Synthesis, molecular docking, dynamic simulations, kinetic mechanism, cytotoxicity evaluation of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl} butanamides as tyrosinase and melanin inhibitors: In vitro, in vivo and in silico approaches

Article abstract of DOI:10.1016/j.bioorg.2019.103445

In the current research work, different N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides have been synthesized according to the protocol described in scheme 1. The synthesis was initiated by reacting various substituted anilines (1a-e) with 4-chlorobutanoyl chloride (2) in aqueous basic medium to give various electrophiles, 4-chloro-N-(substituted-phenyl)butanamides (3a-e). These electrophiles were then coupled with 1-[(E)-3-phenyl-2-propenyl]piperazine (4) in polar aprotic medium to attain the targeted N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides (5a-e). The structures of all derivatives were identified and characterized by proton-nuclear magnetic resonance (¹H NMR), carbon-nuclear magnetic resonance (¹³C NMR) and Infra-Red (IR) spectral data along with CHN analysis. The in vitro inhibitory potential of these butanamides was evaluated against Mushroom tyrosinase, whereby all compounds were found to be biologically active. Among them, 5b exhibited highest inhibitory potential with IC₅₀ value of 0.013 ± 0.001 μM. The same compound 5b was also assayed through in vivo approach, and it was explored that it significantly reduced the pigments in zebrafish. The in silico studies were also in agreement with aforesaid results. Moreover, these molecules were profiled for their cytotoxicity through hemolytic activity, and it was found that except 5e, all other compounds showed minimal toxicity. The compound 5a also exhibited comparable results. Hence, some of these compounds might be worthy candidates for the formulation and development of depigmentation drugs with minimum side effects.

Full text of DOI:10.1016/j.bioorg.2019.103445

Journal Pre-proofs
Synthesis, molecular docking, dynamic simulations, kinetic mechanism, cyto-
toxicity evaluation of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propen-
yl]-1-piperazinyl} butanamides as tyrosinase and melanin inhibitors: In vitro,
in vivo and in silico approaches
Hussain Raza, Muhammad Athar Abbasi, Aziz-ur-Rehman, Sabahat Zahra
Siddiqui, Mubashir Hassan, Qamar Abbas, Hansol Hong, Syed Adnan Ali
Shah, Muhammad Shahid, Sung-Yum Seo
PII:
S0045-2068(19)31426-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.103445
YBIOO 103445
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
30 August 2019
29 October 2019
13 November 2019
Please cite this article as: H. Raza, M. Athar Abbasi, Aziz-ur-Rehman, S. Zahra Siddiqui, M. Hassan, Q. Abbas,
H. Hong, S. Adnan Ali Shah, M. Shahid, S-Y. Seo, Synthesis, molecular docking, dynamic simulations, kinetic
mechanism, cytotoxicity evaluation of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}
butanamides as tyrosinase and melanin inhibitors: In vitro, in vivo and in silico approaches, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103445
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Synthesis, molecular docking, dynamic simulations, kinetic mechanism, cytotoxicity
evaluation of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}
butanamides as tyrosinase and melanin inhibitors: In vitro, in vivo and in silico approaches
Hussain Razaa, Muhammad Athar Abbasib,*, Aziz-ur-Rehmanb, Sabahat Zahra Siddiquib,
Mubashir Hassanc, Qamar Abbasd, Hansol Honga, Syed Adnan Ali Shahe, Muhammad Shahidf,
and Sung-Yum Seoa,*
a College of Natural Science, Department of Biological Sciences, Kongju National University,
Gongju, 32588, South Korea.
b Department of Chemistry, Government College University, Lahore-54000, Pakistan.
cInstitute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Lahore-
54000,, Pakistan.
d Department of Physiology, University of Sindh, Jamshoro Pakistan.
e Faculty of Pharmacy and Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns),
Level 9, FF3, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia.
f Department of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan.
*Corresponding Authors: aProf. Dr. Sung-Yum Seo, E-mail: dnalove@kongju.ac.kr Tel: (+82)-
416-8508503 and bProf. Dr. Muhammad Athar Abbasi, E-mail: abbasi@gcu.edu.pk Tel: (+92)-
42-111000010 Ext. 266.
1

Abstract
In the current research work, different N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-
propenyl]-1-piperazinyl}butanamides have been synthesized according to the protocol described
in scheme 1. The synthesis was initiated by reacting various substituted anilines (1a-e) with 4-
chlorobutanoyl chloride (2) in aqueous basic medium to give various electrophiles, 4-chloro-N-
(substituted-phenyl)butanamides (3a-e). These electrophiles were then coupled with 1-[(E)-3-
phenyl-2-propenyl]piperazine (4) in polar aprotic medium to attain the targeted N-(substituted-
phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides (5a-e). The structures of all
derivatives were identified and characterized by proton-nuclear magnetic resonance (1H-NMR),
carbon-nuclear magnetic resonance (13C-NMR) and Infra-Red (IR) spectral data along with CHN
analysis. The in vitro inhibitory potential of these butanamides was evaluated against Mushroom
tyrosinase, whereby all compounds were found to be biologically active. Among them, 5b
exhibited highest inhibitory potential with IC50 value of 0.013 ± 0.001 µM. The same compound
5b was also assayed through in vivo approach, and it was explored that it significantly reduced the
pigments in zebrafish. The in silico studies were also in agreement with aforesaid results.
Moreover, these molecules were profiled for their cytotoxicity through hemolytic activity, and it
was found that except 5e, all other compounds showed minimal toxicity. The compound 5a also
exhibited comparable results. Hence, some of these compounds might be worthy candidates for
the formulation and development of depigmentation drugs with minimum side effects.
Keywords: 1,4-Piperazine, butanamide, tyrosinase inhibitors, depigmentation, cytotoxicity.
2

