Design, synthesis, kinetic mechanism and molecular docking studies of novel 1-pentanoyl-3-arylthioureas as inhibitors of mushroom tyrosinase and free radical scavengers

Article abstract of DOI:10.1016/j.ejmech.2017.09.059

A series of novel 1-pentanoyl-3-arylthioureas was designed as new mushroom tyrosinase inhibitors and free radical scavengers. The title compounds were obtained in excellent yield and characterized by FTIR, ¹H NMR, ¹³C NMR and X-ray crystallography in case of compound (4a). The inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed that 1-Pentanoyl-3-(4-methoxyphenyl) thiourea (4f) showed tyrosinase inhibitory activity (IC₅₀ 1.568 ± 0.01 mM) comparable to Kojic acid (IC₅₀ 16.051 ± 1.27 mM). Interestingly compound 4f exhibited higher antioxidant potential compared to other derivatives. The docking studies of synthesized 1-Pentanoyl-3-arylthioureas analogues were also carried out against tyrosinase protein (PDBID 2ZMX) to compare the binding affinities with IC₅₀ values. The predicted binding affinities are in good agreement with the IC₅₀ values as compound (4f) showed highest binding affinity (?7.50 kcal/mol) compared to others derivatives. The kinetic mechanism analyzed by Line-weavere Burk plots exhibited that compound (4f) inhibit the enzyme inhibits the tyrosinase non-competitively to form an enzyme inhibitor complex. The inhibition constants Ki calculated from Dixon plots for compound (4f) is 1.10 μM. It was also found from kinetic analysis that derivative 4f irreversible enzyme inhibitor complex. It is proposed on the basis of our investigation that title compound (4f) may serve as lead structure for the design of more potent tyrosinase inhibitors.

Full text of DOI:10.1016/j.ejmech.2017.09.059

Accepted Manuscript
Design, synthesis, kinetic mechanism and molecular docking studies of novel 1-
pentanoyl-3-arylthioureas as inhibitors of mushroom tyrosinase and free radical
scavengers
Fayaz Ali Larik, Aamer Saeed, Pervaiz Ali Channar, Urooj Muqadar, Qamar Abbas,
Mubashir Hassan, Sung-Yum Seo, Michael Bolte
PII:
S0223-5234(17)30774-2
10.1016/j.ejmech.2017.09.059
EJMECH 9774
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 November 2016
Revised Date: 25 September 2017
Accepted Date: 27 September 2017
Please cite this article as: F.A. Larik, A. Saeed, P.A. Channar, U. Muqadar, Q. Abbas, M. Hassan,
S.-Y. Seo, M. Bolte, Design, synthesis, kinetic mechanism and molecular docking studies of novel 1-
pentanoyl-3-arylthioureas as inhibitors of mushroom tyrosinase and free radical scavengers, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.059.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
1
Design, Synthesis, kinetic mechanism and molecular docking studies of novel 1-Pentanoyl-
3-arylthioureas as inhibitors of mushroom tyrosinase and free radical scavengers
Fayaz Ali Larika, Aamer Saeed*a, Pervaiz Ali Channara, Urooj Muqadara, Qamar Abbasb,
Mubashir Hassanb, Sung-Yum Seob , Michael Boltec
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
bDepartment of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of Korea
cInstitut für Anorganische Chemie, J.W.-Goethe-Universität, Max-von-Laue-Str.7, D-60438
Frankfurt/Main, Germany.
Email: aamersaeed@yahoo.com, Tel +92-51-9064-2128; Fax: +92-51-9064-2241
1

ACCEPTED MANUSCRIPT
2
Highlights
‹ A small library of novel 1-Pentanoyl-3-arylthioureas (4a-4j) synthesized.
‹ Mushroom tyrosinase inhibition and free radical scavenging activity were evaluated
‹ Most of the compounds show excellent activity, particularly 4f higher than the standard.
‹ The kinetic mechanism proposed 4f is non-competitive inhibitor of mushroom tyrosinase.
‹ Molecular docking, druglikeness, Ramachandran graph, Chemo-informatics and
Lipinski’s rule were studied.
2

ACCEPTED MANUSCRIPT
3
Graphical abstract
Abstract
A series of novel 1-pentanoyl-3-arylthioureas was designed as new mushroom tyrosinase
inhibitors and free radical scavengers. The title compounds were obtained in excellent yield and
3

ACCEPTED MANUSCRIPT
4
characterized by FTIR, 1H NMR, 13C NMR and X-ray crystallography in case of compound (4a).
The inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed
that 1-Pentanoyl-3-(4-methoxyphenyl) thiourea (4f) showed tyrosinase inhibitory activity (IC50
1.568 ± 0.01 mM) comparable to Kojic acid (IC50 16.051 ± 1.27 mM). Interestingly compound
4f exhibited higher antioxidant potential compared to other derivatives. The docking studies of
synthesized 1-Pentanoyl-3-arylthioureas analogues were also carried out against tyrosinase
protein (PDBID 2ZMX) to compare the binding affinities with IC50 values. The predicted
binding affinities are in good agreement with the IC50 values as compound (4f) showed highest
binding affinity (-7.50 kcal/mol) compared to others derivatives. The kinetic mechanism
analyzed by Line-weavere Burk plots exhibited that compound (4f) inhibit the enzyme inhibits
the tyrosinase non-competitively to form an enzyme inhibitor complex. The inhibition constants
Ki calculated from Dixon plots for compound (4f) is 1.10 µM. It was also found from kinetic
analysis that derivative 4f irreversible enzyme inhibitor complex. It is proposed on the basis of
our investigation that title compound (4f) may serve as lead structure for the design of more
potent tyrosinase inhibitors.
Keywords: Thiourea derivatives, Synthesis, Mushroom Tyrosinase inhibitor, Kinetic
mechanism, Molecular docking, Crystal structure.
1. Introduction
It is believed that the browning of fruits, vegetables and beverages poses serious threat to agro-
economic countries [1]. The food industry has recently witnessed loss of quality during
4

