Synthesis and biological evaluation of novel hydroxybenzaldehyde-based kojic acid analogues as inhibitors of mushroom tyrosinase

Article abstract of DOI:10.1016/j.bmcl.2016.12.027

Two series of novel kojic acid analogues (4a–j) and (5a–d) were designed and synthesized, and their mushroom tyrosinase inhibitory activities was evaluated. The result indicated that all the synthesized derivatives exhibited excellent tyrosinase inhibitor

Full text of DOI:10.1016/j.bmcl.2016.12.027

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel hydroxybenzaldehyde-
based kojic acid analogues as inhibitors of mushroom tyrosinase
a
a
a
a
a
a
Wenlin Xie a,b,c, , Huilin Zhang , Jingjing He , Jingai Zhang , Qiuyan Yu , Chunxiang Luo , Shangru Li
⇑
a School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, China
b Key Laboratory of Theoretical Organic Chemistry and Function Molecule of Ministry of Education, Hunan University of Science and Technology, Xiangtan 411201, China
c Hunan Provincial Key Laboratory of Controllable Preparation and Functional Application of Fine Polymers, Xiangtan 411201, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel kojic acid analogues (4a–j) and (5a–d) were designed and synthesized, and their
mushroom tyrosinase inhibitory activities was evaluated. The result indicated that all the synthesized
derivatives exhibited excellent tyrosinase inhibitory properties having IC50 values in the range of
1.35 2.15–17.50 2.75 lM, whereas standard inhibitor kojic acid have IC50 values 20.00 1.08 lM.
Specifically, 5-phenyl-3-[5-hydroxy-4-pyrone-2-yl-methylmercap-to]-4-(2,4-dihydroxyl-benzylamino)-
Received 9 August 2016
Revised 17 November 2016
Accepted 8 December 2016
Available online xxxx
1,2,4-triazole (4f) exhibited the most potent tyrosinase inhibitory activity with IC50 value of
1.35 2.15 lM. The kinetic studies of the compound (4f) demonstrated that the inhibitory effects of
the compound on the tyrosinase were belonging to competitive inhibitors. Meanwhile, the structure-
activity relationship was discussed.
Keywords:
Kojic acid
Tyrosinase inhibitors
Benzaldehyde
1,2,4-Triazole
Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
tyrosinase inhibitors have been found application in cosmetics
and pharmaceutical products.7,8 A large number of tyrosinase inhi-
Tyrosinase (EC 1.14.18.1) is copper-containing metalloenzyme
that are widespread among microorganisms, plants, and animals.
They catalyze the ortho-hydroxylation of monophenols, which
leads to the corresponding o-diphenols, as well as the oxidation
of catechols, which leads to the corresponding o-quinones,1 under
many conditions, these then react further to result in the formation
of various pigments.2 Although the melanin production in human
skin is a major defense mechanism against UV light, excessive
accumulations of epidermal pigmentation can cause various
hyperpigmentation disorders. Therefore, the regulation of melanin
synthesis via the inhibition of tyrosinase is an important research
topic.3 In clinical usage, tyrosinase inhibitors are used for treat-
ments of dermatological disorders related to melanin hyperaccu-
mulation and are essential in cosmetics for depigmentation,4
such as age spots and freckle, caused by the accumulation of an
excessive level of epidermal pigmentation.5 Inhibition of tyrosi-
nase is equally important commercially. In most fruits and vegeta-
bles, the enzyme is responsible for undesired browning that takes
place during senescence or damage during post-harvest handling,
leading to faster degradation and shorter shelf life.6 Taking into
account the key role of tyrosinase in melanin production, many
bitors have been reported but only a few are used because of their
limitations with regards to cytotoxicity, selectivity and stability.
Thus, it is great need of developing new tyrosinase inhibitors with-
out causing adverse reactions.
So far, a number of kojic acid and benzaldehyde analogues have
been reported as mushroom tyrosinase inhibitors,9,10 such as Rho
et al. reported that Kojyl thioether 2-((4-Hydroxyphenylthio)
methyl)-5-hydroxy-4H-pyran-4-one inhibited mushroom tyrosi-
nase with an IC50 of 0.54
lM which is better kojic acid
(IC50 = 48.52
l
M),11 Li et al. reported that chlorobenzaldehyde
thiosemicarbazones had inhibitory activity on tyrosinase.12
Previously, we have also reported the synthesis of 1,2,4-triazoles-
containing kojic acid derivatives by reactions of 5-hydroxy-2-
chloromethyl-4H-pyran-4-one with 5-substituted-3-mercapto-4-
amino-1,2,4-triazole,13 and most of them showed significant
tyrosinase inhibitory activity. As a part of our continuous project
aimed at searching for new effective mushroom tyrosinase inhibi-
tors, we hybridized the kojic acid with Hydroxybenzaldehyde by a
linker 1,2,4-triazole to form 5-substituted-3-[5-hydroxy-4-pyrone-
2-yl-methylmercapto]-4-aryl-methylamino-1,2,4-triazole (4) and
5-substituted-3-[5-hydroxy-4-pyrone-2-yl-methylmercapto]-4-
arylmethyleneamino-1,2,4-triazole (5).
The synthetic procedure employed to obtain the target com-
pounds 4a–j and 5a–d was depicted in Scheme 1. The intermediate
⇑
Corresponding author at: School of Chemistry and Chemical Engineering,
Hunan University of Science and Technology, Xiangtan 411201, China.
E-mail address: xwl99zsu@163.com (W. Xie).
http://dx.doi.org/10.1016/j.bmcl.2016.12.027
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xie W., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.027