1. Introduction
The biologically active nitrogen in piperazine ring and amide groups have a great
importance in the therapeutical, pharmaceutical, material sciences, and pesticidal research fields
[1]. Piperazine is an efficient functional moiety in biological and pharmacological investigations.
It has represented its potential as anti-microbial [2], antiviral [3], anticoagulant, and anticancer
agent [4]. It has also manifested insecticidal [5], herbicidal, and fungicidal potential [6] along with
its plant growth regulator activity [7]. The various amide derivatives with heterocyclic rings
showed excellent antibacterial in vitro and in vivo activity. The presence of lipophilic groups, like
methoxy, methyls etc., enhanced the antimicrobial activity [8]. In order to enhance their
therapeutic potential, the various other small functional groups have also been introduced in these
derivatives [8]. Therefore, a large number of derivatives containing heterocyclic piperazine ring
and amide groups have been synthesized which showed a large number of biological activities [9].
Animals, microorganisms and plants distributed copper bound enzyme known as tyrosinase. In
plants and many organisms its reaction sequences monophenols to o-diphenols and oxidation
reaction of ortho-diphenols to ortho-quinones. The tyrosinase enzyme is responsible for the
alteration of texture, test and nutritional requirements of vegetables and fruits. In higher animals,
humans and fungi this enzyme speeds up the transformation of tyrosine into melanin. Melanin is
a pigment which is accountable for skin color and acts as a defense against ultraviolet radiations
[10]. The elevated level of melanin pigment production is accountable for various skin disorders
in females and males such as dark color, acne, melasma and ephelides. Therefore these type of
studies encouraged scientists and researchers to synthesize the bioactive inhibitors of tyrosinase
for the development of depigmentation drugs [11].
In the literature some piperazine or amide containing molecules have been reported as
effective tyrosinase inhibitors [12-14], so the rationale in the present study was to synthesize some
hybrid compounds bearing piperazine and butanamide moieties together in the same skeleton in
order to explore their accumulative therapeutic effect for searching some new drug candidates for
skin disorders.
2. Results and Discussion
3

2.1.
Chemistry
A unique series of piperazine bearing butanamides (5a-e) was synthesized according to a
protocol outlined in Scheme 1. The procedures of synthesis and conditions of reactions are
described in the experimental section. The synthesis of targeted compounds was initiated by
reacting substituted anilines (1a-e) with 4-chlorobutanoyl chloride (2) in the presence of aqueous
alkaline medium under 4-5 hours stirring at room temperature to acquire the respective
electrophiles, 4-chloro-N-(substituted-phenyl)butanamides (3a-e), in amorphous powder forms
with different colors. Then, these electrophiles (3a-e) were coupled, one by one, with a
nucleophilic 1-[(E)-3-phenyl-2-propenyl]piperazine (4) in the presence of acetonitrile and K2CO3
by refluxing the mixture for 4-5 hours. In this way, the targeted compounds, N-(substituted-
phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides (5a-e), were obtained in
1
13
good yields. The structures of these compounds were identified by using IR, H-NMR, and C-
NMR spectral data along with CHN analysis data. The structural assignment of one of the
compounds (5a-e) is described hereby for clear understanding of interpretational decorum of the
synthesized compounds. The molecule 5e, was obtained as light pink amorphous powder in 71%
yield with a melting point of 125-126 oC. The molecular formula, C25H33N3O, of this compound
was predicted with its CHN analysis data. Counting the number of protons in 1H-NMR spectrum
and carbon resonances in its 13C-NMR spectrum was also in agreement with the deduced molecular
formula. The speculated functionalities present in this molecule were affirmed by various
vibrational bands in IR spectrum, at υ 3243 (N-H, str.), 3047 (Ar C-H, str.), 1711 (C=O, str.), 1635
1
(Ar C=C, str.), and 1592 (C=C, str.) cm-1. In its H-NMR spectrum, (Figure 1a), the most
deshielded singlet at  9.23 was rational for N-arylated amidic proton (Ar-NH-CO). A cinnamyl
[(E)-3-phenyl-2-propenyl] moiety was depicted by two resonances, integrated for five protons of
phenyl group, in aromatic region (Figure 1b) appearing at  7.44 (br.d, J = 7.5 Hz, 2H, H-2''' & H-
6'''), and 7.33-7.31 (m, 3H, H-3''', H-4''' & H-5''') along three typical signals of (E)-2-propenyl
moiety at  6.53 (br.d, J = 15.9 Hz, 1H, H-3''), 6.29 (td, J = 6.6, 15.9 Hz, 1H, H-2''), and 3.09 (br.d,
J = 6.3 Hz, 2H, CH2-1''). The 4-ethylphenyl ring attached with nitrogen of acetamido group was
represented by a peculiar A2B2 spin system in aromatic region, signified by two ortho-coupled
doublets at  7.22 (br.d, J = 8.0 Hz, 2H, H-2'''' & H-6''''), and 7.12 (br.d, J = 8.0 Hz, 2H, H-3'''' &
H-5'''') along with symbolic signals for an ethyl group in aliphatic region at  2.58 (q, J = 7.5 Hz,
2H, 4''''-CH2CH3), and 1.11 (t, J = 7.5 Hz, 3H, 4''''-CH2CH3). The proton signals of symmetrical
4