ACCEPTED MANUSCRIPT
5
postharvest handling and processing of fruits and vegetables [2]. The plausible mechanism of
browning of food is well recognized can be enzymatic or non-enzymatic in origin [3]. The
enzymatic browning is catalyzed by tyrosinase (copper containing multifunctional glycosylated
enzyme) also known as polyphenol oxidase (PPO; 1,2 benzenediol oxygen oxidoreductase; EC
1.10.3. l), polyphenolase, phenolase, catechol oxidase, cresolase, or catecholase is widely found
in nature [4]. PPO is typically present in the majority of microorganisms, plants and animals. In
plants, tyrosinase acts by oxidizing mono phenols into diphenols (monophenolase activity) and
the oxidation of o-diphenols into o-quinones (diphenolase activity) followed by quinones into
dark brown pigments [5]. These results in deterioration of flavor, color, and nutritional quality
and continue during postharvest handling and marketing of vegetables [6]. In fungi and
vertebrates, tyrosinase catalyzes the rate-limiting step in the formation of the pigment melanin
from tyrosine [7]. Melanin is responsible for skin color and plays an important role in the
protection of the skin against UV light induced damage [8]. During the melanin biosynthesis
pathway, tyrosinase (EC 1.14.18.1) is the rate-limiting enzyme that hydroxylates L-tyrosine to L-
3,4-dihydroxyphenylalanine (L-DOPA), and L-DOPA is further oxidized to the corresponding o-
dopaquinone [9, 10].These phenomena have encouraged researchers to seek new potent
tyrosinase inhibitors, for use in cosmetics and foods.
The literature survey reveals that researchers are devoted to design and synthesize novel
tyrosinase inhibitors such as Seo et al.[11] reviewed the importance of mushroom tyrosinase and
defined its biochemical character and inhibition and activation by several inhibitors from natural
and synthetic origin. Zhu et al.[12] also reported that tyrosinase enzyme has also been linked to
Parkinson’s disease. Loizzo et al.[13] reviewed the tyrosinase inhibitors from both natural and
5

ACCEPTED MANUSCRIPT
6
synthetic origin for their use in food and cosmetic industry. Khan also reviewed the molecular
design of tyrosinase inhibitors from synthetic origin [14].
Recently, Chang et al reported that thio-containing compounds could react with dopaquinone, to
form colorless products [15,16]. Then the melanogenetic process is slowed down, until all the
scavengers are consumed. Thus, the development of safe and effective tyrosinase inhibitors is of
great concern in the medical, food and cosmetic industries.
Our research group has fine contributions in the field of medicinal chemistry and recently we
have designed and synthesized novel enzyme inhibitors [17-19]. The above mentioned literature
survey prompted us to synthesize thio-containing compounds in order to be utilized as safe and
effective inhibitors of tyrosinase enzyme.
Aryl thioureas found a wide diversity of applications in heterocyclic syntheses, metal complexes
and molecular electronics and exhibit a vast array of biological activities [20-22]. Arylthioureas
containing both carbonyl and thiocarbonyl groups can coordinate to metals using both sulphur
and oxygen atoms, the presence of these hard and soft donor sites offer a huge bonding potential
for metals such as copper [23,24]. Thiourea possess more than one binding sites it provoked us to
design and synthesize novel 1-Pentanoyl-3-aryl thioureas as the potent inhibitors of tyrosinase
enzyme.
2. Results and discussion
2.1 Chemistry
A novel series of thioureas derivatives (4a-j) were synthesized by forming isothiocyanate
intermediate using the well-known protocol (treating potassium thiocyanate in dry acetone for 30
min and followed by the addition of acid chloride, after cooling the reaction mixture different
aromatic substituted amines were incorporated) in scheme 1.
6

ACCEPTED MANUSCRIPT
7
NH2
S
O
R
i). SOCl2
reflux, 2h
O
HN
N
O
H
3a-3j
OH
N
(2)
C
S
ii). KSCN
dry acetone
1.5 h
R
(1)
reflux
4a-4j
R= 3a-3j and 4a-4j
4a 2,4,6-tri-CH3
4b 2,4-di-NO2
4c 3-NO2
4d 4-NO2
4e 2-F, 4-Br
4f 4-OCH3
4g 2,3-dichloro
4h 2,6-di-Br-4-F
4i 4-Cl
4j 4-Br
Scheme 1. Synthetic path to novel 1-pentanoyl-3-arylthioureas (4a-4j)
2.2 Spectroscopic characterization
The synthesized aromatic substituted 1-pentanoyl-3-aryl thioureas have been characterized on the
basis of spectroscopic data.1H and 13C NMR were recorded in deutrated DMSO-d6 solvent.
Three different types of protons gave distinct signals in 1H NMR, two N-H protons at 11.87 ppm
and 11.34 ppm appeared singlet, most deshielded signals because of intra molecular hydrogen
1
bonding, in H NMR intramolecular hydrogen bonding shifts signals towards higher ppm value
justifying presence of thio core in thiourea, signals at 7-8 ppm region clearly indicated the
aromatic ring. Two types of methyl groups are present, one methyl group was attached with acyl
7

