Evaluation of novel pyrimidine derivatives as a new class of mushroom tyrosinase inhibitor

Article abstract of DOI:10.2147/DDDT.S209324

Background and aim: Tyrosinase (EC 1.14.18.1) is responsible for enzymatic browning in fruits and vegetables. Its inhibitors may be applied to efficiently treat hyperpigmentation and are widely used in pharmaceutical and cosmetic products, food supplements and insecticides. Previous studies have shown that heterocyclic compounds with an amino group can inhibit tyrosinase activity. The present study aims to evaluate the inhibitory effect of some novel 2,6-diamino-4-chloropyrimidine derivatives (1a-e) and 2,4,6-triami-nopyrimidine (2a–e) including bioactive aniline moiety on the activity of the mushroom tyrosinase. Methods: In practice, the azo salt was initially synthesized from aniline derivatives and combined subsequently with the 2,4,6-triaminopyrimidine and 2,6-diamino-4 chloropyrimidine followed by crystallization. The structures of resulting compounds were confirmed by FT-IR,¹³C NMR, and¹ H NMR. The derivatives (0–100 μM) were evaluated for their inhibitory effect on tyrosinase activity using l-3,4-dihydroxyphenylalanine (l-DOPA) as substrate. Results: All compounds showed inhibitory effects against the activity of the enzyme. About 23.72–55.08% inhibition was observed in the presence of 30 μM of each compound. The IC₅₀ values of the synthesized compounds were measured, and their inhibition properties were also visualized by zymography. Based on the results, the compounds 1a-e and 2a-e showed moderate inhibitory activities. Notably, pyrimidine derivatives 1a (IC₅₀=24.68) and 1d (IC₅₀=24.45) also exhibited similar inhibitory activities when compared with the positive control, kojic acid (IC₅₀=25.24 μM). Kinetic studies indicated that the type of inhibition was noncompetitive. Conclusion: All results suggest that pyrimidine derivatives, especially 1d and 1a, can be considered as safe and efficient tyrosinase inhibitors.

Full text of DOI:10.2147/DDDT.S209324

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O R I G I N A L R E S E A R C H
Evaluation of novel pyrimidine derivatives as a new
class of mushroom tyrosinase inhibitor
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
S Shohreh Mirmortazavi1
Background and aim: Tyrosinase (EC 1.14.18.1) is responsible for enzymatic browning
Mahdieh Farvandi1
in fruits and vegetables. Its inhibitors may be applied to efficiently treat hyperpigmenta-
Hossein Ghafouri1
tion and are widely used in pharmaceutical and cosmetic products, food supplements and
Asadollah Mohammadi2
insecticides. Previous studies have shown that heterocyclic compounds with an amino
Mostafa Shourian1
group can inhibit tyrosinase activity. The present study aims to evaluate the inhibitory
effect of some novel 2,6-diamino-4-chloropyrimidine derivatives (1a-e) and 2,4,6-triami-
1Department of Biology, Faculty of
nopyrimidine (2a–e) including bioactive aniline moiety on the activity of the mushroom
Sciences, University of Guilan, Rasht,
Iran; 2Department of Chemistry, Faculty
of Sciences, University of Guilan, Rasht,
Iran
tyrosinase.
Methods: In practice, the azo salt was initially synthesized from aniline derivatives and
combined subsequently with the 2,4,6-triaminopyrimidine and 2,6-diamino-4 chloropyrimi-
dine followed by crystallization. The structures of resulting compounds were confirmed by
FT-IR, 13C NMR, and 1H NMR. The derivatives (0–100 µM) were evaluated for their
inhibitory effect on tyrosinase activity using l-3,4-dihydroxyphenylalanine (l-DOPA) as
substrate.
Results: All compounds showed inhibitory effects against the activity of the enzyme. About
23.72–55.08% inhibition was observed in the presence of 30 µM of each compound. The
IC50 values of the synthesized compounds were measured, and their inhibition properties
were also visualized by zymography. Based on the results, the compounds 1a-e and 2a-e
showed moderate inhibitory activities. Notably, pyrimidine derivatives 1a (IC50=24.68) and
1d (IC50=24.45) also exhibited similar inhibitory activities when compared with the positive
control, kojic acid (IC50=25.24 µM). Kinetic studies indicated that the type of inhibition was
noncompetitive.
Conclusion: All results suggest that pyrimidine derivatives, especially 1d and 1a, can be
considered as safe and efficient tyrosinase inhibitors.
