Design, synthesis and bioactivity of chalcones and its analogues

Article abstract of DOI:10.1016/j.cclet.2017.03.018

The Vernohia anthelmintica L.'s extract is one of the most popular Uygur medicines used for vitiligo. It is believed that the chalcone compounds of the plant play an important role in the treatment since they may activate tyrosinase and improve melanin production. In this study, twenty-one chalcones and nine analogues were synthesized in view of three different components of chalcone (A, B ring and α, β-unsaturated carbonyl). After biological evaluation of their activity on tyrosinase in cell-free systems, the result showed that most compounds (except polyhydroxy chalcones) possess activator effect on the tyrosinase, especially for 13a–15a, 20a and 1b, which bearing a comparable activity to the positive control 8-MOP. SAR of these tyrosinase activator was summed up for the first time as well. Finally, compound 13a was found to increase melanin contents and tyrosinase activity 1.75 and 1.3 fold, respectively, compared with that of untreated murine B16 cells at the concentration of 40?μg/mL.

Full text of DOI:10.1016/j.cclet.2017.03.018

Accepted Manuscript
Title: Design, synthesis and bioactivity of chalcones and its
analogues
Authors: Chao Niu, Adila Tuerxuntayi, Gen Li, Madina
Kabas, Chang-Zhi Dong, Haji Akber Aisa
PII:
S1001-8417(17)30096-7
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Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.03.018
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18-1-2017
9-3-2017
Please cite this article as: Chao Niu, Adila Tuerxuntayi, Gen Li, Madina Kabas, Chang-
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Original article
Design, synthesis and bioactivity of chalcones and its analogues
Chao Niu a, b, Adila Tuerxuntayi a, b, Gen Li a, b, Madina Kabas a, b, Chang-Zhi Dong c *, Haji Akber Aisa a, b

a The Key Laboratory of Plant Resources and Chemistry of Arid Zone, Chinese Academy of Sciences, Urumqi 830011, China
b State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese
Academy of Sciences, Urumqi 830011, China
c Université Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, 15 rue J-A de Baïf, 75205 Paris Cedex 13, France
ARTICLE INFO
Article history:
Received 7 December 2016
Received in revised form 19 January 2017
Accepted 24 February 2017
Available online
 Corresponding author.
E-mail address: haji@ms.xjb.ac.cn