ACCEPTED MANUSCRIPT
8
group that appeared at 2.35 ppm and other methyl group which is directly attached to aromatic
ring appeared slightly shielded than the methyl protons attached with acyl group. In all of the
synthesized thioureas, the –NH proton situated in between phenyl ring and C=S, is maximum
deshielded due to the intramolecular hydrogen bonding and thus gave a singlet at around 12 ppm
and the –NH proton located in between the C=O and C=S is deshielded to a lesser extent thus
providing a singlet in the region of 10-11 ppm [8,15-17]. The aromatic ring protons gave their
signals between 7 and 8 ppm. In 13C NMR, carbon bonded with sulfur provided a strong signal at
about 180 ppm and the carbonyl carbon appeared in the range of 172-175 ppm. Signals for
aromatic carbons appeared in the region between 120-140 ppm and the signals for ipso carbons
were found weak in intensity due to less number of hydrogen. In FTIR, -NH of thioureas gave a
broad band above 3200 cm-1 due to the intramolecular hydrogen bonding between the oxygen of
the carbonyl and -NH. Just around 3000 cm-1 Ar-H stretch was present and carbonyl group
appeared as an intense band in the region of 1700-1600 cm-1. C=S was available between 1050
cm-1 and 1250 cm-1 for all the compounds.
2.3 Bioassay for tyrosinase inhibition activity
1-Pentanoyl-3-arylthioureas have been designed to evaluate their inhibitory effects on mushroom
tyrosinase activity. Kojic acid a competitive tyrosinase inhibitor was used as standard for
comparison purpose. The aryl thioureas tyrosinase inhibitors as described in the preceding
section are of special interest because of their high activity (IC50 < 1.578 mM). Novel aryl
thioureas derivatives (4a-4j) have been synthesized by incorporation of substituted anilines to
isothiocyanate. The synthesis 1-pentanoyl-3-arylthioureas was carried out to explore the role
electron donating and electron withdrawing groups in tyrosinase inhibition. It has been exposed
8

ACCEPTED MANUSCRIPT
9
from our bioassay results (Table 1) that the major determining factor of inhibitory activity is the
position and not the number of the electron donating groups.
Table 1 The inhibitory effects of compounds (4a-4j)
on mushroom tyrosinase
Compound
Tyrosinase inhibition activity
IC50 ± SEM (µM)
4a
19.658 ± 1.25
10.079 ± 0.87
16.599 ± 0.98
7.986 ± 0.21
6.461 ± 0.05
1.568 ± 0.01
7.750 ± 0.87
19.537 ± 1.25
2.316 ± 0.08
3.371 ± 0.05
16.051 ± 1.27
4b
4c
4d
4e
4f
4g
4h
4i
4j
Kojic acid
SEM = Standard error of the mean; values are expressed
in mean ± SEM.
2.3.1 Free radical scavenging
All of the synthesized compounds (4a-4j) were evaluated for DPPH free radical scavenging
ability. The compound 4f showed good 80% scavenging potency, other nine compounds did not
show significant radical scavenging potential even at high concentration (100µg/mL) Figure 1
9

ACCEPTED MANUSCRIPT
10
150
100
50
0
Figure.1 The % radical scavenging activity of synthetic inhibitors. The values are expressed in
mean ± SEM. All inhibitors and Vitamin C concentration were 100µg/mL. SEM= standard error
of mean
2.4 Kinetic Mechanism
To understand the inhibitory mechanism of synthetic compounds on mushroom tyrosinase
inhibition kinetic study were performed. Based upon our IC50 results we select our most potent
compound 4f to determine their inhibition type and inhibition constant. The kinetic results of the
enzyme by the Line weaver-Burk plot of 1/V versus 1/[S] in the presence of different inhibitor
concentration gave a series of straight lines, the result of compound 4f showed that all of which
intersected at the same point on the x-axis (Figure 2A). The analysis showed that 1/Vmax
increased to new value while that of a Km remains the same as a result increase in the
concentration of compound 4f. This behavior indicated that compound 4f inhibits the tyrosinase
non-competitively to form an enzyme inhibitor complex. Second plot (Figure 2B) of slope
against concentration of 4f showed EI dissociation constant.
10

ACCEPTED MANUSCRIPT
11
Table 2. The kinetic parameters of the mushroom Tyrosinase for L-DOPA activity in the
presence of various concentration of 4f compound.
Compound
Concentration 1/Vmax
Km
(mM)
Inhibition
Type
Ki
(µM)
(µM)
(∆A/Sec)
4f
0.271
28571.428
0.389
0.389
0.389
0.389
0.389
0.542
1.084
2.18
52380.952
90476.190
109523.809
185714.285
Non-competitive
1.10
4.336
11

ACCEPTED MANUSCRIPT
12
1400000
1200000
1000000
800000
600000
400000
200000
0
0.271 µM
A
0.542 µM
1.084 µM
2.18 µM
4.336 µM
-5
0
5
10
15
20
1/[S] mM-1
80000
70000
60000
50000
40000
30000
20000
10000
B
0
0
-1
1
2
3
4
5
[I] µM
12

ACCEPTED MANUSCRIPT
13
Figure 2. Lineweaver-Burk plot for inhibition of tyrosinase in the presence of various
concentrations of compound 4f. (A). The insets represent the plot of the slope versus inhibitor 4f
to determine the inhibition constant (B). The lines were drawn using linear least square fit.
2.5 Structure Activity relationship
Structure Activity Relationships (SAR) are relations between the molecular structure and
biological or physicochemical activity of chemicals. The SAR allows to design and modify the
structure in order to obtain effective drugs. A series of 1-pentanoyl-3-aryl thioureas was designed
and synthesized as potent inhibitors of tyrosinase enzyme. The pharmacophore of thiourea
comprised of three parts (Figure 3).
Figure 3. Design of novel 1-pentanoyl-3-arylthioureas (4a-4j)
The results inhibitory effects of compounds revealed that the compound 4f was found to most
potent in the series and exhibited approximately 15 time higher inhibition compared to standard
Kojic acid. In compound 4f, the methoxy group at para-position could facilitate to occupy the
whole pocket and lead to stronger hydrophobic interactions (figure 4). The compound 4a bears
three methyl groups which creates crowding/steric hindrance and was found to be least potent
inhibitor in the series.
13