Keywords: mushroom tyrosinase, inhibitor, pyrimidine derivatives, noncompetitive
inhibition
Introduction
Tyrosinase is a copper-dependent enzyme from the oxidoreductase family considered as
the rate-limiting enzyme in the melanin production pathway. Melanin is a dark brown to
black pigment that effectively absorbs light. It is found in plants and fungi as well as in
hair, skin, and iris of the eye in human and animals.1,2 Tyrosinase is one of the multi-
functional enzymes that catalyze two different reactions resulting from the binding of
dioxygen to two copper atoms in the strongly conserved active center: the hydroxylation
of monophenols (tyrosine) to o-diphenols (monophenolase activity) and the oxidation of
o-diphenols to o-quinones (diphenolase activity). The process ultimately leads to the
production of melanin through several reactions from o-quinones.2,3 This type of
Correspondence: Hossein Ghafouri
Department of Biology, Faculty of
Sciences, University of Guilan, Namjoo
Ave, P.O. Box 41335-1914, Rasht, Iran
Tel/Fax +98 133 333 3647
Email h.ghafoori@guilan.ac.ir
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tyrosinase activity is due to four possible oxidation states heterocyclic compounds have anticancer, antiviral, anti-
(deoxy-, oxy-, met- and deact-tyrosinase). At the met state, inflammatory, antibacterial, and antiparkinson properties.15
both copper atoms present in the active site are in Cu (II) state. Considering that several aromatic amines can play a role as
In the case of the deoxy state, copper atoms in Cu (I) can be tyrosinase substrates, and the fact that amines and anilines are
attached to molecular oxygen. At the oxy state, the molecular its inhibitors, we decided to synthesize new derivatives based
oxygen in peroxy conformation is surrounded by both copper on pyrimidine and aniline derivatives.14,17 In this study, for the
atoms in a Cu (II) state, in which case it can be linked to first time, we synthesized a series of novel azo-pyrimidine
phenols and catechols, as substrate, converting them into dyes and evaluated the inhibitory activities of these low-cost
ortho-quinones by different oxidative cycles. The deact state and easy-to-prepare compounds against mushroom tyrosinase
is an inactivated form of tyrosinase with one of the copper based on dopaquinone production by an enzyme assay with
atoms in the Cu (0) state and the other in the Cu (II) state.3 L-dopa as substrate. The IC50 was obtained and compared
Given this background, while pigmentation under normal with kojic acid as positive control. Also, zymography was
conditions protects skin against harmful UV injury, melanin used to show the inhibitory effects of these compounds on
increment due to the tyrosinase hyperactivity can give rise to tyrosinase activity. The kinetic analysis revealed the Ki value
hyperpigmentation disorders, which is followed by freckles, and inhibition mechanism, and finally, the docking study of
wrinkles, age spots, skin irritation, postinflammatory hyper- these compounds was performed by the auto dock software
pigmentation, melanoderma and melasma in extreme cases and Swiss dock web server.
that are particularly common among women.4,5 Tyrosinase is
regarded as a key agent in the risk of malignant melanoma
Materials and equipment
cancer and might play a significant role in neuromelanin
All chemicals and reagents used in this study were purchased
production responsible for the neurodegeneration associated
from Sigma-Aldrich Co., St Louis, MO, USA and Merck
with Parkinson’s and Huntington’s diseases.6,7 Tyrosinase has
Chemical companies and used without further purification.
also been confirmed to be one of the most important causes of
Silica gel thin layer chromatography (TLC) using n-hexane
browning in vegetables and fruits during post-harvest, hand-
and ethyl acetate as mobile phase was used to monitor the
ling, and storage, leading to quick degradation.8,9 Finally,
progress of reactions. The solutions were prepared using dis-
tyrosinase has a different biochemical process in insects,
tilled water. Melting points (°C) of the synthesized compounds
including wound healing, sclerotization, melanin synthesis,
were recorded on Electrothermal IA9000 equipment (Cole-
and defensive encapsulation.10,11 Therefore, the production
Parmer, Stone, UK) and are uncorrected. The UV-visible
of useful and safe inhibitors of tyrosinase as insecticides and
absorption spectra were recorded using a Pharmacia Biotech
skin lightening agents is important for the agricultural, cos-
Spectrophotometer in the range of 200–800 nm. Fourier trans-
metic and medical industries. While there have been many
form infrared (FT-IR) spectra were recorded on a Shimadzu
efforts to discover effective inhibitors, only a few compounds
8400 FT-IR spectrophotometer (Shimadzu, Kyoto, Japan).
have been used commercially for therapeutic and cosmetic
1H NMR and 13C NMR spectra were obtained on an FT-
uses; these include kojic acid, tropolone, arbutin and 1-phenyl-
NMR (400 MHz) Bruker apparatus (Bruker AXS Inc.,
2-thiourea (PTU).12,13 Tyrosinase can oxidize several aromatic
Madison, WI, USA), the chemical shifts were expressed in δ
amines and o-aminophenols as substrates, which are analogs
of monophenols and o-diphenols, respectively.13,14
ppm using TMS as an internal standard and J values in Hz.
