1,2,3-Triazole-linked 5-benzylidene (thio)barbiturates as novel tyrosinase inhibitors and free-radical scavengers

Article abstract of DOI:10.1002/ardp.202000058

In this study, benzyl-1,2,3-triazole-linked 5-benzylidene (thio)barbiturate derivatives 7a–d and 8a–h were designed as potential tyrosinase inhibitors and free-radical scavengers. The twelve derivatives were synthesized via the [3+2] cycloaddition reactio

Full text of DOI:10.1002/ardp.202000058

Received: 25 February 2020
Revised: 14 June 2020
Accepted: 15 June 2020
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DOI: 10.1002/ardp.202000058
F U L L P A P E R
1,2,3‐Triazole‐linked 5‐benzylidene (thio)barbiturates as
novel tyrosinase inhibitors and free‐radical scavengers
Sara Ranjbar1 | Parisa‐sadat Shahvaran2 | Najmeh Edraki3
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Mahsima Khoshneviszadeh3 | Mahdieh Darroudi4 | Yaghoub Sarrafi4
Mahshid Hamzehloueian5 | Mehdi Khoshneviszadeh2,3
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1Pharmaceutical Sciences Research Center,
Shiraz University of Medical Sciences,
Abstract
Shiraz, Iran
In this study, benzyl‐1,2,3‐triazole‐linked 5‐benzylidene (thio)barbiturate derivatives
7a–d and 8a–h were designed as potential tyrosinase inhibitors and free‐radical
scavengers. The twelve derivatives were synthesized via the [3+2] cycloaddition
reaction of the corresponding benzyl azide as a dipole and the corresponding alkyne
as a dipolarophile in the presence of copper(I) species, generated in situ from copper
(II)/ascorbate. The thiobarbiturate derivative 8h and the barbiturate derivative 8b
bearing 4‐fluoro and 4‐bromo groups on the benzyl–triazole moiety were found to
be the most potent tyrosinase inhibitors with IC50 values of 24.6 0.9 and
26.8 0.8 μM, respectively. Almost all the compounds showed a good radical
scavenging activity with EC50 values in the range of 29.9–324.9 μM. Derivatives 7a,
8f, and 8h were the most potent free‐radical scavengers with EC50 values of
29.9 0.8, 36.8 0.9, and 39.2 1.1 μM, respectively. The kinetic analysis revealed
that compound 8h was a mixed‐type tyrosinase inhibitor. The molecular docking
analysis indicated that 8b and 8h were well accommodated in the active site of the
tyrosinase enzyme and possessed the most negative binding energy values of −8.55
and −8.81 kcal/mol, respectively. Moreover, it was found that the two residues,
Asn81 and Glu322, played a significant role in forming stable enzyme–inhibitor
complexes.
2Department of Medicinal Chemistry, School
of Pharmacy, Shiraz University of Medical
Sciences, Shiraz, Iran
3Medicinal and Natural Products Chemistry
Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
4Department of Organic Chemistry, Faculty of
Chemistry, University of Mazandaran,
Babolsar, Iran
5Department of Chemistry, Jouybar Branch,
Islamic Azad University, Jouybar, Iran
Correspondence
Mehdi Khoshneviszadeh, Department of
Medicinal Chemistry, School of Pharmacy,
Shiraz University of Medical Sciences,
PO Box 71345‐3388, Shiraz, Iran.