ACCEPTED MANUSCRIPT
14
H
O
S
O
S
R
R
N
H
O
N
H
O
N
H
O
N
H
O
Receptors
Receptors
Increasing activity
Receptors
H
Electron
donaiting
O
O
S
No effect
group-EDG
enhance the inhibition
N
H
O
N
H
O
Most potent inhibitor 4f
Receptors
Figure 4. Structure activity relationship and the plausible binding sites
3. Docking studies
3.1 Structural assessment of target protein
Mushroom tyrosinase (Agaricus bisporus) is a class of oxidoreductase copper containing protein
comprises 391 residues. The structural architecture of mushroom tyrosinase showed that, it
consists of 39% helices (154 residues) and 14% β sheets (57 residues) and 46% coils (180
residues). The X-Ray diffraction study confirmed its resolution 2.78Ȧ, R-value 0.238 and unit
cell crystal dimensions like length and angles of coordinates. The unit cell length values were
observed for a=103.84, b=104.82 and c=119.36 with angles 90°, 110.45° and 90° for all α, β and
γ dimensions respectively. The Ramachandran plots and values indicated that 95.90% of residues
were in favored regions and 100.0% residues were lies in allowed regions (Figure. S14).
3.2 Chemo-informatics properties and Lipinski’s rule of synthesized compounds (4a-4j)
The predicted properties such as molar volume and refractivity, density, polarizability and
surface tension were evaluated by computational approach. Literature study showed a standard
14

ACCEPTED MANUSCRIPT
15
value for molar refractivity (40 to 130), molecular weight (160 to 480) and number of atoms (20
to 70) [25]. Results showed that 4a-4j predicted values are better than standard values.
Moreover, the Lipinski’s rule of five (RO5) results showed that compounds (4a-4j) possess
HBA, HBD and logP values which are significantly justified its drug like behavior. Moreover,
their molecular weight of 4f (266.11 g/mol) was also much better than standard value
(<5000g/mol). The RO5 justifies that molecules with poor absorption are more likely to have-
more than 5 HBD, MWT over 500, logP over 5 and more than 10 HBA. However, there are
plenty of examples available for RO5 violation amongst the existing drugs [26]. In overall
results, these predicted values justify the significance of the 4f synthesized compound as good
candidate molecule (Table. 2).
3.3 Molecular docking analyses
The docked complexes of all the synthesized compounds (4a-4j) were analyzed on the basis of
lowest binding energy values and hydrogen bonding analyses. Results showed that 4f was the
most active compound with best binding energy value (-7.50 kcal/mol) compared to others
derivatives. The graphical depiction of 4f docking complex is mentioned in Figure 5.
15

ACCEPTED MANUSCRIPT
16
Figure 5. Docking interaction between 4f and mushroom tyrosinase. The 4f is mentioned in
purple color with nitrogen, oxygen and sulphur functional groups in blue, red and yellow colors.
The target protein is highlighted in line ribbon format with light gray color. The active binding
site amino acids are highlighted in red color. Two copper ions are also mentioned in black color.
Two hydrogen bonds were observed between 4f and receptor amino acids like His85 and Gly245
with bonding distances 2.20Å and 3.37Å, respectively. The black dotted lines show the binding
distance in angstrom (Å).
The bonding analysis of all compounds against mushroom tyrosinase showed that 4f compound
is directly interacts with active residues of targeted protein. The structure activity relationship
analysis shows that 4f builds two hydrogen bonds at specific residues His85 and Gly245 against
target protein. The carbonyl nitrogen of benzene ring of 4f interacts with His85, while methyl
group of benzene ring interacts with Gly245 having bonds lengths 2.20Å and 3.37Å,
16

ACCEPTED MANUSCRIPT
17
respectively. Literature study also justified that these interacted residues are significant in the
downstream signaling pathways [27]. The graphical representations of all other interacted
compounds (4a-4e and 4g-4j) against the target protein are mentioned in (Supplementary Figure.
S15-S24). Moreover, comparative energy binding analysis showed that 4f has good energy
values (-7.50 kcal/mol) as compared other derivatives. The other candidate compounds (4a-4e
and 4g-4j) also possess good binding energy values -6.70, -6.60, -7.30, -7.60, -6.60 and -7.00, -
6.70, -6.70, -6.60 Kcal/mol binding energy values respectively (Figure S25). Here, it is
noticeable that most of candidates compounds bind in the active binding regions but little
fluctuated with their conformational positions. The computational and bioassay studies showed
that 4f is the most active and significant compound as compared to other synthesized
compounds.
Literature study showed that different mushroom tyrosinase inhibitors can be further employed
for human tyrosinase inhibitions [28]. The computational and assay based approach depicts the
significance of these synthesized ligands in tyrosinase inhibition activity.
4. Conclusion
The 1,3-disubstitued thioureas (4a-4j) having aryl and alkyl are synthesized. The molecular
docking investigation revealed that thiourea derivative (4f) possesses maximum binding affinity
(-7.50 kcal/mol) with the target protein (PDBID 2ZMX). The wet lab results are in good
agreement with the calculated docking scores. The most potent activity against mushroom
tyrosinase was exhibited by the compound (4f) having IC50 15.2 mM. The presence of the
methoxy group at para-position on phenyl ring which play very significant role in the tyrosinase
inhibition activity. The kinetic analysis of compound (4f) shown that it is non-competitive type
inhibitor with Ki value 1.10 mM and formed an irreversible drug receptor complex with
17

