive. Subse-
quently, cell-based experiments were carried out on B16F10 melanoma
cells, the results reveal that compound 9e is non-toxic even at high
concentration (0–50 µM) and confirms exceptional anti-melanogenic
effect validated using western blot findings. Additionally, the most
effective compound 9e passionately associates with essential amino acid
for example Met 280 and His 244 was proven by docking analysis. Also,
the molecular dynamic simulation experiment was performed to
recognize the residual backbone stability of protein structure. Overall,
one can be concluded from the entire study that, all the synthesized
novel 1,3,4-oxadiazole compounds were considered as a good thera-
peutic agent. Though, among the series compound 9e served as a potent
tyrosinase inhibitor and hence in future, it could be employed as a great
contender of the drug in a medicinal field for the expansion of the new
medication.
4.1.2. General synthetic procedure for the key intermediate 5(a-c):
The intermediate 5(a-c) were synthesized by two step reaction.
Firstly, to a 100 mL flask was added the intermediate compound 3(a-b)
(1 mmol) and milled potassium hydroxide (1.1 mmol) in ethanol (5 mL),
stirred for 10 min under nitrogen atmosphere. Later, slowly added car-
bon disulphide (2 mmol) to the reaction mixture, solid will observed (if
necessary, add ethanol) and heated to reflux for 4–5 h. The reaction
progress was detected by using TLC technique. After completion of the
reaction, the reaction mass was cooled to room temperature and
concentrated under reduced pressure to afford a crude solid 4(a-d). The
obtained crude solid was taken as such for further step.
The obtained crude intermediate 4(a-d) was taken in water and
heated to reflux at 70–80 ◦C for 7–8 h. After completion of the reaction,
the reaction mixture was gradually brought to room temperature fol-
lowed cooled to 0 to 5 ◦C. Then neutralised with 10% hydrochloric acid
solution, the precipitated solid was filtered, and several times washed
with cold water. The obtained solid was dried and used as it is for next
step.
4.1.3. General procedure for the synthesis of the target compounds 9(a-l):
As displayed in Scheme 1, an intermediate compound 5(a-l) (1
mmol) and potassium carbonate (1.5 mmol) in dimethylformamide
(DMF) were stirred under nitrogen atmosphere at room temperature for
15–20 min. Afterwards, added intermediate 8(a-d) (1 mmol) to the
above reaction mixture and stirred for 4–5 h. After completion of the
reaction, ice cold water was added to the reaction mixture and stirred for
20 min (solid was obtained). The obtained solid was filtered and several
times washed with cold water and dried. The acquired crude solid was
purified by column chromatography skill, when eluting with the hexane
and ethyl acetate as a solvents to afford a pure 1,3,4-oxadiazole com-
pounds 9(a-l) with an excellent yield.
4. Materials and methods
4.1. Chemistry
The required chemicals were purchased from Sigma Aldrich
(Munich, Germany) and Samchun chemicals (Daejeon, South Korea) and
used without purification. 1H NMR and 13C NMR spectra were taken in
(CD3)2SO at 400 & 100 MHz spectrophotometers, by using a Bruker
Avance (Germany). The chemical shift and coupling constant (J) values
mentioned in ppm and Hz, respectively. The mass analysis (LC-MS) was
recorded on 2795/ZQ2000 (waters) spectrometer and HRMS spectra
was recorded on UHPLC/High Resolution Mass Spectrometer, Model:
1290 infinity II/ Triple TOF 5600 plus (USA). The FT-IR spectra were
recorded on Frontier IR spectrophotometer (Perkin Elmer, USA).
Melting points of the target compounds were determined in open
capillary tubes and are uncorrected (Pittsburgh, PA, and USA). The
melting points of the compounds 9(a-l) were determined by using Fisher
Scientific (USA) melting point instrument and are uncorrected. The
improvement of the reactions was monitored by a thin layer chroma-
tography (TLC) technique.