Email: m.khoshneviszadeh@gmail.com
Funding information
Vice‐Chancellor for Research, Shiraz
University of Medical Sciences, Iran,
Grant/Award Numbers: 95‐01‐103‐12178,
98‐01‐36‐20126
K E Y W O R D S
antioxidant, barbituric acid, diphenolase activity, docking, kojic acid, triazole
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1
INTRODUCTION
to generate melanin; hence, tyrosinase has a crucial role in melano-
genesis and many tyrosinase inhibitors have been developed for the
Tyrosinase (EC 1.14.18.1) is a copper‐containing oxidase enzyme
distributed widely in nature. It is a rate‐limiting enzyme that con-
tributes to melanin biosynthesis by catalyzing the two consecutive
oxidation steps of L‐tyrosine.[1] Initially, tyrosinase interacts with
L‐tyrosine to produce 3,4‐dihydroxy‐L‐phenylalanine (L‐DOPA;
monophenolase activity), and in the second step, the enzyme
catalyzes the oxidation of L‐DOPA to dopaquinone (diphenolase
activity).[2] Dopaquinone is a reactive substance that can polymerize
remedy of hyperpigmentation‐related skin disorders, such as mel-
asma, freckles, ephelides, and senile lentigines.[3,4] Tyrosinase is also
associated with neurodegenerative disorders including Parkinson's
and Huntington's diseases, as the excessive formation of dopaqui-
none in the brain results in neuronal damage and cell death.[5]
Moreover, tyrosinase plays an important role in enzymatic browning
of fruits and vegetables, which leads to fast degradation during the
postharvest and handling processes.[6] Therefore, tyrosinase is an
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Arch Pharm. 2020;e2000058.
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© 2020 Deutsche Pharmazeutische Gesellschaft
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evolving target in the fields of medicine, cosmetics, agriculture, and
food industry, and several natural and synthetic tyrosinase inhibitors,
including hydroquinone, ascorbic acid derivatives, azelaic acid, re-
tinoids, arbutin, kojic acid, resveratrol, and polyphenolic compounds,
have been applied.[4] However, some common tyrosinase inhibitors,
such as hydroquinone, kojic acid, and arbutin, have been reported to
cause undesirable side effects, including dermatitis, cytotoxicity, and
carcinogenicity.[4,7,8] Besides, tyrosinase inhibitors have some lim-
itations such as low efficacy, poor bioavailability, and instability
during storage, and they can also cause off‐flavor, off‐donor, and
allergic reactions.[4,9] Therefore, the identification and development
of new, effective, and safe tyrosinase inhibitors with drug‐like
properties are greatly needed.
as tyrosinase inhibitors. The synthesized compounds were screened for
tyrosinase inhibitory and antioxidant (radical scavenging) activities. The
inhibition kinetic analysis was carried out for the most potent deriva-
tive. Moreover, to gain some information on the ligand–receptor in-
teractions, a molecular docking analysis was performed.
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2
RESULTS AND DISCUSSION
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2.1
Design approach
The structure of target benzyl‐1,2,3‐triazole‐linked 5‐benzylidene
(thio)barbiturates was designed by applying molecular hy-
a
Barbituric acid and its derivatives have attracted the attention of
scientists due to their therapeutic importance. Barbiturates have ex-
hibited various biological activities such as antibacterial,[10,11] sedative,
hypnotic,[12,13] antispasmodic,[14] anticonvulsant,[15] anticancer,[16,17]
anti‐inflammatory,[18] antiurease,[19] antitubercular,[20] and anti-
oxidant[21] ones. It is also reported that some arylidene barbiturates
are potent tyrosinase inhibitors.[22–24] Also, the 1,2,3‐triazole hetero-
cyclic nucleus has emerged as a versatile pharmacophore due to its
potency to establish hydrogen bonding and dipole–dipole interactions
that have made it very stable to hydrolysis and oxidative/reductive
conditions.[25] 1,2,3‐Triazole‐containing compounds have shown
bridization strategy based on the structures of some potent tyr-
osinase inhibitors, as reported in the literature (Figure 1). It has
been reported that hydroxyl/methoxy‐substituted 5‐benzylidene
(thio)barbiturates exhibited
a tyrosinase inhibitory activity
(IC50 = 1.50–100.0 μM). Benzylidene barbiturate and benzylidene
thiobarbiturate derivatives bearing 4‐methoxy function possessed
IC50 values of 100.0 and 70.1 μM, respectively (Figure 1a).[23]
Moreover, Khan et al.[34] introduced a series of 5‐benzylidene
barbiturates containing different substitutions on benzylidene
moiety as a class of DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) radical
scavengers with IC50 values in the range of 6.2–406.7 μM. In our
previous study, we proved that some phthalimide–1,2,3‐triazole
(Figure 1b) hybrid structures were potent tyrosinase inhibitors.[32]
On the basis of these findings, 5‐benzylidene (thio)
barbiturate–1,2,3‐triazole hybrids bearing different benzyl moi-
eties were designed (Figure 1).