ACCEPTED MANUSCRIPT
18
mushroom tyrosinase. It was concluded from our results that the title compound (4f) may serve
as lead structure for the design of more potent tyrosinase inhibitors.
5. Experimental part
5.1 General methods and materials
Melting points were recorded using a digital Gallenkamp (Tokyo, Japan) model MPD.BM 3.5
apparatus and are uncorrected. 1H NMR spectra were determined as CDCl3 solutions at 300 MHz
13
using a Bruker AM-300 spectrophotometer using TMS as an internal reference and C NMR
spectra were determined at 75 MHz using a Bruker 75 MHz NMR spectrometer in DMSO
solution. FTIR spectra were recorded on an FTS 3000 MX spectrophotometer. Mass Spectra (EI,
70 eV) on a MAT 312 instrument, and elemental analyses were conducted using a LECO-183
CHNS analyzer.
5.2 General method for the synthesis of 1-pentanoyl-3-arylthiourea (4a-j).
All chemicals, organic solvents and reagents were purchased from Sigma-Aldrich, Fluka and E.
Merck. Organic solvents, acetone (99.9 % pure), and n-hexane (95 % pure) were distilled
purified & dried according to reported methods. Potassium thiocyanate (98% pure), pentanoic
acid (99 % pure), 4-nitroaniline (99 % pure), 4-chloro-3-nitroaniline (99 % pure), 2-methyl-4-
nitroaniline (99 % pure) and 2-methoxy-4-nitroaniline (99 % pure) were used without further
purification.
5.2.1 General procedure for the synthesis of title compounds
A freshly prepared solution of pentanoyl chloride was synthesized from pentanoic acid by
treating the reaction mixture with thionylchloride.The acid chloride synthesized (10 mmol) in
anhydrous acetone (50 ml) was added dropwise to a suspension of potassium thiocyanate (10
18

ACCEPTED MANUSCRIPT
19
mmol) in acetone (30 ml) at room temperature. The reaction mixture was refluxed and cooled to
room temperature. A solution of the substituted anilines (10 mmol) in anhydrous acetone (20 ml)
was added dropwise and the reaction mixture was refluxed further for 2-3 h. On completion of
reaction (TLC) the mixture was poured into cold water and the precipitated thioureas were
filtered and recrystallized from methanol.
5.3 Experimental Data
5.3.1 1-Pentanoyl-3-(2,4,6-trimethylphenyl)thiourea (4a)
White crystalline solid, Yield=90%, M.P=199 oC, Rf=0.52 (n-Hexane: Ethyl acetate 1:1) FTIR υ
(cm-1) 3184.2 (N-H, stretching), 2982.5 (Aromatic C-H, stretching), 1679.2 (C=O, stretching),
1573 (N-O, bending), 1509.5 (C-C, stretching), 1411.8 (C-H, bending), 1332.89 (N-O, bending),
1255.4 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.77 (s, 1H, NH), 11.56 (s, 1H, NH), 6.94
(2H Ar-H), 2.24 (s, 6H Ar 2x CH3),2.10 (s, 3H Ar-CH3); 2.3 (t, 2H, J=2.4), 2.0 (2H,quintet, J=
1.8), 1.9 (sex,2H, J=2.1),1.4 (t, 3H, J= 2.5); 13C NMR: δ 180 (C=S), 172.9 (C=O), 136.9, 135.0,
133.8, 128.9 (Ar-C) ,28(CH3, Ar), 25(CH3, Ar), 22 (CH3-Ar), 40, 24, 20, 17 (CH3) (Anal.Calcd.
for C14H20N2OS: C, 62.99; H, 7.82; N, 12.85; S, 13.57 found: C, 63.97; H, 7.84; N, 12.84; S,
13.58.
5.3.2 1-Pentanoyl-3-(2,4-dinitrophenyl)thiourea (4b)
Yellow crystalline solid , Yield=89%, M.P=255 oC, Rf=0.68 (n-Hexane: Ethyl acetate 1:1) FTIR
υ (cm-1) 3185.3 (N-H, stretching), 2996.4 (Aromatic C-H, stretching), 1685.3 (C=O, stretching),
1575 (N-O, bending), 1509.7 (C-C, stretching), 1417.3 (C-H, bending), 1345.97 (N-O, bending),
1259.3 (C=S, stretching); 1H NMR (DMSO-d6); δ 11.61 (s, 1H, NH), 11.41 (s, 1H, NH), 7.35-6.9
(m, 3H Ar-H),2.4 (t, 2H, J=2.8), 1.8 (quintet,2H,J=2.1), 1.3 (sex, 2H, J=1.8), 0.9 (t,3H, J=2.2);
19