4.1.3.1. Synthesis of the N-phenyl-2-(5-phenyl-1,3,4-oxadiazol-2-ylthio)
acetamide (9a). White solid; isolated yield: 80%; Rf = 0.65 (n-hexane:
ethyl acetate, 1:1); mp-141–145 ◦C. Fig. S1: 1H NMR (400 MHz,
(CD3)2SO) δ = 10.44 (s, 1H), 7.99 – 7.95 (m, 2H), 7.69–7.53 (m, 5H),
7.37–7.30 (m, 2H), 7.12 – 7.06 (m, 1H), 4.36 (s, 2H). Fig. S2: 13C NMR
(100 MHz, (CD3)2SO) δ = 165.5, 165.3, 163.8, 139.0, 132.4, 129.8,
129.33, 126.8, 124.14, 123.4, 119.5 and 37.3. Fig. S3: IR (KBr) cmꢀ 1
:
2
3
–
–
3267 (Ar-NHCO-), 2983 (SP C H), 2927 (SP C H), 1681 (Ar-NH-CO-
3
–
–
), 1600, 1553 (aromatic C C bending), 1474 (SP C H, bending), 1197
–
(Ar-O-CH3,) 705. Fig. S4: LC-MS: 312 m/z. Fig. S5: HRMS (m/z):
calculated (312.0801, M+H); observed (312.0805, M+1).
4.1.1. General synthetic procedure for the key intermediate (2a-2 g), (3a-
3b) and (8a-8d):
4.1.3.2. Synthesis of the N-(4-fluorophenyl)-2-(5-phenyl-1,3,4-oxadiazol-
The intermediate 2(a-b), 3(a-b) and 8(a-d) were prepared by
2-ylthio) acetamide (9b). Off-white solid; isolated yield: 82%; Rf = 0.61
following previously reported method.2,34
1
(n-hexane: ethyl acetate, 1:1); mp-137–141 ◦C. Fig. S6: H NMR (400
MHz, (CD3)2SO) δ = 10.50 (s, 1H), 8.01 – 7.91 (m, 1H), 7.80 (d, J = 7.4
9

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
Hz, 1H), 7.69 – 7.52 (m, 3H), 7.52 – 7.30 (m, 3H), 7.22 – 7.11 (m, 1H),
4.19 (s, 2H). Fig. S7: 13C NMR (150 MHz, (CD3)2SO) δ = 165.1, 164.8,
163.3, 158.9, 157.3, 135.0, 135.0, 132.0, 129.4, 126.3, 122.9, 121.0,
120.9, 115.9, 115.5, 115.3 & 36.6. Fig. S8: IR (KBr) cmꢀ 1: 3269 (Ar-
0.69 (n-hexane: ethyl acetate, 1:1); mp-144–149 ◦C. Fig. S31: 1H NMR
(400 MHz, (CD3)2SO) δ = 10.58 (s, 1H), 7.59 – 7.57 (m, 1H), 7.56 – 7.54
(m, 1H), 7.53 (s, 2H), 7.52 – 7.47 (m, 2H), 7.46 – 7.43 (m, 1H), 7.23 –
7.18 (m, 1H), 4.36 (s, 2H), 3.83 (s, 3H). Fig. S32: 13C NMR (100 MHz,
(CD3)2SO) δ = 165.6, 165.5, 163.8, 160.1, 138.4, 132.1, 131.2, 124.5,
2
3
–
–
NHCO-), 3140 (aromatic SP C H), 2926 (SP C H), 1676 (Ar-NH-CO-
3
), 1600, 1552 (aromatic C C bending), 1473 (SP C H, bending), 1196
121.5, 119.1, 118.5, 115.7, 111.6, 55.9, 37.2. Fig. S33: IR (KBr) cmꢀ 1
:
–
–
–
2
3
–
–
(Ar-O-CH3); 705. Fig. S9: LC-MS: 330 m/z. Fig. S10: HRMS (m/z):
3266 (Ar-NHCO-), 3059 (aromatic SP C H), 2926 (SP C H), 1681
3
–
–
C H,
calculated (330.0707, M+H); observed (330.0703, M+1).