a
broad range of biological activities such as anticancer,[26]
antibacterial,[27] anti‐inflammatory,[28] antifungal,[29] antitubercular,[30]
antiacetylcholinesterase,[31] and antityrosinase activities.[32,33]
Considering the above‐mentioned pharmacophores, 1,2,3‐triazole‐
linked 5‐benzylidene (thio)barbiturates were designed and synthesized
FIGURE 1 The design of proposed
tyrosinase inhibitors using the molecular
hybridization approach

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2.2
Chemistry
Finally, the cycloadducts 7 and 8 were obtained from the [3+2] cy-
cloaddition reaction of the corresponding benzyl azide (6a–d) as a
dipole and the corresponding alkyne (4a–f) as a dipolarophile in the
presence of copper(I) species, generated in situ from copper(II)/as-
corbate in dimethyl sulfoxide (DMSO).
The procedure for the synthesis of 1,2,3‐triazole‐linked 5‐
benzylidene (thio)barbiturates (7a–d and 8a–h) is illustrated in
Scheme 1. Initially, 2‐(prop‐2‐yn‐1‐yloxy)benzaldehyde (2a), 4‐(prop‐
2‐yn‐1‐yloxy)benzaldehyde (2b), 5‐bromo‐2‐(prop‐2‐yn‐1‐yloxy)
benzaldehyde (2c), and 5‐bromo‐4‐methyl‐2‐(prop‐2‐yn‐1‐yloxy)
benzaldehyde (2d) were synthesized from the reaction of the cor-
responding hydroxybenzaldehyde (1a–d) and propargyl bromide in
the presence of K2CO3. Then, the alkynes 4a–f were acquired
through the Knoevenagel condensation of the related (thio)barbituric
derivative (3a–c) and corresponding propargylated hydro-
xybenzaldehyde (2a–d) under reflux conditions in good yields. The
reaction of different benzyl bromides 5a–d and sodium azide in di-
methylformamide (DMF) at 100°C afforded organic azides 6a–d.
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2.3
Biological activity
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2.3.1
Mushroom tyrosinase inhibitory activity
Synthesized compounds were screened for their potential inhibitory
effect on the diphenolase activity of mushroom tyrosinase. According
to the IC50 values listed in Table 1, thiobarbiturate derivative 8h and
barbiturate derivative 8b were the most potent tyrosinase inhibitors
SCHEME 1 The synthesis of 1,2,3‐triazole‐linked 5‐benzylidene (thio)barbiturates. DMF, dimethylformamide; DMSO, dimethyl sulfoxide

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TABLE 1 The tyrosinase inhibitory activity and free‐radical scavenging potential of synthesized compounds
Tyrosinase
inhibitory activitya
DPPH radical
scavenging activitya
Compounds
X
O
O
O
O
O
O
O
S
R
R′
–
Y
Z
IC50 (µM)b
EC50 (μM)
29.9 0.8
>100
7a
–
2‐Cl
4‐Br
H
H
>50
7b
–
–
H
>50
7c
–
–
CH3
CH3
H
>50
134.3 2.3
67.8 0.7
1,000.1 3.6
239.6 1.5
324.9 2.8
74.8 0.6
195.0 3.5
36.8 0.9
122.1 2.4
39.2 1.1
–
7d
–
–
4‐F
H
>50
8a
H
H
H
H
H
H
CH3
CH3
–
H
H
Br
H
H
H
Br
Br
–
49.1 2.2
26.8 0.8
>50
8b
4‐Br
4‐F
H
H
8c
H
8d
H
44.4 0.4
44.2 1.3
>50
8e
S
4‐Br
4‐F
H
H
8f
S
H
8g
S
H
>50
8h
S
4‐F
–
H
24.6 0.9
16.5 0.3
–
Kojic acid
Quercetin
–
–
–
–
–
–
–
9.4 0.6
Abbreviation: DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl.
aValues represent means standard error of three to four independent experiments.
b50% inhibitory concentration (IC50).