2
W. Xie et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
O
OH
O
S
c
N
N
SH
N
N N
SH
O
R2
N
N
N
N
a
NH2
N N
R1
R1
OH
R2
5
O
SH
S
R1
b
c
N
N
N
N
N N
H
N NH
2
R2
1
R2
R1
R1
3
4
Scheme 1. General synthesis of compounds (4a–j; 5a–d). Reagents and conditions: (a) substituted benzaldehyde/NaOAc, AcOH; (b) NaBH4, stir 1 h; (c) 5-hydroxy-2-
chloromethyl-4H-pyran-4-one, Et3N/EtOH, reflux 0.5 h.
Table 1
Schiff bases 2 were prepared using 5-substituted-3-mercapto-4-
amino-1,2,4-triazoles 1 were reacted with Hydroxylbenzaldehyde
by condensation reaction with TsOH as catalyst in anhydrous etha-
nol, and the compound 2 was reduced to amine 3 using NaBH4 in
Tyrosinase inhibitory activities of the synthesized compounds.
a
Compounds
R1
R2
IC50 ( SD, lmol/L)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
CH3
CH3
CH2CH3
H
CH3
Ph
4-CH3Ph
3-ClPh
4-ClPh
Ph
2-ClPh
H
2,4-di-OH
2-OH
2-OH
2-OH
3-OH
2,4-di-OH
2-OH
2-OH
2-OH
2-OH
5.80 0.28
10.20 0.56
7.60 1.50
5.85 0.60
12.5 1.08
1.35 2.15
3.80 0.78
2.50 0.89
1.71 1.56
1.50 1.25
5.20 0.69
8.54 2.38
9.60 1.78
17.50 2.75
20.00 1.08
methanol. And then, the compound
3 was reacted with 5-
hydroxy-2-chloromethyl-4H-pyran-4-one13 by nucleophilic sub-
stitution reaction in DMF medium in the presence of Et3N to give
5-substituted-3-[5-hydroxy-4-pyrone-2-yl-methylmercapto]-4-
arylmethylamino-1,2,4-triazole 4 with good yields. Similarly, the
compound 2 was reacted with 5-hydroxy-2-chloromethyl-4H-
pyran-4-one to obtain 5-substituted-3-[5-hydroxy-4-pyrone-2-yl-
me-thylmercapto]-4-arylmethyleneamino-1,2,4-triazole 5. The
structures of all the newly synthesized compounds 4a–j and
5a–d were confirmed by 1H NMR, 13C NMR, HRMS, and IR spectral
analysis.
3,4-di-OH
4-OH
2,4-di-OH
3-CH3O-4-OH
H
H
Kojic acidb
For evaluating the tyrosinase inhibitory activity, all the synthe-
sized compounds were subjected to tyrosinase inhibition assay
with l-DOPA as substrate using kojic acid as the positive control,
according to a method described by Kubo14 with some slight mod-
ifications. Fig. 1 showed that the remaining enzyme activity rapidly
decreased with the increasing concentrations of compound 4f.
The IC50 values of the titled compounds 4a–j and 5a–d against
tyrosinase were summarized in Table 1. From our preliminary
investigation, we found that all of the synthesized compounds
exhibited potent inhibitory effect on mushroom tyrosinase with
a
IC50 values: the concentration of the inhibitor required to produce 50% inhi-
bition of tyrosinase, results are the mean SD from three independent experiments.
b
Standard inhibitors of the tyrosinase.
IC50 values ranged from 1.35 2.15 to 17.50 2.75 lM. They dis-
played higher inhibitory activities on tyrosinase in vitro than the
reference standard inhibitor kojic acid. When the Schiff bases were
reduced to obtain the amines 4, by comparing two classes of com-
pounds no clear superiority could be observed, but when the aryl
groups were introduced to 5-position in triazole ring, it can be
observed that the inhibitory activities of the compounds 4 were
more potent than the compounds 5, such as compounds 4f, 4g,
4h, 4i, 4j. Among the compounds (4a–j) and (5a–d), compounds
4a, 4d, 4f, 4g, 4h, 4i, 4j and 5a demonstrated the most potent inhi-
bitory activities. In particular, compound 4f exhibited greater inhi-
bitory activities with IC50 1.35 2.15 lM. From the inhibitory
activities on mushroom tyrosinase, preliminary structure-activity
relationships of the synthesized compounds were achieved. In gen-
eral, compared with compounds (4a–j) and (5a–d), the aromatic
substituents on 1,2,4-triazole ring had great effect on the inhibi-
tory activities, for example, the compounds 4f, 4g, 4h, 4i, 4j and
5a displayed the more potent inhibitory activities than the com-
pounds with no substituent and aliphatic hydrocarbon on 1,2,4-tri-
azole ring (4a, 4b, 4c, 4d, 4e, 5b, 5c, and 5d). But when the
substituents (such as methyl, chlorine) were introduced to ben-
zene ring in 5-position, we found that the inhibitory activities of
these compounds (4g, 4h, 4i) were less than compound 4f and
4j. Among compounds 5a–d, comparing the activities of the com-
pound 5b and 5d it can be observed that the introduction of meth-
oxyl group to position 3 of benzene ring on aromatic aldehyde
decreased the activity.
Fig. 1. Dose-dependent inhibitory effects of compound 4f on tyrosinase activity.
Tyrosinase activity was measured using l-DOPA as the substrate.
Please cite this article in press as: Xie W., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.027