1,4-piperazine heterocycle and one other methylene group were merged and identified through
two resonances at  3.38-3.34 (m, 4H, CH2-3' & CH2-5'), and 2.33-2.25 (m, 6H, CH2-2, CH2-2' &
CH2-6'). The connecting butanamide moiety was characterized by typical peaks in aliphatic region
at  2.48-2.47 (m, 2H, CH2-4), and 1.74 (quint. J = 7.1 Hz, 2H, CH2-3) while the signal of a
methylene (CH2-2) was intermixed with those of piperazine heterocycle as a multiplet, as
mentioned above. All these assignments were verified with its 13C-NMR spectrum, in which the
(E)-3-phenyl-2-propenyl moiety was embodied by four signals of phenyl ring at  137.16 (C-1'''),
129.01 (C-3''' & C-5'''), 127.65 (C-4'''), and 126.58 (C-2''' & C-6'''), in addition to three signals for
a 2-propenyl unit at 132.36 (C-2''), 126.25 (C-3''), and 60.65 (C-1''). 4-Ethylphenyl ring attached
with nitrogen of amido group was deduced by resonances at  138.42 (C-1''''), 136.26 (C-4''''),
128.86 (C-3'''' & C-5'''') for a phenyl ring while 4-ethyl moiety was depicted by two signals at 
24.23 (4''''-CH2CH3), and 14.68 (4''''-CH2CH3). Four methylenes of a symmetrical 1,4-piperazine
ring were unified by two signals at  53.29 (C-3' & C-5') and 53.20 (C-2', & C-6') [15]. The linking
butanamido entity was specified by a downfield amidic carbonyl carbon at  171.76 (NH-CO)
along with three methylene resonances at  57.81 (C-4), 34.17 (C-2), and 23.01 (C-3). Based upon,
these cumulative evidences, the structure of 5e was confirmed and named as N-(4-ethylphenyl)-4-
{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide. A similar strategy was followed for
1
13
structural interpretation of other butamides in the series. The H-NMR and C-NMR spectra of
all compounds are shown in Fig. S5-S14.
5

3"" R1
5'
O
3"
1"
N
5a-e
N
5"'
II
1"'
2"
1""
3'
1'
5""
C
2
N
4
3"'
NH
4
3
N
H
1
R2
R1
1-[(E)-3-Phenyl-2-propenyl]piperazine
R1
H2N
O
C
O
C
I
+
Cl
Cl
Cl
N
H
R2
R2
1a-e
2
3a-e
CH3
CH3
H3C
H3C
O
C
O
N
N
N
C
N
N
N
H
N
H
5b
5a
CH3
CH3
O
C
O
C
N
N
N
N
H
N
H
5d
5c
CH3
O
C
N
N
N
H
5e
Scheme 1. Outline for the synthesis of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-
piperazinyl}butanamides. Reagents & Conditions: (I) Aq. Na2CO3 soln./pH 9-10/stirring at RT
for 4-5 hrs. (II) Acetonitrile/K2CO3/refluxing of 4 for 0.5 hrs for its activation, followed by addition
of respective electrophile, 3a-3e, and then refluxing for 4-5 hrs.
2.2.
Biology
2.2.1. Enzyme inhibition and structure-activity relationship
The synthesized compounds (5a-5e) were screened to corroborate their significance as tyrosinase
enzyme inhibitors. The standard compound used for the screening was kojic acid and the
evaluations of half-maximal inhibitory concentration (IC50) are summarized in Table 1.
Interestingly, all synthesized compounds showed potent inhibition towards mushroom tyrosinase.
Compared to kojic acid, the compound 5b (0.013 ± 0.001) was dominant tyrosinase inhibitors as
compared to other analogs in the series. The presence of two methyl groups at 1 and 4 positions in
N-aryl moiety greatly affected the tyrosinase inhibition. The less sterically hindered substituents
at these positions in N-aryl part probably made this molecule prone to interact strongly and occupy
the pocket of the receptor in a superb manner. The compound 5a was identified as the second most
6

potent inhibitor. Again it was attributed with similar two methyl groups but at 2 and 3 positions in
the N-aryl part, however, this substitution pattern of methyl groups resulted in a very little decrease
in the inhibitory effect, presumably due to the close proximity of these groups. The compound 5e
exhibited the least inhibitory potential comparatively in the synthetic series, probably due to
relatively bulky ethyl group in it.
Table 1
IC50 values of compounds (5a-5e) were calculated by nonlinear regression using GraphPad Prism
5.0.
Compound
Tyrosinase activity Compound
IC50 ± SEM (µM)
Tyrosinase activity
IC50 ± SEM (µM)
0.051 ± 0.037
5a
5b
5c
0.019 ± 0.013
0.013 ± 0.001
0.039 ± 0.006
5d
5e
0.682 ± 0.112
Kojic Acid
16.841 ± 1.146
2.2.2. Kinetic analysis
Depending on our results, the most potent compound 5b was selected to determine the
inhibition type and inhibition constant on tyrosinase. The potential of this compound to inhibit free
enzyme and enzyme-substrate complex was determined 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 different compound’s concentrations gave a series of straight lines (Fig. 1A).
The results of 5b showed that the compound intersected within the second quadrant. The analysis
showed that Vmax decreased with new increasing doses of inhibitors while Km remains the same.
This behavior indicates that 5b inhibit the tyrosinase non-competitively to form the enzyme-
inhibitor complex. The Secondary plot of slope against the concentration of inhibitors showed
enzyme inhibitor dissociation constant (Ki) (Fig. 1B).
The kinetic results are presented in the Table 2. (Kinetic parameter table).
7

Table 2
Kinetic parameters of the mushroom tyrosinase for L-DOPA activity in the presence of various
concentration of 5b.
Concentration
(µM)
Vmax
(ΔA /Sec)
9.16×10-5
Km
(mM)
0.4
Ki
(µM)
Inhibition Type
0.00
4.07×10-5
2.92×10-5
2.45×10-5
0.4
0.4
0.4
Non-Competitive 0.017
0.007
0.013
0.026
Vmax is the reaction velocity, Km is the Michaelis-Menten constant, Ki is the EI dissociation
constant.
Fig 1. Lineweaver–Burk plots for inhibition of tyrosinase in the presence of Compound 5b. (A)
Concentrations of 5b were 0.00, 0.007, 0.013 and 0.026 µM, respectively. Substrate L-DOPA
Concentrations 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 5b concentrations to determine inhibition constant. The lines
were drawn using linear least squares fit.
2.2.3. Cytotoxicity
The cytotoxic potential of all synthesized compounds, 5a-e, was evaluated through
hemolytic assay. The percentage of cell hemolysis values of all synthesized compounds are given
in Table 3. It is evident from the results that all compounds exhibited little toxicity towards red
8