with IC50 values of 24.6 0.9 and 26.8 0.8 μM, respectively. Com-
pounds 8a, 8d, and 8e demonstrated a moderate tyrosinase in-
hibitory activity with IC50 values in the range of 44.2–49.1 μM. In
general, Group 8 compounds containing benzyl–triazolyl–methoxy
substitutions at the ortho position of 5‐benzylidene (thio)barbiturate
moiety showed a superior inhibitory activity on tyrosinase than
Group 7 compounds bearing benzyl–triazolyl–methoxy groups at the
para position. It seems that substitutions on the benzyl ring have no
considerable effect on the inhibitory potential of Class 7 compounds;
however, this does not apply to Group 8 compounds. Interestingly,
introducing a 4‐Br function on the benzyl core of Group 8 barbitu-
rates, as in 8b, led to a remarkable increase in the activity as com-
pared with 8a (IC50 = 49.1 2.2 μM). Changing the barbiturate ring in
compound 8b to thiobarbiturate, as in compound 8e (IC50 = 44.2
1.3 μM), reduced the tyrosinase inhibitory activity. However, repla-
cing 4‐Br with 4‐F in the case of thiobarbiturate 8h (IC50 = 24.6 0.9)
improved the inhibitory potential.
(thio)barbiturate (IC50 = 70.1–100.0 μM); however, most of the
hydroxyl‐substituted benzylidene (thio)barbiturates (IC50 = 1.5–17.1
μM) were more effective inhibitors than compounds 8h and 8b.[23,24]
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2.3.2
Free‐radical scavenging activity
All the compounds except 8a exhibited a good radical scavenging
activity with EC50 values in the range of 29.9–324.9 μM (Table 1).
Compounds 7a, 8f, and 8h were the most potent free‐radical sca-
vengers with EC50 values of 29.9 0.8, 36.8 0.9, and 39.2 1.1 μM,
respectively. Derivatives 7d and 8d showed a moderate activity with
EC50 values of 67.8 0.7 and 74.8 0.6 μM, respectively.
Considering the data listed in Table 1, a brief structure–activity re-
lationship can be presented for benzyl‐1,2,3‐triazole‐linked 5‐benzylidene
(thio)barbiturates as free‐radical scavengers. Generally, compounds
bearing a chloro (compound 7a) or a fluoro (compounds 8f and 8h) group
on benzyl–triazole moiety showed a greater free‐radical scavenging ac-
tivity than compounds having a hydrogen or a bromo substitution. The
introduction of bromo or methyl substituent on benzylidene moiety, as in
8g and 8h in comparison with their counterparts 8d and 8f, would reduce
It can be stated that benzyl‐1,2,3‐triazole‐linked 5‐benzylidene
(thio)barbiturate derivatives 8h and 8b (IC50 = 24.6 0.9 and
26.8 0.8 μM, respectively) were better tyrosinase inhibitors than
(thio)barbituric acid (IC50 = >200 μM) and 4‐methoxybenzylidene

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the activity. Comparing EC50 values of 8d and 8e with 8a and 8b, re-
spectively, it can be deduced that thiobarbiturates were better radical
scavengers than their barbiturate counterparts.
enzyme, a molecular docking analysis was carried out. Docking re-
sults and binding affinities of the enzyme–inhibitor complexes are
listed in Table 2. The more negative estimated free energy of binding
(ΔG) indicated the more stable complex that was formed between
the ligand and target enzyme. The docking results are in good
agreement with the experimental outcomes. Docking studies in-
dicated that all the active derivatives were well accommodated
within the tyrosinase binding pocket with binding energy values
ranging from −6.95 to −8.81 kcal/mol. The most active benzyl‐1,2,3‐
triazole‐linked 5‐benzylidene (thio)barbiturate derivatives 8b and 8h
possessed the most negative binding energy values of −8.55 and
−8.81 kcal/mol, respectively.