(Ar-NH-CO-), 1601, 1552 (aromatic C C bending), 1443 (SP
–
bending), 1177 (Ar-O-CH3), & 705. Fig. S34: LC-MS: 420 m/z. Fig. S35:
HRMS (m/z): calculated (421.9992, M+H, 81Br); observed (421.9980,
M+1).
4.1.3.3. Synthesis of N-(4-bromophenyl)-2-(5-phenyl-1,3,4-oxadiazol-2-
ylthio) acetamide (9c). Grey solid; isolated yield: 81%; Rf 0.63 (n-
=
hexane: ethyl acetate, 1:1); mp-169–173 ◦C. Fig. S11: 1H NMR (400
MHz, (CD3)2SO) δ = 10.58 (s, 1H), 7.96 (d, J = 7.2 Hz, 2H), 7.66 – 7.60
(m, 2H), 7.57 (d, J = 8.7 Hz, 3H), 7.52 (d, J = 8.9 Hz, 2H), 4.35 (s, 2H).
Fig. S12: 13C NMR (100 MHz, (CD3)2SO) δ = 165.6, 163.8, 163.7, 138.5,
132.5, 132.1, 129.8, 126.8, 123.4, 121.5, 115.7, & 37.2. Fig. S13: IR
4.1.3.8. Synthesis of N-(4-iodophenyl)-2-(5-(2-methoxyphenyl)-1,3,4-
oxadiazol-2-ylthio) acetamide (9h). Brown solid; isolated yield: 92%; Rf
= 0.71 (n-hexane: ethyl acetate, 1:1); mp-138–142 ◦C. Fig. S36: 1H NMR
(400 MHz, (CD3)2SO) δ = 10.56 (s, 1H), 7.70 – 7.67 (m, 1H), 7.67 – 7.64
(m, 1H), 7.56 – 7.47 (m, 2H), 7.46 – 7.44 (m, 2H), 7.43 – 7.39 (m, 1H),
7.20 (ddd, J = 7.9, 2.6, 1.4 Hz, 1H), 4.35 (s, 2H), 3.83 (s, 3H). Fig. S37:
13C NMR (150 MHz, (CD3)2SO) δ = 165.0, 165.0, 163.3, 159.6, 138.4,
137.5, 130.7, 124.0, 121.3, 118.6, 118.0, 111.1, 837.2, 55.4 & 36.8.
2
–
3
(KBr) cmꢀ 1: 3322 (Ar-NHCO-), 2982 (aromatic SP C H), 2927 (SP
–
–
C
H), 1664 (Ar-NH-CO-),1599, 1527 (aromatic C C bending), 1445
–
3
–
(SP C H, bending), 1198 (Ar-O-CH3); 704. Fig. S14: LC-MS: 392 m/z.
Fig. S15: HRMS (m/z): calculated (391.9886, M+H, 81Br); observed
(391.9880, M+1).
Fig. S38: IR (KBr) cmꢀ 1: 3318 (Ar-NHCO-), 2925 (SP C H), 1662 (Ar-
–
3
–
–
–
C H,
NH-CO-), 1590, 1557 (aromatic C C bending), 1466 (SP
bending), 1178 (Ar-O-CH3); & 722. Fig. S39: LC-MS: 468 m/z. Fig. S40:
4.1.3.4. Synthesis of N-(4-iodophenyl)-2-(5-phenyl-1,3,4-oxadiazol-2-
ylthio) acetamide (9d). Brown solid; isolated yield: 88%; Rf = 0.65 (n-
hexane: ethyl acetate, 1:1); mp-152–156 ◦C. Fig. S16: 1H NMR (400
MHz, (CD3)2SO) δ = 10.54 (s, 1H), 7.98 – 7.94 (m, 1H), 7.90 (dd, J =
10.3, 5.1 Hz, 1H), 7.80 (d, J = 7.3 Hz, 1H), 7.75 – 7.63 (m, 2H), 7.58
(ddd, J = 12.4, 11.5, 6.9 Hz, 2H), 7.51 – 7.37 (m, 2H), 4.35 (s, 2H).