blood cell membranes, compound 5e showed little bit higher hemolysis activity (33.38 ± 0.07%),
as compared to other compounds, but it was much less than the positive control, (Triton-X) having
a value of 99.56 ± 0.02%. So, in general, all of these compounds can be regarded as harmless
therapeutic agents.
Table 3
Cytotoxicity studies of the synthesized amides, 5a-e.
Compound
% Hemolysis
6.73 ± 0.05
3.94 ± 0.03
0.58 ± 0.01
Compound
5d
% Hemolysis
5.42 ± 0.04
5a
5b
5c
5e
33.38 ± 0.07
99.56 ± 0.02
Triton X
Note: PBS (% Hemolysis) = 1.03 ± 0.01.
2.2.4. In vivo depigmentation zebrafish assay
Zebrafish is a vital research model against various diseases, due to gene makeup similarity
with human [11]. Owing to these benefits, the zebrafish seeds were used to determine the
depigmentation efficacy of 5b through in vivo assay. The inhibition efficacy of 5b on the coloring
of zebrafish was investigated with the treatment of 5, 10, 20 and 40 µM of inhibitor 5b and equal
doses were tested for kojic acid. The pigment level of zebrafish significantly decreased P<0.05,
(Fig. 2) to about 53.55% while positive control kojic acid showed 25.53% at 40 µM. Additionally,
inhibitor 5b exhibited good depigmenting effects at 20 µM treatment, relative to those of kojic
acid.
9

Fig 2. Depigmentation efficacy of 5b in zebrafish (A) showed the rapid decrease in fish coloring
from black brown to whitish at various doses (B) Pixel comparison of the depigmenting potency
of 5b and kojic acid. The fish image color intensity was measured using Image J software and
compared using the t-test. *P <0.05.
2.2.5. Molecular docking analysis
2.2.5.1.
Binding energy evaluation of synthesized compounds
To predict the best-fitted conformational position of synthesized ligands (5a-e) within the
active region of the target protein. The generated docked complexes were analyzed based on Glide
docking energy values (kcal/mol) and bonding interaction (hydrogen/hydrophobic) behavior. The
lowest 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 (5a-e)
bound within the active site of the target protein with different conformational poses and energy
values, respectively. The binding pattern of all synthesized compounds showed their similar
conformational behavior within the active region of the target protein (Fig. 3). The compound 5a
exhibited -6.34 (kcal/mol), whereas, 5b, 5c, 5d and 5e possessed -6.20, -5.32, 4.90 and 6.23
kcal/mol, respectively (Fig. 4). The comparative results showed that compounds exhibited good
10

docking energy values. The basic skeleton of all the synthesized compounds was similar therefore,
no big energy value difference was observed in all docking results.
Fig 3. Docking complex of 5a-e within the active region of target protein along with binding
energy values (kcal/mol).
Figure 4. Docking complex of 5a-e within the active region of target protein along with binding
energy values (kcal/mol).
11

2.2.5.2.
Binding analysis of ligands against tyrosinase
Based on in vitro results, the 5b-docking complex was evaluated to understand their
binding conformational analysis within the active site of the target protein. In detail, in silico
docking analysis revealed that three π-π interactions were observed in the 5b-docking complex.
The 3D and 2D depictions of most active compounds 5b has been mentioned in Fig. 5 A, B. The
benzene ring formed π-π interaction with His85, whereas, the other two interactions were observed
at aromatic residues His259 and His263.
Literature data also ensured the importance of these residues in bonding with other tyrosinase
inhibitors which strengthened our docking results [16-18].
Figure 5 (A and B). 3D and 2D docking depictions of 5b complex against tyrosinase.
12

2.2.6. Molecular dynamic simulations
2.2.6.1. Root mean square deviation and fluctuation (RMSD/RMSF) analysis
To evaluate the residual flexibility of the receptor (c-alpha and backbone) through MD
simulation RMSD graph was generated to evaluate the protein structural behavior. The RMSD is
used to measure the average change in displacement of a selection of atoms for a particular frame
with respect to a reference frame. The RMSD graph result of 5b interprets the protein residual
deviation in a 100 ns simulation time frame. Initially, the graph line showed an increasing trend
from 0-40 ns having an RMSD value range from 1.00 and 1.75 Å for backbone residues. The
generated RMSD graph showed little stability with fluctuations from 40 to 60 ns having RMSD
value range from 1.75 -2.00 Å. A Similar trend was observed from 60-100 ns with fluctuations.
The overall RMSD analysis showed that fluctuations in the graph in the whole simulation are
within the standard range of RMSD 1-2.25 Å which showed backbone stability in docking (Fig.
6). The RMSF is useful for characterizing local changes along with the protein structure. The
generated plot indicated protein behavior fluctuations during the simulation in Cα and backbone
residues. The overall results showed that N- and C-terminals loop regions showed little fluctuations
whereas, residues around 170-200 depicted high fluctuations in 100 ns simulation (Fig. 7).
Figure 6. RMSD graph of 5b docking complex at 100 ns.
13

Figure 7. RMSF graph of 5b docking complex at 100 ns.
3. Conclusion
A structurally unique series of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1
piperazinyl}butanamides (5a-e) was synthesized successfully and their structural characterization
was carried through spectral analysis lucidly. The title compounds were screened for their
mushroom tyrosinase inhibitory activity, compound 5b showed excellent activity with 0.013 ±
0.001 µM while IC50 value of standard kojic acid is 16.841 ± 1.146 µM. The computational
molecular docking performed against tyrosinase protein (PDB ID: 2Y9X) showed that 5b which
is bound in active binding site of the protein. The molecular modeling demonstrates that the oxygen
atom of the compound 5b synchronized with the key residues in the energetic site of the enzyme.
Zebrafish result showed the 5b significantly reduced the pigment without harming the zebrafish.
Most of the compounds also displayed mild cytotoxicity. The compound 5a also exhibited
promising results. These results revealed that compounds 5b and 5a could be the lead chemical
entities in developing the most potent tyrosinase inhibitors for the removal of extra pigment from
humans and the prevention of browning in fruits with modest cytotoxicity.
4. Experimental
14