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2.3.3
Determining the inhibitory mechanism on
mushroom tyrosinase
The most potent tyrosinase inhibitor, derivative 8h, was selected to
determine its inhibition type on mushroom tyrosinase diphenolase
activity. The kinetic studies of the enzyme by the Lineweaver–Burk
plot of 1/V versus 1/[S] in the presence of different inhibitor con-
centrations gave a series of straight lines intersected within the
second quadrant, as shown in Figure 2. With the increase in the
concentration of 8h, there was an increase in Km values and a de-
crease in Vm values. The result indicated that 8h inhibits tyrosinase
by two different pathways: competitively forming enzyme–inhibitor
complex and interrupting enzyme–substrate–inhibitor complex in a
noncompetitive manner. Therefore, it could be stated that 8h was a
mixed‐type tyrosinase inhibitor. The equilibrium constants for its
binding with free enzyme, KI, and with the enzyme–substrate com-
plex, KIS, were determined from the second plots of Km/Vm and 1/Vm
versus the concentration of 8h, respectively. The values of KI and KIS
were calculated to be 60.27 and 143.51 µM, respectively.
The binding poses and interactions of 8b and 8h at the binding
site of mushroom tyrosinase are depicted in Figures 3a and 3b, re-
spectively. The pyrimidine‐trione ring in 8b compound and the
thioxo‐dihydropyrimidine‐dione ring of derivative 8h were similarly
oriented in the enzyme active site so that the two rings formed three
key hydrogen bonds with His85, Asn81, and Glu322, and also one
Pi–H interaction with His85. However, the presence of different
substituents on phenyl rings caused other moieties of 8a and 8h to
have dissimilar orientations. The triazole ring in compound 8a formed
a hydrogen bond with Ser282 residue and 4‐Br substituent inter-
acted with copper ions; however, in the case of 8h, the triazole core
formed a hydrogen bond with His244 and 4‐fluorophenyl ring par-
ticipated in a Pi–alkyl interaction with Val283.
From the docking results presented in Table 2, it can be stated
that the orientations of pyrimidine‐trione and thioxo‐
dihydropyrimidine‐dione rings, and their ability to form hydrogen
bonds with Asn81 and Glu322 had a significant role in the inhibitory
potency of these compounds. In the case of the less potent tyrosinase
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2.4
Molecular docking study
To inquire about the binding mode and potential interactions of
presented tyrosinase inhibitors at the active site of tyrosinase
(a)
(b)
200
180
160
140
120
100
80
25
4
20
15
10
3
5
0
0
20
40
60
[I] µM
2
1
(c)
25
20
15
10
5
60
40
20
0
0
-4
-2
0
2
4
6
8
10
0
20
40
60
-20
1/[S] (mM)-1
[I] µM
FIGURE 2 (a) Lineweaver–Burk plots for tyrosinase inhibition in the presence of 8h as an inhibitor and L‐DOPA as a substrate. The
reciprocal tyrosinase inhibitory activity was plotted against the reciprocal substrate concentration (double‐reciprocal plot, n = 3).
Concentrations of 8h for curves 1–4 were 0, 10, 25, and 50 μM, respectively. (b, c) Plots of slope and intercept versus concentration of 8h for
determining the inhibition constants KI and KIS, respectively

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TABLE 2 Docking results of the suggested tyrosinase inhibitors at the tyrosinase binding site
Code
8a
ΔG (kcal/mol)
−6.95
Ki (μM)
Interactions
Atom of ligand
Amino acid
Distance (Å)
8.07
Pi–alkyl
Pi–H
Phenyl ring
Val283
His244
Pyrimidine‐trione ring
8b
−8.55
0.54
H‐bonding
H‐bonding
H‐bonding
H‐bonding
C═O of pyrimidine‐trione ring
C═O of pyrimidine‐trione ring
N–H of pyrimidine‐trione ring
N of triazole ring
His85
Asn81
Glu322
Ser282
Cu
1.76
2.14
2.01
1.97
2.83
2.90
Br
Br
Cu
Pi–H
Pyrimidine‐trione ring
His85
8d
8e
−7.78
−8.15
1.97
1.07
H‐bonding