4-Aminophenylalanine, 4-aminotoluene, 4-aminoacetanilide,
Synthesis of pyrimidine-based azo dyes
(1a-e and 2a-e)
and several o-aminophenolic derivatives of benzoic acid
served as substrates for the enzyme.14 Because of the wide-
spread presence of a pyrimidine ring in biological and organic
compounds such as nucleic bases, vitamins, enzymes, chlor-
ophyll, hemoglobin, and hormones, and, moreover, the broad
spectrum of pharmaceutical properties subsequently has
attracted much attention in drug industries.15,16 Pyrimidine
and its derivatives with an active heterocyclic structure having
two nitrogen atoms in the ring are highly regarded as interest-
ing bioactive compounds in medicinal chemistry. These
Pyrimidine-based azo dyes (1a-e and 2a-e) were synthe-
sized in high yield via diazotization and coupling reac-
tions as shown in Figure 1. One mmol of diazonium
salts from donor- and acceptor-substituted anilines was
prepared according to a method previously described.18
To 1 mmol of solution containing coupling reagent
(2,4,6-triaminopyrimidine or 2,6-diamino-4-chloropyri-
midine) was added dropwise from diazonium salt from
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Mirmortazavi et al
+
NH2
N N
HNO2
0-5 °C
X
X
H2N
N
+
NH2
N
N N
N
N
DMF
NH2
N
+
0-5 °C
X
Y
N
Y
NH2
X
1a: X=o-NO2;
Y=Cl
2a: X=p-NHCOCH3;
2b: X=p-CH3;
Y=NH2
Y=NH2
Y=NH2
Y=NH2
Y=NH2
1b: X=p-NO2;
1c: X=p-CH3;
Y=Cl
Y=Cl
2c: X=p-NO2;
1d: X=p-NHCOCH3;Y=Cl
1e: X=p-OCH3;
2d: X=p-OCH3;
2e: X=o-NO2;
Y=Cl
Figure 1 Synthesis of diazonium salts from aniline derivatives. Reagents and conditions: aniline derivatives, H2SO4, NaNO2, stirring at 0–5°C. Synthesis of pyrimidine-based
azo dyes (Y=Cl: 2, 6-diamino-4-chloropyrimidine) Y=NH2: 2, 4, 6-triaminopyrimidine. Reagents and conditions: Diazonium salts from aniline derivatives, TAP or DATP, DMF,
stirring in 0–5°C.
the previous step over 30 min with stirring at 0–5°C.
The stoichiometry ratio was 1:1. The pH of the solution
was slowly adjusted to 7 with HCl (0.5 M). The reaction
was continued by addition of 20 ml distilled water to
precipitate. The precipitate was then filtered and washed
with water (three times). The crude products were iso-
lated by recrystallization from dimethylformamide
(DMF)/H2O at 80°C. Structures of the obtained azo
dyes were confirmed by FT-IR and NMR (1H and 13C)
spectroscopy.
Compound 1c (6-chloro-5-(p-tolyldiazenyl)
pyrimidine-2,4-diamine)
Light brown solid; yield 84%; MP 234–236°C. FT-IR
(KBr, cm−1): 3,385 (NH), 3,312 (NH), 3,205 (NH), 3,028
(=CH), 2,919 (CH), 1,643 (C=C), 1,601 (C=N), 1,551
(N=N). 1H NMR (400 MHz, DMSO-d6, 298 K), δ
(ppm): 2.38 (s, 3H, CH3), 7.29 (s, 1H, NH), 7.33 (d, 2H,
J=8.0 Hz, Ar-H), 7.51 (s, 1H, NH), 7.71 (d, 2H, J=8.4 Hz,
Ar-H), 8.08 (s, 1H, NH), 9.28 (s, 1H, NH). 13C NMR
(DMSO-d6), δ (ppm): 20.4, 118.4, 121.9, 130.3, 139.7,
150.8, 156.5, 161.5, 164.6.
Compound 1a (6-chloro-5-((2-nitrophenyl) diazenyl)
pyrimidine-2,4-diamine)
Compound 1d (N-(4-((2, 4-diamino-
6-chloropyrimidin-5-yl) diazenyl) phenyl) acetamide)
Dark red solid; yield 74%; MP 286–288°C. FT-IR
(KBr, cm−1): 3,425 (NH), 3,390 (NH), 3,127 (NH), 1,691
(C=O), 1,658 (C=C), 1,598 (C=N), 1,502 (N=N). 1H NMR
(400 MHz, DMSO-d6, 298 K), δ (ppm): 2.09 (s, 3H, CH3),
7.24 (s, 1H, NH), 7.34 (s, 1H, NH), 7.75 (m, 4H, Ar-H),
8.05 (s, 1H, NH), 9.27 (s, 2H, NH), 10.19 (s, 1H, NH).