W. Xie et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
4f acted as a competitive inhibitor of mushroom tyrosinase. Pre-
liminary structure activity relationships analysis indicated that
aromatic substituents on the triazole ring might play an important
role in determining their inhibitory activities, but the substituent
on the benzene ring in 5-position was somewhat unfavorable to
their biological activity. Further investigations into the structure-
activity relationships of these compounds are currently in
progress.
Acknowledgments
The authors thank the National Natural Science Foundation of
China (No. 21571058), the Natural Science Foundation of Hunan
Province (2015JJ4026), and the Scientific Research Fund of Hunan
Provincial Education Department (No. 13A028) for the financial
support of this work.
Fig. 2. Lineweaver–Burk plots for the inhibition of Mushroom tyrosinase by
compound 4f. Concentrations of compound 4f for curves were (j) 0 M, (N) 0.6
and (r) 1.2 M respectively.
l
lM
l
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.12.
027.
Among the tested target compounds, 5-phenyl-3-[5-hydroxy-4-
pyrone-2-yl-methylmercap-to]-4-(2,4-dihydroxyl-benzylamino)-
1,2,4-triazole 4f showed the highest inhibitory activity, therefore,
we carried out the kinetic analysis of 4f for tyrosinase inhibition
using l-DOPA as a substrate. The Lineweaver-Burk plots for the
inhibition of tyrosinase by 4f were obtained with variable concen-
trations of 4f and the substrate (Fig. 2). The intersection of these
lines on the vertical axis indicated that 4f was a competitive inhi-
bitor of tyrosinase with respect to l-DOPA as a substrate, thus, we
speculated that this type of compound could enter into the active
center of tyrosinase.
In summary, we designed and synthesized two series of new
kojic acid analogues 5-substituted-3-[5-hydroxy-4-pyrone-2-yl-
methylmercapto]-4-arylmethylamino-1,2,4-triazole (4) and
5-substituted-3-[5-hydroxy-4-pyrone-2-yl-methylmercapto]-4-
arylmethyleneamino-1,2,4-triazole (5), and their activity as tyrosi-
nase inhibitors has been examined. The results displayed that all
the synthesized compounds exhibited higher inhibitory activity
on tyrosinase than kojic acid (1). Among the compounds synthe-
sized, compound 4f showed the strongest inhibitory activity with
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IC50 value of 1.35 2.15 lM. Kinetic study revealed that compound
Please cite this article in press as: Xie W., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.12.027