H‐bonding
N–H of thioxo‐dihydropyrimidine‐dione
C═S of thioxo‐dihydropyrimidine‐dione
His244
His85
1.81
3.16
H‐bonding
H‐bonding
N–H of thioxo‐dihydropyrimidine‐dione
His244
His85
Cu
1.81
3.07
2.58
2.64
C═S of thioxo‐dihydropyrimidine‐dione
Br
Br
Cu
8h
−8.81
0.35
H‐bonding
H‐bonding
H‐bonding
H‐bonding
Pi–H
C═S of thioxo‐dihydropyrimidine‐dione
C═S of thioxo‐dihydropyrimidine‐dione
N–H of thioxo‐dihydropyrimidine‐dione
N of triazole ring
His85
1.96
1.83
2.05
2.02
Asn81
Glu322
His244
His85
Thioxo‐dihydropyrimidine‐dione
Thioxo‐dihydropyrimidine‐dione
4‐Fluorophenyl ring
Pi–H
His85
Pi–alkyl
Val283
FIGURE 3 The binding orientation of (a) 8b
and (b) 8h within the active site of tyrosinase
enzyme (PDB: 2Y9X). The inhibitors and amino
acids are displayed as cyan and gray sticks,
respectively

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inhibitors 8d and 8e, the thioxo‐dihydropyrimidine‐dione formed two
hydrogen bonds with His85 and His244, but the other two key hy-
drogen bonds with Asn81 and Glu322 were not observed. The least
potent inhibitor 8a possessed the least favorable binding energy.
Moreover, the orientation of 8a was very different from that of the
other compounds and the pyrimidine‐trione ring formed no
hydrogen‐bonding interaction with the active site residues.
operating at an ionization potential of 70 eV. Elemental analyses were
realized using a PerkinElmer 2400II CHNS/O elemental analyzer.
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as Supporting Information Data.
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4.1.2
Synthesis
All triazoles incorporating barbituric motifs were prepared according
to the described procedure.[35]
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3
CONCLUSIONS
The 5‐benzylidene (thio)barbiturate–1,2,3‐triazole hybrids bearing
different benzyl moieties were designed according to the structures
of 4‐methoxybenzylidene (thio)barbiturate derivatives and
phthalimide–triazole hybrid analogs reported in the literature as
tyrosinase inhibitors. The compounds were synthesized to evaluate
their tyrosinase inhibitory activity and DPPH free‐radical scavenging
potential. Two derivatives, 8h and 8b, showed a promising inhibitory
effect on the tyrosinase diphenolase activity (IC50 = 24.6 0.9 and
26.8 0.8 μM, respectively). The compounds exhibited a better tyr-
osinase inhibitory activity than each of their primary components,
based on which they were designed. Most of the compounds ex-
hibited a good radical scavenging activity; however, 7a, 8f, and 8h
were the most potent derivatives with EC50 values of 29.9 0.8,
36.8 0.9, and 39.2 1.1 μM, respectively. The kinetic analysis
showed that compound 8h was a mixed‐type tyrosinase inhibitor.
Docking analysis results of the derivatives showed that the tyr-
osinase inhibitory activity was in agreement with the biological
evaluation. 8b and 8h possessed the most negative binding energy
values of −8.55 and −8.81 kcal/mol, respectively. Moreover, it was
found that the Asn81 and Glu322 amino acids played a significant
role in forming stable enzyme–inhibitor complexes. Consequently,
compound 8h could be a promising lead with dual biological activities
as a potent tyrosinase inhibitor and a potential antioxidant in the
field of drug discovery, and further development of such a compound
in cosmetics, medicine, or food industry may be of interest.
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4.1.3
Propargylation of hydroxybenzaldehyde
derivatives 2a–d
Propargyl bromide (6 mmol) was added to a stirred solution of cor-
responding hydroxybenzaldehydes 1a, 1b, 1c, or 1d (5 mmol) and
potassium carbonate (5 mmol) in DMF (15 ml). The mixture was
stirred for 4–24 hr, water was added, and the precipitate was filtered
and washed with water.