13C NMR (DMSO-d6), δ (ppm): 24.5, 118.9, 122.7, 141.0,
148.3, 156.5, 161.4, 162.8, 164.3, 169.0.
Red solid; yield 79%; melting point (MP) 301–303°C. FT-
IR (KBr, cm−1): 3,467 (NH), 3,308 (NH), 3,138 (NH),
1
1,636 (C=N), 1,565 (NO2), 1,509 (N=N). H NMR (400
MHz, DMSO-d6, 298 K), δ (ppm): 7.58–7.62 (m, 2H),
7.76 (s, 1H, NH), 7.79–7.85 (m, 2H, Ar-H), 8.05 (d, 1H,
J =8.0 Hz, Ar-H), 8.42 (s, 1H, NH), 9.22 (s, 1H, NH).
13C NMR (DMSO-d6), δ (ppm): 118.1, 120.4, 125.1,
129.7, 134.2, 144.7, 146.1, 156.2, 161.9, 166.1.
Compound 1b (6-chloro-5-((4-nitrophenyl) diazenyl)
pyrimidine-2,4-diamine)
Compound 1e (6-chloro-5-((4-methoxyphenyl)
diazenyl) pyrimidine-2,4-diamine)
Orange solid; yield 82%; MP 305–307°C. FT-IR
(KBr, cm−1): 3,502 (NH), 3,327 (NH), 3,186 (NH), 1,632
Dark red solid; yield 72%; MP 270–272°C. FT-IR
(KBr, cm−1): 3,366 (NH), 3,313 (NH), 3,201 (NH), 3,002
(=CH), 2,907 (CH), 1,648 (C=C), 1,602 (C=N), 1,550
(N=N). 1H NMR (400 MHz, DMSO-d6, 298 K), δ
(ppm): 3.35 (s, 3H, CH3), 7.0 (s, 1H, NH), 7.08 (d, 2H,
J =8.8 Hz, Ar-H), 7.23 (s, 1H, NH), 7.79 (d, 2H, J =8.8
Hz, Ar-H), 8.01 (s, 1H, NH), 9.23 (s, 1H, NH). 13C NMR
1
(NO2), 1,569 (C=N), 1,514 (N=N). H NMR (400 MHz,
DMSO-d6, 298 K), δ (ppm): 7.55 (s, 1H, NH), 7.73 (s, 1H,
NH), 7.90 (d, 2H, J=8.8 Hz, Ar-H), 8.35 (d, 2H, J =7.6 Hz,
Ar-H), 8.39 (s, 1H, NH), 9.41 (s, 1H, NH). 13C NMR
(DMSO-d6), δ (ppm): 120.1, 122.5, 125.4, 147.0, 156.4,
161.8, 162.8, 166.2.
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1
(DMSO-d6), δ (ppm): 39.4, 107.9, 114.6, 118.2, 123.4, 1,513 (N=N). H NMR (400 MHz, DMSO-d6, 298 K), δ
147.1, 157.4, 158.2, 160.0
(ppm): 7.27 (s, 2H, NH), 7.46 (t, 1H, J=7.6, Ar-H), 7.71 (t,
1H, J=7.6 Hz, Ar-H), 7.95 (s, 2H, NH), 7.97 (d, 1H, J=8.0
Hz, Ar-H), 8.28 (d, 1H, J=8.4 Hz, Ar-H), 9.39 (s, 2H,
NH). 13C NMR (DMSO-d6), δ (ppm): 111.3, 118.4, 121.3,
123.8, 124.7, 127.6, 133.8, 145.1, 145.7.
Compound 2a N-(4-((2,4,6-triaminopyrimidin-5-yl)
diazenyl) phenyl) acetamide
Red solid; yield 75%; MP 284–286°C. FT-IR (KBr, cm−1):
3,389 (NH), 3,174 (NH), 2,960 (=CH), 2,929 (CH), 1,650
1
(C=O), 1,605 (C=N), 1,501 (N=N). H NMR (400 MHz,
Mushroom tyrosinase inhibition assay
(IC50)
DMSO-d6, 298 K), δ (ppm): 2.09 (s, 3H, CH3), 7.06 (s,
2H, NH), 7.61 (s, 2H, NH), 7.67 (d, 2H, J =8.0 Hz, Ar-H),
7.85 (d, 2H, J =8.0 Hz, Ar-H), 8.66 (s, 2H, NH), 10.10 (s,
1H, NH). 13C NMR (DMSO-d6), δ (ppm): 24.5, 108.2,
119.6, 122.1, 122.4, 139.9, 148.5, 168.9.