ACCEPTED MANUSCRIPT
20
13C NMR: δ 182.3 (C=S), 175.6 (C=O), 140.9, 137.1, 135.1, 131.5, 127.7, 125.5 (Ar-C), 37, 24,
22,17 (CH3), Anal.Calcd. for C14H18N4O5S:C, 47.15; H, 4.17; N, 15.62 S, 9.04 found: C, 48.17;
H, 5.78; N, 16.65 S, 10.05
5.3.3 1-Pentanoyl-3-(3-nitrophenyl)thiourea (4c)
o
Yellow crystalline solid, Yield=98%, M.P=215 C, Rf=0.61 (n-Hexane: Ethyl acetate 1:1) FTIR
υ (cm-1) 3199.1 (N-H, stretching), 2984.1 (Aromatic C-H, stretching), 1685.8 (C=O, stretching),
1589 (N-O, bending), 1509.8 (C-C, stretching), 1419.7 (C-H, bending), 1337.87 (N-O, bending),
1299.2 (C=S, stretching). 1H NMR (DMSO-d6); δ 12.80 (s, 1H, NH), 11.88 (s, 1H, NH), 7.89-
7.32 (m, 4H, ArH),2.4 (t, 2H, J=2.5), 2.0 (2H,quintet, J=2.3),1.8 (sex,2H, J=2.1),1.5 (t,3H,
J=1.8) ;13C NMR: δ 189.1 (C=S), 170.5 (C=O), 146.4, 139.2, 136.7, 132.2, 129.2, 127.6 (Ar-C),
40.5, 24.5, 20.4,16.4, (CH3), Anal.Calcd. for C11H13N3O3S: C, 49.17; H, 4.76; N, 15.59; S, 12.41
found: C, 50.19; H, 5.77; N, 16.58; S, 13.43
5.3.4 1-Pentanoyl-3-(4-nitrophenyl)thiourea (4d)
White crystalline solid , Yield=90%, M.P=199.5 oC, Rf=0.71 (n-Hexane: Ethyl acetate 1:1) FTIR
υ (cm-1) 3185.2 (N-H, stretching), 2998.3 (Aromatic C-H, stretching), 1691.2 (C=O, stretching),
1581 (N-O, bending), 1507.7 (C-C, stretching), 1415.8 (C-H, bending), 1337.51 (N-O, bending),
1297.2 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.78 (s, 1H, NH), 11.59 (s, 1H, NH), 7.75-
6.92 (m, 4H, ArH), 2.4 (t, 2H, J=2.8), 2.0 (2H,quintet, J=2.4),1.8 (sex,2H, J=1.9),1.5 (t,3H,
13
J=1.5) C NMR: δ 183 (C=S), 173.5 (C=O), 137.1, 135.5, 129.7, 128.2, 126.2 (Ar-C), 40.3,
20

ACCEPTED MANUSCRIPT
21
23.4, 20.2, 18.8; Anal.Calcd. forC11H13ClN2OS: C, 51.71; H, 5.45; N, 10.17; O, 7.08; S, 12.04
found: C, 52.30; H, 6.55; N, 11.27; S, 13.08.
5.3.5 1-Pentanoyl-3-(4-bromo-2-fluorophenyl)thiourea (4e)
Yellow crystalline solid , Yield=89%, M.P=205 oC, Rf=0.68 (n-Hexane: Ethyl acetate 1:1) FTIR
υ (cm-1) 3185.3 (N-H, stretching), 2996.4 (Aromatic C-H, stretching), 1685.3 (C=O, stretching),
1575 (N-O, bending), 1509.7 (C-C, stretching), 1417.3 (C-H, bending), 1345.97 (N-O, bending),
1259.3 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.61 (s, 1H, NH), 11.41 (s, 1H, NH), 7.35-6.9
(m, 3H Ar-H),2.4 (t, 2H, J=2.4), 2.0 (2H,quintet, J=2.1),1.8 (sex,2H, J=1.8),1.5 (t,3H, J=1.4) ;
13C NMR: δ 182 (C=S), 175.6 (C=O), 140.9, 137.1, 135.1, 131.5, 127.7, 125.5 (Ar-C), 40.2 22.3
18.5, 14.4 Anal.Calcd. for C11H12BrFN2OS:C, 41.15; H, 3.77; N, 9.62 S, 11.04 found: C, 42.17;
H, 4.78; N, 10.65, S, 12.05
5.3.6 1-Pentanoyl-3- (4-methoxyphenyl)thiourea (4f)
Brown crystalline solid , Yield=89%, M.P=180 oC, Rf=0.80(n-Hexane: Ethyl acetate 1:1) FTIR υ
(cm-1) 3179.5 (N-H, stretching), 2985.5 (Aromatic C-H, stretching), 1675.5 (C=O, stretching),
1569 (N-O, bending), 1504.2 (C-C, stretching), 1416.2 (C-H, bending), 1337.61 (N-O, bending),
1292.8 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.99 (s, 1H, NH), 11.77 (s, 1H, NH), 7.12 (d,
2H, J= 7.6 Hz ArH), 6.83(d,2H, J= 7.6 Hz ArH), 3.88 (s,3H),2.4 (t, 2H, J=2.6), 2.0 (2H,quintet,
J=2.4),1.8 (sex,2H, J=2.1),1.5 (t,3H, J=1.8) ;13C NMR: δ 181.3 (C=S), 169.2 (C=O), 131.1,
130.1, 127.2, 124.8 (Ar-C), 40.5, 23.7, 18.9, 12.5 Anal.Calcd. for C12H16N2O2S: C, 57.58; H,
6.41; N, 11.51; S, 12.31 found: C, 58.53; H, 7.45; N, 12.52; S, 13.33.
21