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4.1.4
General procedure for the preparation of
5‐[prop‐2‐yn‐1‐yloxy)benzylidene] (thio)
barbiturates 4a–f
5‐[4‐(Prop‐2‐yn‐1‐yloxy)benzylidene]pyrimidine‐2,4,6(1H,3H,5H)‐trione (4a),
5‐[2‐(prop‐2‐yn‐1‐yloxy)benzylidene]pyrimidine‐2,4,6(1H,3H,5H)‐trione (4b),
5‐[5‐bromo‐2‐(prop‐2‐yn‐1‐yloxy)benzylidene]pyrimidine‐2,4,6(1H,3H,5H)‐
trione (4c), 5‐[2‐(prop‐2‐yn‐1‐yloxy)benzylidene]‐2‐thioxodihydropyrimidine‐
4,6(1H,5H)‐dione (4d), and 5‐[5‐bromo‐4‐methyl‐2‐(prop‐2‐yn‐1‐yloxy)
benzylidene]‐2‐thioxodihydropyrimidine‐4,6(1H,5H)‐dione (4e):
Propargylated aldehydes 2a–d (1 mmol) were added to a stirred
solution of (thio)barbituric acid 3a,b (1.2 mmol) in aqueous HCl
(25 ml, 10%; 1.0 mmol) at room temperature. After the mixture was
stirred for 2–10 hr, the precipitated material was filtered and washed
with water and ethanol.
1,3‐Dimethyl‐5‐[4‐(prop‐2‐yn‐1‐yloxy)benzylidene]pyrimidine‐
2,4,6(1H,3H,5H)‐trione (4f):
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4
EXPERIMENTAL
Propargylated aldehyde 2b (1.0 mmol) was added to a stirred
solution of N,N‐dimethylbarbituric acid (3c, 1.2 mmol) in water
(20 ml) containing (NH4)2HPO4 (20 mol%) at room temperature.
After stirring the mixture for 4–12 hr, the yellow precipitate was
filtered and washed with water and ethanol.
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4.1
Chemistry
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4.1.1
General
All reagents and solvents were purchased from commercial suppliers.
Mushroom tyrosinase (EC1.14.18.1), kojic acid, DMSO, L‐DOPA, and
DPPH were purchased from Sigma Chemical Co. (St. Louis, MO).
Melting points were measured on a Kofler hot‐stage apparatus and
were uncorrected. The nuclear magnetic resonance spectra were re-
corded on a Bruker DRX‐400 Avance instrument (400.1 MHz for 1H,
100.6 MHz for 13C) using DMSO as the solvent. The infrared (IR) spectra
were recorded on an FT‐IR Bruker Vector 22 spectrometer. The mass
spectra were recorded on a Finnigan MAT 8430 mass spectrometer
|
4.1.5
General procedure for the preparation of
alkyl azides; 1‐(azidomethyl)‐2‐chlorobenzene (6a),
1‐(azidomethyl)‐4‐bromobenzene (6b), (azidomethyl)
benzene (6c), 1‐(azidomethyl)‐4‐fluorobenzene (6d)
Sodium azide (1.2 mmol) was added to a solution of the required
benzyl bromide derivative 5a–d (1 mmol) in DMF. The mixture was

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Radical scavenging activity (%) = 100 × [(Abscontrol − Abscompound
)
heated at 100°C, and after completion of the reaction (3 hr), the
reaction mixture was quenched with an aqueous solution of NH4Cl
(15 ml) and extracted with ethyl acetate (3 × 20 ml). The combined
organic extracts were washed with brine (3 × 20 ml) and dried over
MgSO4. After evaporation of the solvent at reduced pressure, the
pure azides were isolated.
/Abscontrol].
The EC50 values were calculated from a linear regression plot
between the test compounds' concentrations and percent radical
scavenging activity. Each concentration was analyzed in three in-
dependent experiments run in triplicate.
|
4.1.6
General procedure for the preparation of
|
Inhibitory mechanism of 8h on the
7a–d and 8a–h
4.2.3
diphenolase activity of mushroom tyrosinase
Alkynes 4a–f (1.2 mmol) and benzyl azide 6a–d (1 mmol) were added
to a solution of CuSO4 (0.2 equiv.) in DMSO (10 ml) in a capped flask
at room temperature. The reaction mixture was stirred at 80°C, and
after completion of the reaction (12 hr), the reaction mixture was
quenched with a saturated aqueous solution of NH4Cl (30 ml) and
extracted with ethyl acetate (3 × 40 ml). The combined organic ex-
tracts were washed with brine (3 × 30 ml), dried over Na2SO4, and
concentrated under vacuum.