Tyrosinase was assayed in the presence of synthesized
products using a previously described method with some
modification.6 Briefly, reaction mixtures (total volume of
100 μL) with 70 μL of phosphate buffer (pH 6.8, 100 mM)
and 0, 10, 30, 60, 100 μM synthesized compounds dis-
solved in DMSO (final concentration 7% v/v) were firstly
prepared. This was followed by the addition of 3 μL mush-
room tyrosinase (0.0003 mg/mL), mixing well and pre-
incubation for 5 min at 37°C. The final concentration of
DMSO (7% v/v) did not interfere with the enzyme activity.
Twenty μL of L-DOPA (10 mM) were then added as the
substrate and incubated for 30 min at 37°C. Subsequently,
the mixed solution was placed in a 96-well plate, and the
absorbance of dopachrome was measured at 490 nm by
ELISA reader. The half maximal inhibitory concentration
of each tested compound (IC50) was determined by inter-
polation of the dose–response curves. All the experiments
were carried out in triplicate for each compound, and con-
centration and values were expressed as the means of three
experiments before further calculation. Kojic acid and phos-
phate buffer were used as a reference inhibitor and negative
control, respectively. The percent inhibition of the enzyme
reaction was calculated as follows:
Compound 2b (5-p-tolyldiazenyl)
pyrimidine-2,4,6-triamine
Orange solid; yield 83%; MP 250–252°C. FT-IR
(KBr, cm−1): 3,428 (NH), 3,338 (NH), 3,150 (NH), 2,958
(=CH), 2,925 (CH), 1,660 (C=C), 1,599 (C=N), 1,501
1
(N=N). H NMR (400 MHz, DMSO-d6, 298 K), δ (ppm):
2.35 (s, 3H, CH3), 6.45 (s, 2H, NH), 7.14 (s, 2H, NH), 7.23
(d, 2H, J =7.6 Hz, Ar-H), 7.70 (d, 2H, J =6.8 Hz, Ar-H), 7.97
(s, 2H, NH). 13C NMR (DMSO-d6), δ (ppm): 21.24, 109.9,
121.4, 129.9, 137.2, 151.3, 162.9, 162.9, 163.
Compound 2c (5-((4-nitrophenyl) diazenyl)
pyrimidine-2,4,6-triamine)
Orange solid; yield 85%; MP 307–309°C. FT-IR
(KBr, cm−1): 3,428 (NH), 3,316 (NH), 3,110 (NH), 1,695
(NO2), 1,508 (N=N). 1H NMR (400 MHz, DMSO-d6, 298
K), δ (ppm): 7.36 (s, 2H, NH), 7.97 (s, 2H, NH), 8.12 (d,
2H, J=8.8 Hz, Ar-H), 8.27 (d, 2H, J=9.2 Hz, Ar-H), 9.05
(s, 2H, NH). 13C NMR (DMSO-d6), δ (ppm): 122.3, 124.1,
125.2, 145.9, 157.4, 161.3, 162.8.
Inhibition rate (%)=(B−S)/B×100
Compound 2d (5-((4-methoxyphenyl) diazenyl)
pyrimidine-2,4,6-triamine)
where B and S are the absorbance values for the blank and
sample, respectively.
Yellow solid; yield 73%; MP 278–280°C. FT-IR
(KBr, cm−1): 3,463 (NH), 3,379 (NH), 3,280 (NH), 3,134
(NH), 1,503 (N=N). 1H NMR (400 MHz, DMSO-d6, 298 K),
δ (ppm): 3.82 (s, 3H, CH3), 7.01 (d, 2H, J=9.2 Hz, Ar-H),
7.05 (s, 2H, NH), 7.60 (s, 2H, NH), 7.88 (d, 2H, J=8.8 Hz,
Ar-H), 8.65 (s, 2H, NH). 13C NMR (DMSO-d6), δ (ppm):
39.4, 118.8, 123.1, 146.9, 156.5, 161.3, 164.1, 165.9.
Determination of the inhibition type and
the inhibition constants
In order to obtain kinetic parameters, the activity of tyrosi-
nase was measured at different concentrations of substrate
(0, 0.1, 0.3, 0.75, 1.25 and 2 mM) in the absence and
presence of 30 μM concentration of inhibitors. The protocol
was the same as discussed in mushroom tyrosinase inhibi-
Compound 2e (5-((2-nitrophenyl) diazenyl)
pyrimidine-2,4,6-triamine)
Orange solid; yield 74%; MP 322–324°C. FT-IR tion assay. Using the value of enzyme activity obtained
(KBr, cm−1): 3,398 (NH), 3,324 (NH), 3,170 (NH), 3,069 from the assay in different concentrations of the substrate
(=CH), 2,962–2,930 (CH), 1,666 (C=C), 1,632 (NO2), and inhibitor, Michaelis–Menten and Lineweaver–Burk
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Mirmortazavi et al
plots were obtained. Km and Vmax were calculated from for obtaining λmax of all compounds was between 200 and
the Lineweaver–Burk plot linear equation and the inhibition 800 nm. Chemical properties of synthetic compounds are
constants were determined by the secondary plots of 1/ presented in Table 1.