4.1.
4.1.1. Materials and method
All the chemicals, along with analytical grade solvents, were purchased from Sigma
Chemistry
Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates
were used for TLC with ethyl acetate and n-hexane as solvent system. Spots were detected by
UV254. Gallonkamp apparatus was used to detect melting points in capillary tubes. IR spectra (ν,
1
cm–1) were recorded by the KBr pellet method in the Jasco-320-A spectrophotometer. H-NMR
spectra (δ, ppm) were recorded at 600 MHz (13C-NMR spectra, at 150 MHz) in CDCl3 using the
Bruker Advance III 600 As- cend spectrometer using BBO probe. The 1H-NMR spectral peaks for
interpretation are abbreviated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet;
br.t, broad triplet; q, quartet; quint, quintet; sex, sextet; sep, septet; m, multiplet, dist, distorted.
4.1.2. Procedure for the synthesis of 4-Chloro-N-(substituted-phenyl)butanamides (3a-e)
Substituted aniline (1a-e, 8.8 mmol; one in each respective reaction) was suspended in 25
mL distilled water, stirred for 30 minutes followed by the addition of 10% aqueous Na2CO3 to
adjust pH to 9-10. Then, equimolar quantity of 4-chlorobutanoyl chloride (2, 1mL, 8.8 mmol) was
added with vigorous shaking, and the mixture was then set to stirring further for four to five hours
till completion of each respective reaction. Reaction progress was monitored by TLC until single
spot was achieved. During work-up, respective products were precipitated by lowering the pH up
to 2.0 with conc. HCl. The precipitates of these electrophiles, 4-chloro-N-(substituted-
phenyl)butanamides (3a-e) were filtered out, washed with distilled water, and air-dried.
4.1.3. Procedure for the synthesis of N-(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-
piperazinyl}butanamides (5a-e)
1-[(E)-3-Phenyl-2-propenyl]piperazine (4, 0.2 g; 0.98 mmol) was taken in 10 mL
acetonitrile along with a pinch of K2CO3, and the mixture was refluxed for half an hour for
activation of this nucleophile. Then, calculated amount of one of the substituted electrophiles (3a-
e, 0.98 mmol) was added and reaction mixture was again set to reflux further for 4-5 hrs. Reaction
completion was monitored by TLC. After completion, the reaction mixture was poured on crushed
ice and precipitate of respective product was filtered out, washed and air dried. Thus, pure N-
(substituted-phenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamides (5a-e) were
obtained, which were used for further studies.
4.1.4. Structural characterization
15

4.1.4.1.
N-(2,3-Dimethylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-
piperazinyl}butanamide (5a)
Brown amorphous powder; Yield 72%; m.p. 104-105 ; Molecular Formula: C H N O;
℃
25 33
3
Molecular Mass: 391 gmol-1; IR (KBr, υ, cm-1): 3249 (N-H, str.), 3047 (Ar C-H str.), 1715 (C=O,
1
str.), 1630 (Ar C=C, str.), 1586 (C=C, str.); H-NMR (CDCl3, 600 MHz, δ, ppm): 9.29 (s, 1H, -
NH-CO), 7.44 (br.d, J = 7.4 Hz, 2H, H-2''' & H-6'''), 7.32 (br.t, J = 7.4 Hz, 2H, H-3''' & H-5'''),
7.23 (dist.t, J = 7.2 Hz, 1H, H-4'''), 7.11 (br.d, J = 7.6 Hz, 1H, H-6''''), 7.03 (br.t, J = 7.4 Hz, 1H,
H-5''''), 6.99 (br.d, J = 7.26 Hz, 1H, H-4''''), 6.53 (br.d, J = 15.9 Hz, 1H, H-3''); 6.29 (td, J = 6.5,
15.9 Hz, 1H, H-2''), 3.45-3.44 (m, 4H, CH2-3' & CH2-5'), 3.09 (br.d, J = 6.18 Hz, 2H, CH2-1''),
2.48-2.46 (m, 2H, CH2-4), 2.36-2.28 (m, 6H, CH2-2, CH2-2' & CH2-6'), 2.24 (s, 3H, 3''''-CH3), 2.05
13
(s, 3H, 2''''-CH3), 1.74 (quint. J = 6.9 Hz, 2H, CH2-3); C-NMR (150 MHz, CDCl3, δ, ppm):
171.49 (-NH-CO), 137.22 (C-1''''), 137.17 (C-1'''), 132.72 (C-3''''), 132.36 (C-2''), 129.01 (C-3''' &
C-5'''), 127.81 (C-4''''), 127.66 (C-4'''), 127.18 (C-5''''), 126.67 (C-2''''), 126.63 (C-2''' & C-6'''),
125.50 (C-3''), 124.10 (C-6''''), 60.65 (C-1''), 57.84 (C-4), 53.22 (C-3' & C-5'), 53.20 (C-2' & C-
6'), 34.17 (C-2), 23.04 (C-3), 20.58 (C-3''''), 14.46 (C-2''''); Anal. Calc. for C25H33N3O (391.55):
C, 76.61; H, 8.42; N, 10.70. Found: C, 76.59; H, 8.40; N, 10.68.
4.1.4.2.
N-(2,4-Dimethylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-
piperazinyl}butanamide (5b)
Dark brown solid; Yield 85%; m.p. 121-122
; Molecular Formula: C H N O;
℃
25 33
3
Molecular Mass: 391 gmol-1; IR (KBr, υ, cm-1): 3251 (N-H, str.), 3043 (Ar C-H, str.), 1717 (C=O,
str.), 1635 (Ar C=C, str.), 1590 (C=C, str.); 1H-NMR (CDCl3, 600 MHz, δ, ppm): 9.14 (s, 1H, NH-
CO), 7.44 (br.d, J = 7.4 Hz 2H, H-2''' & H-6'''), 7.32 (br.t, J = 7.5 Hz, 2H, H-3''' & H-5'''), 7.23
(br.t, J = 7.4 Hz, 1H, H-4'''), 7.21 (br.d, J = 7.9 Hz, 1H, H-6''''), 7.00 (br.s, 1H, H-3''''), 6.94 (br.d,
J = 7.7 Hz, 1H, H-5''''), 6.53 (br.d, J = 15.9 Hz, 1H, H-3''), 6.29 (td, J = 6.6, 15.9 Hz, 1H, H-2''),
3.38-3.34 (m, 4H, CH2-3' & CH2-5'), 3.09 (br.d, J = 6.4 Hz, 2H, CH2-1''), 2.48-2.46 (m, 2H, CH2-
4), 2.39-2.30 (m, 6H, CH2-2, CH2-2' & CH2-6'), 2.24 (s, 3H, 4''''-CH3), 2.14 (s, 3H, 2''''-CH3), 1.73
(quint. J = 7.14 Hz, 2H, CH2-3); 13C-NMR (150 MHz, CDCl3, δ, ppm): 171.42 (NH-CO), 137.16
(C-1'''), 137.17 (C-1'''), 134.47 (C-1''''), 134.46 (C-2''''), 132.37 (C-2''), 132.17 (C-4''''), 131.15 (C-
5''''), 129.01 (C-3''' & C-5'''), 127.81 (C-3''''), 127.64 (C-4'''), 126.77 (C-6''''), 126.63 (C-2''' & C-
6'''), 125.69 (C-3''), 60.65 (C-1''), 57.84 (C-4), 53.28 (C-3' & C-5'), 53.19 (C-2' & C-6'), 34.20 (C-
16