A series of experiments was performed to determine the inhibition
kinetics of 8h. The inhibitor concentrations were 0, 10, 25, and
50 μM. Substrate (L‐DOPA) concentrations were 0.125, 0.250, 1.000,
and 2.000 mM in all kinetic studies. Pre‐incubation and measurement
time were the same as described in the tyrosinase inhibition assay
procedure. The tyrosinase inhibition type was then calculated using
Lineweaver–Burk plots of inverse velocities (1/V) versus inverse
substrate concentrations, 1/[S] mM. The Michaelis constant (Km) and
maximal velocity (Vm) were calculated by Lineweaver–Burk plots and
the inhibition constants were determined by the second plot of ap-
parent Km/Vm or 1/Vm versus the concentration of the inhibitor.
The spectroscopic characterization of compounds 7a,b, 8a–e,
and 8g,h is provided as Supporting Information Data.
|
4.2
Biological evaluations
|
|
Molecular docking study
4.2.1
Mushroom tyrosinase inhibition assay
4.3
Mushroom tyrosinase was assayed as reported in our previous
studies.[36–38] L‐DOPA was used as the substrate and enzyme activity
was checked by detecting dopachrome formation at 475 nm. All test
samples were dissolved in DMSO at 40 mM and diluted to the re-
quired concentrations. First, 10 ml of tyrosinase (0.5 mg/ml) was
mixed with 160 ml of phosphate buffer (50 mM, pH 6.8) in 96‐well
microplates, and then 10 ml of the test sample was added. The plates
were incubated at 28°C for 20 min, and then 20 ml of L‐DOPA so-
lution (0.5 mM) was added to the phosphate buffer. Each con-
centration was analyzed in three independent experiments run in
triplicate. The inhibitory activity of the tested compounds was ex-
pressed as the concentration that inhibited 50% of the enzyme ac-
tivity (IC50). The percent inhibition ratio was calculated according to
the following equation:
Docking was performed by AutoDock 4.2 and AutoDock Tools
1.5.4 (ADT). The X‐ray crystal structure of Agaricus bisporus tyr-
osinase containing tropolone as the co‐crystallized ligand in the
binding site (PDB ID: 2Y9X) was taken from Protein Data Bank
(http://www.rcsb.org). Before the docking analysis, tropolone and
water molecules were omitted from 2Y9X, and then hydrogens
were added and nonpolar hydrogens were merged. Finally,
Gasteiger charges were calculated for the protein. The three‐
dimensional structures of ligands were sketched and minimized
using HyperChem software. PDBQT formats of the ligands were
prepared. The grid maps were constructed by AutoGrid and grid
box dimensions were set to 40 × 40 × 40 with 0.375‐Å grid spa-
cing. The active site that comprises copper metal ions was
selected for docking and the grids' centers were placed on the
tropolone's binding site. For the docking parameter file, rigid
macromolecule and Lamarckian genetic search algorithm were
chosen. The number of GA runs was set at 100 and other para-
meters were left at program default values.
Inhibition (%) = 100 × [(Abscontrol − Abscompound)/Abscontrol].
|
4.2.2
DPPH free‐radical scavenging assay
The samples were dissolved in DMSO at 40 mM and diluted to the
required concentrations. A mixture of test compound and DPPH
solution (110 μM in methanol) was shaken in the dark at room
temperature for 30 min. The mixture absorbance was measured at
517 nm. The DPPH solution without test compounds was used as the
control and quercetin was used as the positive control. The percen-
tage of free‐radical scavenging activity was calculated as follows:
ACKNOWLEDGMENTS
The authors wish to thank the Vice‐Chancellor for Research, Shiraz
University of Medical Sciences, Iran (project numbers: 95‐01‐103‐
12178 and 98‐01‐36‐20126).
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.

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ORCID
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Nazreen, A. M. Kalle, C. Kharbanda, Y. Ali, Eur. J. Med. Chem. 2013,
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