Vmax versus the concentration of the inhibitor.
The inhibitory effects of synthetic
compounds on the enzyme activity
Zymogram analysis
The effects of synthetic compounds on tyrosinase activity
were also visualized by native polyacrylamide gel electro-
phoresis (PAGE).19 Synthesized compounds with the
enzyme and the appropriate amount of phosphate buffer
(pH 6.8, 100 mM) were pre-incubated for 10 min and
resolved in 12% PAGE. Preparation of samples was carried
out with a sample buffer without mercaptoethanol without
heat, and the electrophoresis was performed at 4°C. After
electrophoresis, the gel was washed twice with phosphate
buffer at room temperature. An appropriate amount of
L-DOPA (10 mM) was later added in fresh buffer. The
brown color bands of the product appeared after enzymatic
activity with gentle shaking at 37°C for 1 h.
In this study, all compounds were evaluated for their
inhibitory effect on tyrosinase activity according to the
procedure mentioned in the materials and methods section,
and kojic acid was used as the reference inhibitor. The
results indicated that percent inhibition of all compounds
at 30 μM concentration ranged from 23.72% (for 1b) to
55.08% (for 1e) as compared to kojic acid as a positive
control with 57.27% inhibition at 30 μM (Figure 3). The
half maximal inhibitory concentration (IC50) of synthetic
compounds on tyrosinase activity was determined from
logarithmic concentration–inhibition curves (Figure 4).
As shown in Table 2, compounds 1d and 1a revealed the
most potent inhibitory effects on the mushroom tyrosinase
activity with IC50 values of 24.45 and 24.69 μM, respec-
tively. Therefore, it was found that our new compounds
are more potent inhibitors of tyrosinase than the reference
inhibitor, kojic acid (IC50=25.24 μM). In addition, based
on the results, it was found that compound 2d (IC50=43.80
μM) exhibited the lowest inhibitory activity on mushroom
tyrosinase among all synthetic compounds.
Docking
The interaction of compound 1d as a ligand with tyrosinase
was evaluated by molecular docking. The PDB code of the
enzyme from the RCSB Protein Data Bank (http://www.rcsb.
org) was obtained as 2Y9W.20 The REDUCE program were
fixed missing hydrogen of the tyrosinase crystal structure.21
The blind molecular docking was performed by the AutoDock
program.22 The necessary input files were prepared by
MGLTools, and the exhaustiveness parameter was set to
1,000.23 The structure of the ligand was built and optimized
by Hyper-Chem program version 8 using a semi-empirical
method (PM3 force filed).24 The images were created using
Chimera 1.13.1 based on binding modes and scored using their
FullFitness and clustered.25 Also, the molecular interactions
between 1d and tyrosinase was predicted by SwissDock web
service (http://www.swissdock.ch).
Tyrosinase zymography
The effects of synthetic compounds on tyrosinase activity
were visualized by L-DOPA staining assay. Tyrosinase oxida-
tion activity on the gel can be observed as a dark band to
a lighter band after adding L-DOPA solution. Upon incubation
with the synthetic compounds, the band intensity decreased for
each compound as compared to the untreated control. This
phenomenon suggests that all the compounds had an inhibition
effect on tyrosinase. As shown in Figure 5, while the lightest
bands corresponded to 1d and 1a compounds, compound 2d
showed minimal inhibitory effect compared to the other com-
pounds. These results confirm our findings from inhibitory
activity measured by the spectrophotometric method.
The images were created using Python Molecule
Viewer (PMV) and the program LigPlot v.1.0, which gen-
erate schematic two-dimensional representations of pro-
tein–ligand complexes from the input PDB file.23,26
Results
Chemical synthesis
The type of tyrosinase inhibition by
synthetic compounds
The novel bis azo dye compounds (1a-e) and (2a-e) were The steady-state kinetic analysis of all compounds for inhibi-
synthesized (Figure 1). The structures of the synthesized tion of enzyme activity on different concentrations of L-DOPA
compounds were confirmed by FTIR, 1H NMR and (0.1 to 2 mM) was carried out to determine their inhibition type
13C NMR spectroscopic data (Figure 2). Wave scan rang and inhibition constants (Table 2). Lines intersect on
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9.4 9.3 9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 ppm
17 16 15 14 13 12 11 10
9
8
7
6
5
4
3
2
1
ppm
Figure 2 1HNMR spectrum of compound 1a in DMSO.