Fig. S17: 13C NMR (150 MHz, (CD3)2SO) δ = 165.1, 165.1, 163.3, 138.4,
137.5, 132.0, 129.4, 126.3, 122.9, 121.3, 87.2 & 36.8. Fig. S18: IR (KBr)
HRMS (m/z): calculated (467.9873, M+H); observed (467.9857, M+1).
4.1.3.9. Synthesis of 2-(5-(3-methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-
N-phenyl acetamide (9i). Off-white solid; isolated yield: 92%; Rf = 0.64
(n-hexane: ethyl acetate, 1:1); mp-175–180 ◦C. Fig. S41: 1H NMR (400
MHz, (CD3)2SO) δ = 10.43 (s, 1H), 7.93 – 7.90 (m, 1H), 7.90 – 7.87 (m,
1H), 7.60 (s, 1H), 7.58 (s, 1H), 7.34 (dd, J = 10.8, 5.0 Hz, 2H), 7.15 –
7.12 (m, 1H), 7.12 – 7.06 (m, 2H), 4.33 (s, 2H), 3.85 (s, 3H). Fig. S42:
13C NMR (100 MHz, (CD3)2SO) δ = 165.5, 165.4, 163.0, 162.5, 139.1,
129.3, 128.7, 124.1, 119.6, 115.7, 115.3, 56.0, & 37.2. Fig. S43: IR
–
2
3
cmꢀ 1: 3323 (Ar-NHCO-), 2981 (aromatic SP C H), 2927 (SP C H),
–
–
3
–
1664 (Ar-NH-CO-),1599, 1527 (aromatic C C bending), 1445 (SP
–
–
C
H, bending), 1198 (Ar-O-CH3); 704. Fig. S19: LC-MS: 438 m/z.
Fig. S20: HRMS (m/z): calculated (437.9768, M+H); observed
(437.9761, M+1).
(KBr) cmꢀ 1: 3307 (Ar-NHCO-), 2927 (SP3
C
H), 1682 (Ar-NH-CO-
3
–
–
),1611, 1534 (aromatic C C bending), 1473 (SP C H, bending), 1172
–
(Ar-O-CH3); & 704. Fig. S44: LC-MS: 342 m/z. Fig. S45: HRMS (m/z):
calculated (342.0907, M+H); observed (342.0901, M+1).
4.1.3.5. Synthesis of 2-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-
N-phenylacetamide (9e). White solid; isolated yield: 88%; Rf = 0.66 (n-
hexane: ethyl acetate, 1:1); mp-138–143 ◦C. Fig. S21: 1H NMR (400
MHz, (CD3)2SO) δ = 10.44 (s, 1H), 7.62 – 7.57 (m, 2H), 7.55 (dt, J = 7.7
Hz, 1.3, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.45 (dd, J = 2.5, 1.5 Hz, 1H), 7.34
(dd, J = 10.8, 5.1 Hz, 2H), 7.20 (ddd, J = 8.0, 2.6, 1.2 Hz 1H), 7.09 (t, J
= 7.4 Hz, 1H), 4.36 (s, 2H), 3.83 (s, 3H). Fig. S22: 13C NMR (100 MHz,
(CD3)2SO) δ = 165.5, 165.3, 163.9, 160.1, 139.1, 131.2, 129.3, 124.6,
4.1.3.10. Synthesis of N-(4-fluorophenyl)-2-(5-(3-methoxyphenyl)-1,3,4-
oxadiazol-2-ylthio) acetamide (9j). Light yellow solid; isolated yield:
90%; Rf = 0.59 (n-hexane: ethyl acetate, 1:1). mp-182–186 ◦C. Fig. S46:
1H NMR (400 MHz, (CD3)2SO) δ = 10.50 (s, 1H), 7.92 – 7.90 (m, 1H),
7.90 – 7.88 (m, 1H), 7.64 – 7.57 (m, 2H), 7.21 – 7.14 (m, 2H), 7.13 (d, J
= 2.0 Hz, 1H), 7.13 – 7.10 (m, 1H), 4.32 (s, 2H), 3.85 (s, 3H). Fig. S47:
13C NMR (100 MHz, (CD3)2SO) δ = 165.5, 165.3, 162.96, 162.5, 128.7,
124.1, 119.6, 119.1, 118.5, 111.6, 55.9, 37.3. Fig. S23: IR (KBr) cmꢀ 1
:
2
3
3268 (Ar-NHCO-), 3057 (aromatic SP C H), 2927 (SP C H), 1660
121.5, 121.4, 116.0, 115.8, 115.3, 56.0, & 37.1. Fig. S48: IR (KBr) cmꢀ 1
:
–
–
3
2
3
–
–
–
–
(Ar-NH-CO-), 1601, 1547 (aromatic C C bending), 1443 (SP
C
H,
3315 (Ar-NHCO-), 3043 (aromatic SP C H), 2930 (SP C H), 1677
–
3
–
–
H,
bending), 1176 (Ar-O-CH3), & 704; Fig. S24: LC-MS: 342 m/z. Fig. S25:
(Ar-NH-CO-),1612, 1541 (aromatic C C bending), 1476 (SP
C
–
HRMS (m/z): calculated (342.0907, M+H); observed (342.0906, M+1).