2), 23.04 (C-3), 20.91 (C-4''''), 18.23 (C-2''''); Anal. Calc. for C25H33N3O (391.55): C, 76.61; H,
8.42; N, 10.70. Found: C, 76.62; H, 8.41; N, 10.71.
4.1.4.3.
N-(3-Methylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide
(5c)
Pink amorphous semi-solid, Molecular Formula: C24H31N3O; Molecular Mass: 377 gmol-
1; IR (KBr, υ, cm-1): 3256 (N-H, str.), 3047 (Ar C-H, str.), 1713 (C=O, str.), 1630 (Ar C=C, str.),
1588 (C=C, str.); 1H-NMR (CDCl3, 600 MHz, δ, ppm): 9.77 (s, 1H, NH-CO), 7.43 (br.d, J = 7.26
Hz 2H, H-2''' & H-6'''), 7.37 (br.d, J = 8.4 Hz, 1H, H-6''''), 7.32 (dist.t, J = 7.8 Hz, 2H, H-3''' & H-
5'''), 7.24 (m, 1H, H-4'''), 7.15 (br.t, J = 7.8 Hz, 1H, H-5''''), 6.84 (br.d, J = 7.3 Hz, 1H, H-4''''), 6.54
(br.d, J = 15.9 Hz, 1H, H-3''); 6.50 (dist.d, J = 3.2 Hz, 1H, H-2''''), 6.29 (td, J = 6.5, 15.9 Hz, 1H,
H-2''), 3.45-3.43 (m, 4H, CH2-3' & CH2-5'), 3.07 (dist.d, J = 6.5 Hz, 2H, CH2-1''), 2.43-2.39 (m,
2H, CH2-4), 2.37-2.34 (m, 6H, CH2-2, CH2-2' & CH2-6'), 2.21 (s, 3H, 3''''-CH3), 1.73 (quint., J =
13
7.1 Hz, 2H, CH2-3); C-NMR (150 MHz, CDCl3, δ, ppm): 171.52 (NH-CO), 139.77 (C-1''''),
138.66 (C-3''''), 137.16 (C-1'''), 132.37(C-2''), 129.00 (C-3''' & C-5'''), 128.87 (C-5''''), 127.88 (C-
4''''), 127.80 (C-3''), 127.63 (C-4'''), 127.17 (C-2''''), 126.62 (C-2''' & C-6'''), 120.13 (C-6''''), 60.64
(C-1''), 57.78 (C-4), 53.25 (C-3' & C-5'), 53.20 (C-2' & C-6'), 34.87 (C-2), 22.79 (C-3), 21.65 (C-
3''''); Anal. Calc. for C24H31N3O (377.52): C, 76.28; H, 8.21; N, 11.12.Found: C, 76.29; H, 8.22;
N, 11.10.
4.1.4.4.
N-(4-Methylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide
(5d)
Dark brown semi-solid; Yield 80%; Molecular Formula: C24H31N3O; Molecular Mass: 377 gmol-
1; IR (KBr, υ, cm-1): 3253 (N-H, str.), 3049 (Ar C-H, str.), 1719 (C=O, str.), 1633 (Ar C=C, str.),
1594 (C=C, str.); 1H-NMR (CDCl3, 600 MHz, δ, ppm): 9.75 (s, 1H, NH-CO), 7.47 (br.d, J = 8.2
Hz, 2H, H-2'''' & H-6''''), 7.43 (br.d, J = 7.3 Hz, 2H, H-2''' & H-6'''), 7.32 (dist.t, J = 7.5 Hz, 2H, H-
3''' & H-5'''), 7.23 (dist.t, J = 7.3 Hz, 1H, H-4'''), 7.08 (br.d, J = 8.2 Hz, 2H, H-3'''' & H-5''''), 6.51
(br.d, J = 15.9 Hz, 1H, H-3''), 6.28 (td, J = 6.4, 15.9 Hz, 1H, H-2''), 3.45-3.44 (m, 4H, CH2-3' &
CH2-5'), 3.06 (br.d, J = 6.4 Hz, 2H, CH2-1''), 2.46-2.43 (m, 2H, CH2-4), 2.34-2.27 (m, 6H, CH2-2,
13
CH2-2' & CH2-6'), 2.24 (s, 3H, 4''''-CH3), 1.72 (quint. J = 7.1 Hz, 2H, CH2-3); C-NMR (150
MHz, CDCl3, δ, ppm): 171.35 (NH-CO), 137.33 (C-1''''), 137.16 (C-1'''), 132.73 (C-4''''), 132.35(C-
2''), 129.41 (C-3'''' & C-5''''), 129.00 (C-3''' & C-5'''), 127.65 (C-4'''), 126.66 (C-3''), 126.62 (C-2'''
& C-6'''), 119.55 (C-2'''' & C-6''''), 60.64 (C-1''), 57.80 (C-4), 53.26 (C-3' & C-5'), 53.22 (C-2' &
17