Table 1 Structures and properties of synthetic compounds
No.
λ
(nm)
MP °C
MW (g/mol)
Molecular
formula
No.
λ
(nm)
max
MP °C
MW (g/mol)
Molecular
formula
max
1a
1b
1c
1d
1e
402
423
456
402
389
301–303 293.67
305–307 293.67
234–236 262.70
286–288 305.7
270–272 287.70
C10H8ClN7O2
2a
2b
2c
2d
2e
394
423
446
384
402
284–286 286.29
250–252 243.27
307–309 274.24
278–280 259.27
322–324 274.24
C12H14N8O
C11H13N7
C
10H8ClN7O2
C11H11ClN6
C10H10N8O2
C11H13N7O
C10H10N8O
C12H14ClN7O
C
11H11ClN6O
Abbreviations: MP, melting point; MW, molecular weight.
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
100
80
60
40
20
0
Docking
The binding of (1d) to tyrosinase (2Y9W) was computerized
by molecular docking. The results of docking were well clus-
tered around the conformer with the best score. According to
Figure 7, the results of docking showed that hydrophobic
interactions and polar–polar interaction between N3 of the
ligand and carbonyl group of Lys5 play a major role in the
binding of 1d to tyrosinase. Based on the results of the model-
ing, the dominant interaction is hydrophobic.
0
20
40
60
[I](μM)
80
100
Discussion
Figure 3 Percent inhibition curve for oxidation of L-DOPA catalyzed by mushroom
tyrosinase at increasing concentrations of synthetic pyrimidine derivatives.
Inhibitory activity of all synthetic compounds at 30 µM is 23.72–55.08%.
Tyrosinase is a rate-limiting monooxygenase enzyme in
the process of melanin production.2,5 Increasing the activ-
ity of this enzyme causes excessive production of melanin
and, as a result, hyperpigmentation problems such as mel-
asma and skin spots may occur.4,6 Therefore, the discovery
Lineweaver–Burk plots showed that all synthetic compounds
were a noncompetitive inhibitor of the mushroom tyrosinase
(Figure 6).
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50
40
30
20
10
0
Compounds
Figure 4 The IC50 (µM) values of synthetic compounds against mushroom tyrosinase. L-DOPA was used as substrate and concentrations of synthetic compounds were 10,
30, 60, 100 µM. Absorbance of the reaction was taken at 490 nm. (IC50: 2d>1c>2b>2c>1b>2e>2a>1e>kojic acid>1a>1d).
Table 2 The kinetic constants and inhibition constants of all synthetic compounds on mushroom tyrosinase
Compounds
Vmax
Km
αKi
Ki
Alpha(α)
IC50(μM)
Inhibition type
Control
1a
125.58
21.15
24.06
24.78
25.01
23.36
21.88
19.64
23.95
22.41
23.1
0.41
0.63
0.43
0.48
0.39
0.39
0.496
0.62
0.57
0.35
0.46
6.079
7.11
3.601
6.559
5.871
7.837
7.05
4.98
3.4
1.68
1.08
1.25
0.95
0.97
1.27
1.63
1.52
0.85
1.19
24.68
37.191
42.04
24.4
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
Noncompetitive
1b
1c
7.378
7.464
6.857
6.33
1d
1e
28.01
29.77
41.4
2a
2b
5.56
2c
7.07
4.62
7.63
5.66
40.62
43.8
2d
6.51
2e
6.76
34.06
B
A
2a
2d
2c
Control
2b
2e
1c
1a
1b
1d
1e
Control
2200
2000
1800
1600
1400
1200
3000
2700
2400
2100
1800
Compounds
Compounds
Figure 5 The effect of the synthetized compounds (1a-e and 2a-e) on the enzyme activity estimated by zymography using L-DOPA staining (A) based on 2,6-diamino-
4-chloropyrimidine and (B) based on 2,4,6-triaminopyrimidine. (A: 1d>1a>1e>1b>1c) (B: 2a>2e>2c>2b>2d).
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150
100
50
0.10
0.08
0.06
0.04
0.02
[I]=0
[I]=30μm
0
0.0
0.5
1.0
1.5
2.0
[Substrate](μM)
-4
0
4
8
12
1/[S]
Figure 6 Michaelis–Menten and Lineweaver–Burk plots of the enzyme inhibition by the compound 1d.