bending), 1174 (Ar-O-CH3), & 700. Fig. S49: LC-MS: 360 m/z. Fig. S50:
HRMS (m/z): calculated (360.0813, M+H); observed (360.0825, M+1).
4.1.3.6. Synthesis of N-(4-fluorophenyl)-2-(5-(2-methoxyphenyl)-1,3,4-
oxadiazol-2-ylthio) acetamide (9f). Light grey solid; isolated yield: 89%;
Rf = 0.59 (n-hexane: ethyl acetate, 1:1); mp-161–165 ◦C. Fig. S26: 1H
NMR (400 MHz, (CD3)2SO) δ = 10.50 (s, 1H), 7.64 – 7.58 (m, 2H), 7.57 –
7.47 (m, 2H), 7.46 – 7.43 (m, 1H), 7.22 – 7.18 (m, 2H), 7.16 (dd, J = 7.3,
5.1 Hz, 1H), 4.35 (s, 2H), 3.83 (s, 3H). Fig. S27: 13C NMR (100 MHz,
(CD3)2SO) δ = 165.5, 165.3, 163.8, 160.1, 131.2, 124.5, 121.5, 119.1,
118.5, 116.0, 115.8, 111.6, 55.9, & 37.1. Fig. S28: IR (KBr) cmꢀ 1: 3267
4.1.3.11. Synthesis of N-(4-bromophenyl)-2-(5-(3-methoxyphenyl)-1,3,4-
oxadiazol-2-ylthio) acetamide (9k). Light grey solid; isolated yield: 93%;
Rf = 0.61 (n-hexane: ethyl acetate, 1:1); mp-170–175 ◦C. Fig. S51: 1H
NMR (400 MHz, (CD3)2SO) δ = 10.58 (s, 1H), 7.92 – 7.89 (m, 1H), 7.89 –
7.87 (m, 1H), 7.60 – 7.55 (m, 2H), 7.54 – 7.50 (m, 2H), 7.14 – 7.12 (m,
1H), 7.12 – 7.10 (m, 1H), 4.33 (s, 2H), 3.85 (s, 3H). Fig. S52: 13C NMR
(100 MHz, (CD3)2SO) δ = 165.6, 165.6, 162.9, 162.5, 138.4, 132.1,
2
3
(Ar-NHCO-), 3058 (aromatic SP C H), 2927 (SP C H), 1681 (Ar-NH-
128.7, 121.5, 115.7, 115.3, 56.0, & 37.2. Fig. S53: IR (KBr) cmꢀ 1
:
–
–
3
3
–
–
–
CO-), 1601, 1547 (aromatic C C Bending), 1443 (SP C H, bending),
3253.0 (Ar-NHCO-), 2927.8 (SP C H), 1676.9 (Ar-NH-CO-), 1610.0,
–
3
–
–
–
1171 (Ar-O-CH3); 704. Fig. S29: LC-MS: 360 m/z. Fig. S30: HRMS (m/
1544.1 (aromatic C C bending), 1442.3 (SP C H, bending), 1174.2
z): calculated (360.0813, M+H); observed (360.0807, M+1).