C-6'), 34.81 (C-2), 22.81 (C-3), 20.87 (C-4''''); Anal. Calc. for C24H31N3O (377.52): C, 76.28; H,
8.21; N, 11.12. Found: C, 76.26; H, 8.24; N, 11.14.
4.1.4.5.
N-(4-Ethylphenyl)-4-{(4-[(E)-3-phenyl-2-propenyl]-1-piperazinyl}butanamide
(5e)
Light pink amorphous powder; Yield 71%; m.p. 125-126 ; Molecular Formula: C H N O;
℃
25 33
3
Molecular Mass: 391gmol-1; IR (KBr, υ, cm-1): 3243 (N-H, str.), 3047 (Ar C-H, str.), 1711 (C=O,
str.), 1635 (Ar C=C, str.), 1592 (C=C, str.); 1H-NMR (CDCl3, 600 MHz, δ, ppm): 9.23 (s, 1H, NH-
CO), 7.44 (br.d, J = 7.5 Hz 2H, H-2''' & H-6'''), 7.33-7.31 (m, 3H, H-3''', H-4''' & H-5'''), 7.22 (br.d,
J = 8.0 Hz, 2H, H-2'''' & H-6''''), 7.12 (br.d, J = 8.0 Hz, 2H, H-3'''' & H-5''''), 6.53 (br.d, J = 15.9
Hz, 1H, H-3''), 6.29 (td, J = 6.6, 15.9 Hz, 1H, H-2''), 3.38-3.34 (m, 4H, CH2-3' & CH2-5'), 3.09
(br.d, J = 6.3 Hz, 2H, CH2-1''), 2.58 (q, J = 7.5 Hz, 2H, 4''''-CH2CH3), 2.48-2.47 (m, 2H, CH2-4),
2.33-2.25 (m, 6H, CH2-2, CH2-2' & CH2-6'), 1.74 (quint., J = 7.1 Hz, 2H, CH2-3) 1.11 (t, J = 7.5
Hz, 3H, 4''''-CH2CH3);13C-NMR (150 MHz, CDCl3, δ, ppm): 171.76 (NH-CO), 138.42 (C-1''''),
137.16 (C-1'''), 136.26 (C-4''''), 132.36 (C-2''), 129.01 (C-3''' & C-5'''), 128.86 (C-3'''' & C-5''''),
127.81 (C-2'''' & C-6''''), 127.65 (C-4'''), 126.58 (C-2''' & C-6'''), 126.25 (C-3''), 60.65 (C-1''), 57.81
(C-4), 53.29 (C-3' & C-5'), 53.20 (C-2' & C-6'), 34.17 (C-2), 24.23 (4''''-CH2CH3), 23.01 (C-3)
14.68 (4''''-CH2CH3); Anal. Calc. for C25H33N3O (391.55): C, 76.61; H, 8.42; N, 10.70. Found: C,
76.60; H, 8.44; N, 10.72.
4.2.
4.2.1. In vitro methodology
4.2.1.1. Tyrosinase assay
The potency of synthesized amides against tyrosinase was investigated by exactly
Biology
following our published method [19,20]. In the first step, 20 mM with pH 6.8 Phosphate buffer
was prepared and their 140 µL was used in each well, simultaneously 20 µL of target enzyme
mushroom tyrosinase was also added from 30U/mL of stock solution. In a third step, 20 µL of the
compound was poured into the assay plate. After the first incubation of 10 minutes at 25 ºC, the
20 µL of substrate L_DOPA was poured from a stock solution of 0.85 mM, after that reaction
mixture was again incubated 20 minutes at room temperature. Finally, the change in absorbance
was recorded at wavelength 475 nm using SpectraMAX ABS microplate reader. For comparison
and assay validation kojic acid was used as positive control and assay buffer was used as a negative
control. For IC50 calculations all concentration was investigated separately and repeated three
18

times for better accuracy of results. For calculation of IC50 through nonlinear regression GraphPad
Prism was used. By below mentioned equation % inhibition was determined.
푇푦표푠푖푛푎푠푒 % 푖푛ℎ푖푏푖푡푖표푛 = [(퐵푙푎푛푘 ― 푆푎푚푝푙푒)/퐵푙푎푛푘] × 100
4.2.1.2.
Kinetic analysis
The kinetic experiment was carried to know the behavior of compound 5b for inhibition of
tyrosinase. Range of doses was investigated for the identification of the pattern of inhibition of
tyrosinase by 5b we examined the kinetic using our published method [20]. Totally four doses of
5b were investigated 0.00, 0.007, 0.013 and 0.026 µM. various series of substrate L-DOPA doses
were used from 0.0625 to 2mM for all experiments. The first incubation and reading period was
the same as presented in the above inhibition method for IC50 calculations. The maximum first
velocity was determined using the primary linear phase of absorbance for 5 minutes after mixing
enzyme solution at thirty seconds period. The enzyme blockage pattern was determined using the
Lineweaver Burk graph of the opposite of velocities (1/V) against the opposite of used substrate
doses. Another chart was drawn for calculations of enzyme inhibition dissociation constant Ki
through 1/V against the 5b doses.
4.2.1.3.
Hemolytic activity
For a hemolytic activity study, the bovine blood sample was collected in EDTA, diluted
with (0.9% NaCl), and centrifuge the diluted sample at 1000xg for 10 minutes. The erythrocytes
separated, diluted in phosphate buffer of pH 7.4, and suspension was made. 20 µL of synthesized
compounds solution (10 mg/mL) in 180 µL of RBCs suspension was added and then incubated for
30 min at room temperature. PBS was used as negative control and Triton-X was taken as a positive
control [21,22]. The experiment was performed in triplicate. The %age of hemolytic activity
(cytotoxicity) of newly synthesized amides was calculated by using the following formula:
퐴푏푠표푟푏푎푛푐푒 표푓 푆푎푚푝푙푒 ― 퐴푏푠표푟푏푎푛푐푒 표푓 푁푒푔푎푡푖푣푒 퐶표푛푡푟표푙
(%) 표푓 퐻푒푚표푙푦푠푖푠 =
× 100
퐴푏푠표푟푏푎푛푐푒 표푓 푃표푠푖푡푖푣푒 퐶표푛푡푟표푙
4.2.2. In vivo methodology
4.2.2.1.
Determination of pigmentation reducing capacity of 5b in embryos of Zebrafish
The animal experiments were carried out as accordance with the published methods
[20,23].
19