Estimated
FullFitness
ΔG
Show Cluster Element
(kcal/mol)
(kcal/mol)
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
-2198.37
-2198.37
-2198.37
-2198.25
-2198.25
-2197.03
-2197.03
-2197.03
-2194.94
-2194.94
-2194.94
-2194.94
-2194.76
-2194.28
-2194.28
-2194.28
-2183.83
-2183.83
-2183.54
-2183.54
-8.64
-8.64
-8.64
-8.65
-8.65
-8.58
-8.58
-8.58
-8.31
-8.31
-8.31
-8.31
-8.33
-8.13
-8.13
-8.13
-7.77
-7.77
-7.79
-7.79
8
9
10
11
A
B
C
Figure 7 (A) Full fitness (kcal/mol) and estimated ΔG (kcal/mol); (B) 3D secondary structure of tyrosinase that shows the position of 1d at the protein. The important
interacting residues are shown by colored lines and 1d by CPK drawing method; (C) a LigPlot diagram of the interaction between tyrosinase and 1d. Hydrophobic
interaction plays a major role in the binding.
and synthesis of safe and effective inhibitors for treating in the ring and have attracted much attention in medicinal
and preventing skin disorders are very important in the chemistry and pharmacology.15,16 They have been shown
medicinal and cosmetic industries.2,27 On the other hand, to have anticancer, antiviral, anti-inflammatory, antibacter-
this enzyme plays an important role in physiological pro- ial and antiparkinson properties.15 Considering the reports
cesses in insects, including wound healing, sclerotization, on the tyrosinase inhibition ability of aromatic amines and
and defensive encapsulation. Thus, its inhibitors could be anilines, we were encouraged to synthesize some new
useful as insecticides in the agricultural research derivatives based on pyrimidine and aniline derivatives.17
activities.10,11 Tyrosinase is also responsible for the unde- In this study, we synthesized and characterized ten novel
sirable browning of fruits and vegetables during storage, azo-dyes including bioactive pyrimidine moiety and ani-
which reduces the nutritional and commercial value of the line derivatives. Their inhibitory activities against mush-
products.8,9 Synthesis and detection of effective, harmless room tyrosinase were then evaluated. Tyrosinase inhibition
and affordable inhibitors is desirable in order to minimize assay was carried out with L-DOPA as substrate and kojic
these problems. Over recent years, there have been many acid as reference inhibitor. All synthetic compounds (1a-e
studies of screening, discovery, design, and synthesis of and 2a-e) were able to inhibit mushroom tyrosinase activ-
novel tyrosinase inhibitors. Pyrimidine derivatives are ity in a concentration-dependent manner, as shown in
active heterocyclic compounds with two nitrogen atoms Figure 3. The data revealed that compounds with
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Mirmortazavi et al
a Cl-halide group in the R position have more inhibitory aniline, paramethoxyaniline and para-aminoacetanilide
activity than the compounds with an NH2 group in the containing the azo group. Considering the effect of ortho
R position. The 1d (IC50=24.45 μM) and 1a (IC50=24.68 and para position on the benzene ring of synthetic com-
μM) compounds, possessing acetamide and NO2 groups at pounds, it was found that 2e (IC50=34.06) with a nitro
position X, respectively, and with Cl at position R, are group at position ortho had a more potent inhibitory effect
more potent inhibitors of mushroom tyrosinase than the on tyrosinase activity than 2c (IC50=40.62) with a nitro
other synthesized compounds and IC50 values are slightly group at the para position. Bae et al synthesized methoxy-
better than kojic acid (IC50=25.24 μM), the reference aniline derivatives and evaluated as
a tyrosinase
tyrosinase inhibitor. The mechanisms of inhibition of inhibitor.17 The theoretical studies conducted in this
mushroom tyrosinase by all synthetic compounds were study identified the interaction of synthetic compounds
further investigated by steady-state kinetic analysis. with the key amino acids of tyrosinase (PDB =2Y9W).
Inhibition data were then determined using Lineweaver–
Burk double reciprocal plots. The outcome of compounds
Acknowledgment
The authors thank the Research Council of the University
showed that the 1/V vs 1/[S] plot gave two straight lines
with different slopes, but with one intersection on the
of Guilan for the financial support of this study.
horizontal axis, indicating that the values of Vmax
decrease without changing the values of Km when the
Author contributions
concentration of these compounds increased. These data
All authors contributed to data analysis, drafting or revis-
suggest that of all synthetic compounds function as non-
ing the article, gave final approval of the version to be
competitive inhibitors of tyrosinase (Table 2), hence all
published, and agree to be accountable for all aspects of
synthetic compounds bound both the free enzyme mole-
the work.
cule and enzyme-substrate complex. Liu et al28 synthe-
sized several compounds on the basis of thiourea
Disclosure
The authors report no conflicts of interest in this work.
containing NH group in the ring and evaluated their inhi-
bitory potencies on mushroom tyrosinase. According to
their kinetic results, the thiouracil and methylthiouracil
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