(Ar-O-CH3), & 700.0. Fig. S54: LC-MS: 422 m/z. Fig. S55: HRMS (m/z):
calculated (421.9992, M+H, 81Br); observed (422.0010, M+1).
4.1.3.7. Synthesis of N-(4-bromophenyl)-2-(5-(2-methoxyphenyl)-1,3,4-
oxadiazol-2-ylthio) acetamide (9g). Grey solid; isolated yield: 90%; Rf =
10

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
4.1.3.12. Synthesis of N-(4-iodophenyl)-2-(5-(3-methoxyphenyl)-1,3,4-
oxadiazol-2-ylthio) acetamide (9l). Brown solid; isolated yield: 94%; Rf
= 0.64 (n-hexane: ethyl acetate, 1:1); mp-172–176 ◦C. Fig. S56: 1H NMR
(400 MHz, (CD3)2SO) δ = 10.55 (s, 1H), 7.91 – 7.89 (m, 1H), 7.89 – 7.86
(m, 1H), 7.70 – 7.67 (m, 1H), 7.67 – 7.65 (m, 1H), 7.46 – 7.43 (m, 1H),
7.43 – 7.40 (m, 1H), 7.14 – 7.12 (m, 1H), 7.12 – 7.09 (m, 1H), 4.32 (s,
2H), 3.86 (s, 3H). Fig. S57: 13C NMR (100 MHz, (CD3)2SO) δ = 165.6,
conjugated secondary antibody (1:1000) for 2 h at room temperature,
and the bound antibody was visualized using an enhanced chem-
iluminescence solution (Jubiotech, Daejeon, Korea).
4.2.5. Cell viability assay
The cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Cells (0.5 × 105 cells/
well in 96-well plates) were incubated at 37◦ C for 24 h and then MTT
was added with a final concentration of 0.5 mg/mL per well for another
4 h. The reaction product formazan was dissolved in 10% Sodium
dodecyl sulfate (SDS) with 0.01 N HCl. The cell viability was determined
by reading the absorbance at 595 nm by a spectrophotometer (Bio-Rad,
iMark, USA). Results are presented as the mean of three measurements
± standard deviation (n = 3).
165.6, 162.9, 162.5, 138.9, 138.0, 128.7, 121.8, 115.7, 115.3, 56.0, &
3
37.2. Fig. S58: IR (KBr) cmꢀ 1: 3250 (Ar-NHCO-), 2928 (SP C H), 1677
–
3
–
–
–
C H,
(Ar-NH-CO-), 1611, 1539 (aromatic C C bending), 1442 (SP
bending), 1174 (Ar-O-CH3), & 700. Fig. S59: LC-MS: 468 m/z. Fig. S60:
HRMS (m/z): calculated (467.9873, M+H); observed (467.9895, M+1).
4.2. Biology methodology
4.2.6. Statistical analysis
4.2.1. Tyrosinase activity and kinetic analysis
Values are presented as a mean of three different experiments ±
standard deviation (SD). Differences between the calculated means of
each individual group were determined by one-way ANOVA. Any dif-
ference was considered statistically significant at P < 0.05 and P <
0.001.