ACCEPTED MANUSCRIPT
22
5.3.7 1-Pentanoyl-3-(2,3-dichlorophenyl)thiourea (4g)
White crystalline solid , Yield=90%, M.P=190 oC, Rf=0.53(n-Hexane: Ethyl acetate 1:1) FTIR υ
(cm-1) 3189.1 (N-H, stretching), 2994.1 (Aromatic C-H, stretching), 1695.3 (C=O, stretching),
1586 (N-O, bending), 1508.4 (C-C, stretching), 1416.5 (C-H, bending), 1336.57 (N-O, bending),
1298.5 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.80 (s, 1H, NH), 11.66 (s, 1H, NH), 7.71-
6.88 (m, 3H, ArH), 2.4 (t, 2H, J=2.6),2.0 (2H,quintet, J=2.3), 1.8 (sex,2H, J=1.8),1.5 (t,3H,
J=1.5) ;13C NMR: δ 181.3 (C=S), 171.7 (C=O), 137.4, 136.2, 131.7, 129.2, 127.2 (Ar-C),40.8,
21.7, 18.7, 14.7, Anal.Calcd. for C11H12Cl2N2OS: C, 45.03; H, 4.15; N, 9.75; S, 11.15 found: C,
46.09; H, 5.05; N, 10.75; S, 12.17.
5.3.8 1-Pentanoyl-3-(2,6-dibromo-4-fluorophenyl)thiourea (4h)
o
Brown solid, Yield=95%, M.P=209 C, Rf=0.55 (n-Hexane: Ethyl acetate 1:1) FTIR υ (cm-1)
3189.5 (N-H, stretching), 2991.8 (Aromatic C-H, stretching), 1689.8 (C=O, stretching), 1580 (N-
O, bending), 1509.3 (C-C, stretching), 1414.3 (C-H, bending), 1382.19 (N-O, bending), 1257.8
(C=S, stretching). 1H NMR (DMSO-d6); δ 11.76 (s, 1H, NH), 11.55 (s, 1H, NH), 7.50 (2H Ar-
13
H),2.4 (t, 2H, J=2.7),2.0 (2H,quintet, J=2.5), 1.8 (sex,2H, J=2.2),1.5 (t,3H, J=1.4); C NMR: δ
(ppm) 185 (C=S), 178.9(C=O), 139.9,136.0, 134.8, 130.9 (Ar-C),40, 23, 20, 15 (CH3),
Anal.Calcd. for C11H11Br2FN2OS:C, 29.24; H, 1.92; N, 7.57; S, 8.67 found: C, 30.26; H, 2.33; N,
8.59; S, 9.69
22

ACCEPTED MANUSCRIPT
23
5.3.9 1-Pentanoyl-3-(4-chlorophenyl)thiourea (4i)
o
Light Yellow solid , Yield=95%, M.P=188 C, Rf=0.58 (n-Hexane: Ethyl acetate 1:1) FTIR υ
(cm-1) 3186.2 (N-H, stretching), 2994.3 (Aromatic C-H, stretching), 1687.2 (C=O, stretching),
1571 (N-O, bending), 1507.1 (C-C, stretching), 1415.3 (C-H, bending), 1337.91 (N-O, bending),
1295.3 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.77 (s, 1H, NH), 11.55 (s, 1H, NH), 7.73 (d,
2H, J= 7.8 Hz ArH), 7.47(d,2H, J= 7.8 Hz ArH);2.3 (t, 2H, J=2.8),2.0 (2H,quintet, J=2.4), 1.9
13
(sex,2H, J=2.1),1.4 (t,3H, J=2.2); C NMR: δ 187 (C=S), 170.1 (C=O), 134.1, 132.5, 128.7,
126.5 (Ar-C),40.4 24.5, 21.8, 18.5, Anal.Calcd. for C11H13ClN2OS:C, 51.25; H, 5.98; N, 12.25;
S, 14.02 found: C, 52.28; H, 6.95; N, 13.23; S, 15.06.
5.3.10 1-Pentanoyl-3-(4-bromophenyl)thiourea (4j)
White crystalline solid , Yield=95%, M.P=183 oC, Rf=0.47(n-Hexane: Ethyl acetate 1:1) FTIR υ
(cm-1) 3189.5 (N-H, stretching), 2995.5 (Aromatic C-H, stretching), 1685.5 (C=O, stretching),
1579 (N-O, bending), 1509.8 (C-C, stretching), 1419.5 (C-H, bending), 1338.93 (N-O, bending),
1296.5 (C=S, stretching). 1H NMR (DMSO-d6); δ 11.79 (s, 1H, NH), 11.65 (s, 1H, NH), 7.66 (d,
2H, J= 7.7 Hz ArH), 7.43(d,2H, J= 7.7 Hz ArH)2.4 (t, 2H, J=2.7), 2.0 (2H,quintet, J=2.4),1.8
(sex,2H, J=1.9),1.5 (t,3H, J=1.4) ;13C NMR: δ 182 (C=S), 169.2 (C=O), 132.1, 131.1, 128.2,
125.8 (Ar-C),40, 25, 20, 18, Anal.Calcd. for C11H13BrN2OS:C, 43.55; H, 4.38; N, 9.26; S, 10.71
found: C, 43.57; H, 4.39; N, 9.29; S, 10.75.
5.4 Mushroom tyrosinase inhibition assay
The mushroom tyrosinase (Sigma Chemical, USA) inhibition was performed following our
previously reported methods [9,10]. In detail, 140 µL of phosphate buffer (20 mM, pH 6.8), 20
23

ACCEPTED MANUSCRIPT
24
µL of mushroom tyrosinase (30 U/mL) and 20 µL of the inhibitor solution were placed in the
wells of a 96-well micro plate. After pre-incubation for 10 min at room temperature, 20 µL of L-
DOPA (3,4-dihydroxyphenylalanine, Sigma Chemical, USA) (0.85 mM) was added and the
assay plate was further incubated at 25 °C for 20 min. Afterward the absorbance of dopachrome
was measured at 475 nm using a micro plate reader (OPTI Max, Tunable). Kojic acid was used
as a reference inhibitor and phosphate buffer was used as a negative control. The amount of
inhibition by the test compounds was expressed as the percentage of concentration necessary to
achieve 50 % inhibition (IC50). Each concentration was analyzed in three independent
experiments. The IC50 values were determined by the data analysis and graphing software
Origin 8.6, 64-bit.
The % of Inhibition of tyrosinase was calculated as following
Inhibition (%) = [(B-S)/B] ×100
Here, the B and S are the absorbance’s for the blank and samples.
5.5 Kinetic analysis of the inhibition of tyrosinase
On the basis of IC50 we select most potent 4f compound for kinetic analysis. A series of
experiments were performed to determine the inhibition kinetics of 4f by following the already
reported method. [9,10]. The inhibitor concentrations for 4f are 0.271, 0.542, 1.084, 2.18 and
4.336 µM. Substrate L-DOPA concentration was between 0.0625 to 2 mM in all kinetic studies.
Pre-incubation and measurement time was the same as discussed in mushroom tyrosinase
inhibition assay protocol. Maximal initial velocity was determined from the initial linear portion
of absorbance up to five minutes after addition of enzyme at a 30s interval. The inhibition type
on the enzyme was assayed by Lineweaver–Burk plots of inverse of velocities (1/V) versus the
inverse of substrate concentration 1/[S] mM-1. The EI dissociation constant Ki was determined
by secondary plot of 1/V versus inhibitor's concentrations.
5.5 Free radical scavenging assay
Radical scavenging activity was determined by modifying already reported method [9,10] by 2,
2-diphenyl-1-picrylhydrazyl (DPPH) assay. The assay solution consisted of 100 µL of DPPH
24