4.2.2.2.
Zebrafish Farming
The selected animal zebrafishes were obtained from the local market and maintained in our
fish facility laboratory for thirty days. All the conditions were optimized for fish culture as
prescribed in literature for zebrafish maintains. Shrimp larvae were used as the food of fishes and
they were housed in tanks made up of thermostatic material. For respiration, growth and better
health air and water filtration were maintained. The fish seed was obtained using the normal
procedure of fish spawning performed using the light source as a stimulator. All the animal
experiments methods were confirmed by the Departmental Review Board on Kongju National
University (IRB NO. 2011-2).
4.2.2.3.
Compound 5b treatment and depigmentation examination
Firstly the E3 medium was prepared by mixing the 5mM Sodium chloride, 0.17mM of
potassium chloride, 0.33mM calcium chloride and 0.33 mM and magnesium chloride. After that
even fishes were obtained using pipette into an assay plate and poured four to five embryos in each
well. In the second step, 5b solution was prepared in 0.1% DMSO and placed into the embryos
medium for nine to seventy-two hours post-fertilization. For comparison and assay confirmation
kojic acid was used as a reference. After that chorion of embryos was removed and
tricainemethanesulfonate MS-222 was used as anesthesia. Lastly, embryos slide was prepared
using 1% methylcellulose on the whole slide and images were takes using stereomicroscope
purchased from Nikon, Japan.
4.2.3. Computational Methodology
4.2.3.1.
Grid generation and molecular docking
The three dimensional (3D) structure of mushroom tyrosinase (Agaricus bisporus) (PDB
ID: 2Y9X) was accessed from the Protein Data Bank (PDB). Before the molecular docking
experiment, we modified the target protein structure for better docking results. The tyrosinase
structure was prepared by using the "Protein Preparation Wizard" by the Maestro interface in
Schrödinger Suite. Initially, bond orders were assigned and hydrogen atoms were added to the
protein structure. After that, the structure was then minimized to reach the converged root mean
square deviation (RMSD) of 0.30 Å with the OPLS_2005 force field. The active site of the enzyme
(tyrosinase) was defined from the co-crystallized ligands from PDB and literature data [16-18].
The synthesized ligands (5a-e) were sketched in a 2D sketcher in Schrödinger Suite and saved in
the Maestro interface for docking experiments. The molecular docking experiment was performed
20

for all the synthesized ligands against target protein by using a Glide docking protocol [24]. The
predicted binding energies (docking scores) and conformational positions of ligands within the
active region of protein were also performed using the Glide experiment. Throughout the docking
simulations, both partial flexibility and full flexibility around the active site residues are performed
by Glide/SP/XP and induced fit docking (IFD) approaches [25].
4.2.3.2.
Molecular Dynamics
To understand the protein backbone stability in docking complex (5b), a molecular
dynamics simulation experiment was carried out using the Desmond simulation package of
Schrödinger [26]. In all runs, the NPT (isothermal-isobaric) ensemble was applied with a
temperature of 300 K and pressure of 1 bar. The simulation length was 100 ns, with relaxation
time 1 ps. The force field parameters for each simulation were according to OPLS_2005 [27]. The
long-range electrostatic interactions were calculated using the particle mesh Ewald (PME) method
[28]. The cutoff radius in Columb interactions was 9.0 Å. The water molecules were described
using a simple point charge model (SPC) [29]. The Martyna-Tuckerman-Klein chain coupling
scheme [30] with a coupling constant of 2.0 ps was used for pressure control and the Nosé-Hoover
chain coupling scheme for temperature control. Non bonded 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 3 steps. The trajectories were saved at 4.8 ps intervals for analysis. To analyze
the behavior and interactions between the ligands and protein, we used the Simulation Interactions
Diagram tool implemented in the Desmond molecular dynamics package.
Acknowledgement
The present study was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education
(2017R1D1A1B03034948).
Declaration of Competing Interest
The authors declare no conflict of interests.
Authors Contribution
a College of Natural Science, Department of Biological Sciences, Kongju National University,
Gongju, 32588, South Korea.
The study designed, animal, tyrosinase screening were performed, manuscript was completed for
finalized submission.
21

b Department of Chemistry, Government College University, Lahore-54000, Pakistan.
Scheme was designed and synthesis of organic compounds were carried out.
cInstitute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Lahore-
54000,, Pakistan.
Molecular docking study was performed.
d Department of Physiology, University of Sindh, Jamshoro Pakistan.
In silico study and helps in drafting the manuscript.
e Faculty of Pharmacy and Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns),
Level 9, FF3, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia.
Characterization of compounds was performed.
f Department of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan.
Toxicity was assessed.
22

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Highlights
 Butanamides compounds were synthesized for the treatment of melanogenesis.
 The inhibitory activity of synthesized compounds were evaluated against tyrosinase.
 The compounds were evaluated as an antimelanogenesis using zebrafish.
 The cytotoxicity of these butanamides was also screened using hemolytic activity.
 In silico study was performed to check the binding profile against tyrosinase.
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Declaration of interests
☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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