The anti-tyrosinase activity was performed exactly same as our lab
published protocol.6 In this experiment the kojic acid was used as a
standard to compare the results.35 GraphPad Prism 5.0 was used for the
analysis of IC50. All experiments were repeated in triplicate. The % in-
hibition was calculated same as already published equation.33 For the
analysis of kinetic the different concentrations were used for the sub-
strate and inhibitor (L-DOPA = 0.125–2 mM, inhibitor 9e = 0.00,
0.0017, 0.0033 and 0.0066 µM). The inhibition type of ligand against
tyrosinase was analysed by a Lineweaver–Burk plots (1/V). The Ki was
determined from the secondary plot of 1/V against concentrations in-
hibitor. Other all method was followed according to our published
protocol.36
4.3. In-silico methodology
4.3.1. Repossession of mushroom tyrosinase structure from PDB
The crystal structure of mushroom tyrosinase (Agaricus bisporus)
having PDBID: 2Y9X was accessed form the Protein Data Bank (PDB)
(http://www.rcsb.org).2,6 The retrieved protein structure was mini-
mized by using conjugate gradient algorithm and amber force field in
UCSF Chimera 1.10.1.37 The stereo-chemical properties, Ramachandran
graph and values38 of mushroom tyrosinase were assessed by Molpro-
bity server,39 while the Ramachandran graph was generated by Dis-
covery Studio 2.1 Client.40 The protein architecture and statistical
percentage values of helices, beta-sheets, coils, and turns were accessed
by VADAR 1.8.41
4.2.2. Cell culture and treatment of compounds (9e, 9f, 9h, 9i & 9j)
B16F10 cells were obtained from the Korean Cell Line Bank (Seoul,
Korea). Cells were cultured at 37 ◦C under 5% CO2 in DMEM (Sigma, St.
Louis, MO, USA), supplemented with 10% fetal calf serum (FBS, WEL-
GENE, Korea) and 50 unit/mL penicillin (Sigma) and 50 μg/mL strep-
tomycin (Sigma). The media was changed every 2 days. Also, the most
potent target compounds (9e, 9f, 9h, 9i & 9j) dissolved in dimethyl
sulfoxide (DMSO) and further diluted to final concentrations (0, 1, 5, 10,
20, 30, 40, 50 µg/mL). The control groups were administrated with the
same amount of DMSO.
4.3.2. In-silico designing of synthesized compounds and molecular docking
The synthesized ligands 9(a-l) were sketched in ACD/ChemSketch
tool and access in mol format. Furthermore, UCSF Chimera 1.10.1 tool
was employed to energy minimization of each ligand separately having
default parameters such as steepest descent steps 100 with step size 0.02
(Å), conjugate gradient steps 100 with step size 0.02 (Å) and update
interval was fixed at 10. Finally, Gasteiger charges were added using
Dock Prep in ligand structure to obtain the good structure conformation.
Molecular docking experiment was employed on all the synthesized li-
4.2.3. Melanin contents assay
B16F10 cells (3.5 × 104) were cultured in 35 mm dishes at 37 ◦C in
an atmosphere of 5% CO2 for 24 h. After 24 h, cells were harvested and
solubilized in 1 mL of 1 M NaOH containing 10% DMSO for 2 h at 80 ◦C.
After 2 h, cells were centrifuged at 12,000g for 10 min at room tem-
perature and transfer supernatants to 96 well plate. The absorbance of
the supernatants is measured at 470 nm (Molecular Devices, Sunnyvale,
CA, USA).
gands 9(a-l) against α-glucosidase by using PyRx virtual screening tool
with AutoDock VINA Wizard approach.42 The grid box centre values
were adjusted as for X = -2.4528, Y = 21.4728 and Z = -31.9954),
respectively. We have adjusted sufficient grid box size big enough on
biding pocket residues to allow the ligand to move freely in the search
space. The default exhaustiveness value = 8, was adjusted in both
docking to maximize the binding conformational analysis. In all docked
complexes, the ligands conformational poses were keenly observed to
obtain the best docking results. The docked complexes were evaluated
on lowest binding energy (Kcal/mol) values and structure activity
relationship analyses. The graphical depictions of all the docking com-
plexes were carried out using Discovery Studio (2.1.0).