ACCEPTED MANUSCRIPT
25
(150 µM), 20 µL of increasing concentration of test compounds and the volume was adjusted to
200 µL in each well with DMSO. The reaction mixture was then incubated for 30 minutes at
room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The assay
measurements were carried out by using a micro plate reader (OPTI Max, Tunable) at 520 nm.
The reaction rates were compared and the percent inhibition caused by the presence of the tested
inhibitors was calculated. Each concentration was analyzed in three independent experiments run
in triplicate.
5.6 Molecular docking
5.7 Retrieval of receptor protein from PDB
The crystal structure of mushroom tyrosinase (PDBID: 2Y9X) was retrieved from the Protein
Data Bank (PDB) (http://www.rcsb.org). The energy minimization of selected protein was done
by the conjugate gradient algorithm and Amber force field in UCSF Chimera 1.10.1 [25]. The
stereo-chemical properties, Ramachandran graph and values [29] of mushroom tyrosinase were
assessed by Molprobity server [30], while the hydrophobicity graph was generated by Discovery
Studio 4.1 Client [31]. The protein architecture and statistical percentage values of helices, beta-
sheets, coils and turns were accessed by VADAR 1.8 [32].
5.8 Candidate structures
The synthesized candidate molecules (4a-4j) were sketched in drawing ACD/ChemSketch tool.
The designed ligand molecules were further visualized and minimized by UCSF Chimera 1.10.1
in PDB format. The drug assessment properties of these compounds were accessed by various
computational tools like Molinspiration (http://www.molinspiration.com/) and Molsoft
(http://www.molsoft.com/). Lipinski’s rule of five was justified by using Molsoft and
Molinspiraion tools. Furthermore, different molecular properties such as molar refractivity,
density, surface tension and polarizability were also accessed by chemsketch.
5.9 Molecular Docking
The molecular docking experiment was employed on all synthesized ligand molecules (4a-4j)
against mushroom tyrosinase using diverse PyRx tool [33]. The grid box parametric dimension
values (X=61.0781, Y=56.3001 and Z=63.1015) with spacing 1.0Å were adjusted to attain the
25

ACCEPTED MANUSCRIPT
26
finest binding conformational pose of protein-ligand molecules. The maximum docking poses
(100 numbers of run) for each docking were adjusted. All compounds were docked separately
against the crystal structure of mushroom tyrosinase and the obtained docked complexes were
further evaluated on lowest binding energy (Kcal/mol) value. The binding analysis and graphical
depiction was done by Discovery Studio (4.1).
6. X-ray Crystallography studies
Figure 6 shows the molecular structure of (4a) and Table S1 as supplementary material
summarizes the main crystallographic parameters. The molecular conformation is stabilized by
an Intramolecular N-H…O hydrogen bond. The crystal packing shows centosymmetric dimers
connected by N-H…S hydrogen bonds, which are packing in stacks parallel to the a axis.
26

ACCEPTED MANUSCRIPT
27
Figure 6. Crystal structure of 4a
Figure 7. Crystal Packing diagram for 4a viewed along a-axis
27

ACCEPTED MANUSCRIPT
28
Acknowledgement
The authors are thankful to Quaid-i-Azam University, Islamabad, Pakistan, for providing healthy
envoironment for research
Conflict of Interest
The authors declare no any conflict of interest
References
[1] Jung, S.W., Lee, N.K., Kim, S.J., Han, D.S., Korean J. Food Sci. Technol 27 (1995) 891-
896.
[2] Zhu, Y.J., Zhou, H.T., Hu, Y.H., Tang, J.Y., Su, M.X., Guo, Y.J., Chen, Q.X., Liu, B. Food
Chem 124 (2011) 298-302.
[3] Todaro, A., Cavallaro, R., Argento, S., Branca, F., Spagna, G., J. Agricultural. Food Chem,
59 (2011) 11244-11248.
[4] Yoruk, R., Marshall, M.R., J. Food. Biochem 27 (2003) 361-422.
[5] (a) Whitaker, J.R. Principles of Enzymology for the Food Sciences, Second Ed., 1994, 271-
556, Marcel Dekker, New York. (b) Whitaker, J.R. Polyphenol oxidase. In Food Enzymes
Structure and Mechanism, (D.W.S. Wong, ed.) 1995, 271-307, Chapman and Hall, New York.
(c) Whitaker, J.R. Polyphenol oxidase. In Food Chemistry, (O.R. Fennema, ed.) 1996, 492-494,
Marcel Dekker, New York.
[6] Uchida, R., Ishikawa, S., Tomoda, H., Acta Pharmaceutica Sinica B, 4 (2014), 141-145.
28