4.2.4. Western blot analysis
Cells were washed three times with cold- phosphate buffered saline
(PBS). Cells were harvested and lysed in ice-cold RIPA lysis buffer
containing 50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride (NaCl),
1% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS) supplemented
with phosphatase inhibitor and protease inhibitor for 30 min, followed
◦
by centrifugation at 12,000 × g for 10 min at 4 C. Cell lysates were
collected, and protein concentrations were determined using the BCA
Protein assay kit (Thermo Fisher Scientific, Waltham, USA). Equivalent
quantities of protein were loaded on a 9% polyacrylamide gel for elec-
trophoresis and subsequently transferred onto 0.22 µm nitrocellulose
membranes (GE Healthcare, MA, USA). The membranes were blocked
with 5% non-fat dried milk dissolved in Tris-buffered saline containing
0.01% Tween-20 (TBST) for 1 hr at room temperature. The blot was then
probed with the following primary antibodies: tyrosinase (1:1000) and
GADPH (1:2000) antibodies at 37 ◦C for 4 h. After washing three times
with TBST, the membranes were incubated with horseradish peroxidase-
4.3.3. Molecular dynamic simulations (MDS)
To understand the residual backbone flexibility of protein structure,
MD simulations experiment was carried out by Groningen Machine for
Chemicals Simulations43 with GROMOS 96 force field.44 The overall
system charge was neutralized by adding ions. The steepest descent
approach (1000 ps) for protein structure was applied for energy mini-
mization. For energy minimization the nsteps = 50,000 were adjusted
with energy step size (emstep) 0.01 value. Particle Mesh Ewald (PME)
11

B.D. Vanjare et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116222
of the Literature from 2000-2012. Molecules 2012;17. http://dx.doi.10.3390
/molecules170910192.
method was employed for energy calculation and for electrostatic and
van der waals interactions; cut-off distance for the short-range VdW
(rvdw) was set to 14 Å, whereas neighbour list (rlist) and nstlist values
were adjusted as 1.0 and 10, respectively, in em.mdp file.45 It permits
the use of the Ewald summation at a computational cost comparable
with that of a simple truncation method of 10 Å or less, and the linear
constraint solver (LINCS).46 Algorithm was used for covalent bond
constraints and the time step was set to 0.002 ps. Finally, the molecular
dynamics simulation was carried out at 1000 ps in md.mdp file.
Different structural evaluations such as root mean square deviations and
fluctuations (RMSD/RMSF), solvent accessible surface areas (SASA) and
radii of gyration (Rg) of back bone residues were analysed through
Xmgrace software (http://plasma-gate.weizmann.ac.il/Grace/) and
UCSF Chimera 1.10.1 software.
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Declaration of Competing Interest
20 Lam KW, Syahida A, Ul-Haq Z, Rahman MBA, Lajis NH. Synthesis and biological
activity of oxadiazole and triazolothiadiazole derivatives as tyrosinase inhibitors.
Bioorg Med Chem Lett. 2010;20:3755–3759. https://doi.org/10.1016/j.
bmcl.2010.04.067.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
21 Bandgar BP, Adsul LK, Chavan HV, et al. Synthesis, biological evaluation, and
molecular docking of N-{3-[3-(9-methyl-9H-carbazol-3-yl)-acryloyl]-phenyl}-
benzamide/amide derivatives as xanthine oxidase and tyrosinase inhibitors. Bioorg
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22 D’Mello SAN, Finlay GJ, Baguley BC, Askarian-Amiri ME. Signaling pathways in
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Acknowledgement
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by the
Ministry of Education (NRF- 2019R1I1A3A01059089).
melanogenic effect in α-MSH-stimulated B16F10 melanoma cells. Eur J Med Chem.
2019;161:78–92. https://doi.org/10.1016/j.ejmech.2018.10.025.
25 Yu JS, Kim AK. Effect of combination of taurine and azelaic acid on
antimelanogenesis in murine melanoma cells. J Biomed Sci. 2010;17:8–11. https://
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Appendix A. Supplementary material
26 Saeedi M, Eslamifar M, Khezri K. Kojic acid applications in cosmetic and
pharmaceutical preparations. Biomed Pharmacother. 2019;110:582–593. https://doi.
org/10.1016/j.biopha.2018.12.006.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116222.
27 Ashraf Z, Rafiq M, Nadeem H, et al. Carvacrol derivatives as mushroom tyrosinase
inhibitors; synthesis, kinetics mechanism and molecular docking studies. PLoS ONE.
2017;12:1–17. https://doi.org/10.1371/journal.pone.0178069.
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13