Graphical Abstract
Twenty-one chalcones and nine analogues were synthesized and evaluated for their activity on tyrosinase in cell-free system.
Several compounds were more potent than the positive control 8-MOP and 13a was further biologically investigated in B16
cells.
ABSTRACT
The Vernohia anthelmintica L.’s extract is one of the most popular Uygur medicines used for vitiligo. It is believed that the chalcone compounds of the plant
play an important role in the treatment since they may activate tyrosinase and improve melanin production. In this study, twenty-one chalcones and nine
analogues were synthesized in view of three different components of chalcone (A, B ring and α, β-unsaturated carbonyl). After biological evaluation of their
activity on tyrosinase in cell-free systems, the result showed that most compounds (except polyhydroxy chalcones) possess activator effect on the tyrosinase,
especially for 13a-15a, 20a and 1b, which bearing a comparable activity to the positive control 8-MOP. SAR of these tyrosinase activator was summed up for
the first time as well. Finally, compound 13a was found to increase melanin contents and tyrosinase activity 1.75 and 1.3 fold, respectively, compared with that
of untreated murine B16 cells at the concentration of 40 μg/mL.
Keywords:
Vitiligo
Activator of tyrosinase
Chalcone
B16 cells
SAR
1. Introduction
Vitiligo is an acquired pigmentary disorder of unknown etiology that is clinically characterized by the development of white macules
related to the selective loss of melanocytes [1,2]. It was believed that the disease was mainly resulted from destruction of the
melanocyte and obstruction of the melanin synthesis [3]. Tyrosinase is the rate-limiting enzyme in melanin biosynthesis and catalyze
the reaction of hydroxylation and oxidation in the formation of melanin [4-8].
The fruits extract of Vernohia anthelmintica L. (Fig. 1) is one of the most popular Uygur medicines used for vitiligo and initially
recorded in "Yao Yong Zong Ku" around 300 years ago [9-12]. Some important flavonoids are isolated from the plant [13-16] and it is
suggested that these compounds play an important role in this treatments, since they may activate tyrosinase and improve the melanin
production [17,18].
Unfortunately, few flavonoids as activator of tyrosinase have been reported [19-22]. Our group have been dedicated on the drug
development of the vitiligo for many years [23-26]. And some chalcone derivatives with potential activating effect on tyrosinase and
melanin synthesis in B16 cells were discovered as well [27-29]. Based on above research, twenty-one chalcones and nine analogues
were prepared in view of three different parts of chalcone (A, B ring and α, β-unsaturated carbonyl). After that, the activity of them on
tyrosinase were studied and the SAR was summarized as well.
2. Results and discussion
2.1 Chemistry
1
All the compounds were characterized by H NMR, 13C NMR, IR and HRMS (ESI). Firstly, nine mono-, di-, tri- and tetra-
hydroxychalcones (1a-9a) were synthesized to study the impact of the hydroxyl for the activity on tyrosinase. The synthetic route was
described in Scheme 1 (take Butein as example) [30]; Second, with the aim of investigating the significance of different substituents on
phenyl ring for the effect on tyrosinase, compounds 10a-21a substituted in different positions of A ring and para-position of B ring
were prepared as well (Scheme 2); After that, five chalcone analogues (1b-2b and 1c-3c in Fig. 2) with other aromatic A or B rings
were synthesized respectively under the same condition as depicted in Scheme 2. At last, modification on the chain was explored by
preparing four chain-prolonged chalcone analogues (1d-4d in Fig. 2) as represented in Fig. 2. Additionally, 1d and 4d were identified
as novel compounds.

As shown in Fig. 3, the crystals of the compounds 13a and 2c used for X-ray diffraction analysis were obtained by slow evaporation
of acetone-ethanol (1:3) mixed solution at room temperature (13a: CCDC No. 1451159; 2c: CCDC No. 1451160).
2.2 Activity on tyrosinase
The biological activity of polyhydroxy chalcones against tyrosinase was evaluated (Table 1). All the compounds inhibit the
tyrosinase. Generally, the higher inhibitory activity can be observed with more hydroxyl in chalcone. For 2a and 3a, 5a and 6a, the
result showed that the most important factor for their efficacy was the location of the hydroxyl on benzene ring, with a significant
preference to a 4-substituted B ring rather than a substituted A ring. Moreover, a 2,4-substituted resorcinol with more hydroxyl on ring
A improved their activity, such as compounds 7a and 9a.
It was not hard to find that hydroxyl in chalcone skeleton seriously abolished the activator effect on tyrosinase according to the result
above. Therefore, some other groups were introduced to the chalcone to improve the activity. As shown in Table 2, chalcones
substituted with -X (14a, 15a) and -N(CH3)2 (13a) were more potent than ones with -H, -OH, -CH3 and -OCH3, suggesting that the
electron-withdrawing group (EWG) may be favorable to enhance the activity. In the view of best activity of compound 13a, further
modification was performed on this active molecule. Neither changing the location of the -OCH3 on A ring nor replacement of -OCH3
with -NO2, -NH2 or -NHAc enhanced the activator effect, which emphasizes the importance of the para-position of the substituted
group on A ring and the -N(CH3)2 on B ring.
With few exceptions, when A ring was replaced with naphthalene or pyridine ring, similar effect was found with compound 1b and
2b, proving that the benzene for the A ring was not necessary for this effect. However, the activity dramatically decreased once other
aromatic rings were introduced to B ring (compounds 1c-3c), which means that the benzene for B ring was essential for maintaining
the activity (Table 3). For the linker group between A and B ring, the α, β-unsaturated carbonyl was prolonged to give compound 1d-
2d, based on the active compounds synthesized above. Unfortunately, in Table 3, the extended distance between the two groups failed
to retain the biological activity.
2.3 Activity on tyrosinase and melanin synthesis in B16 cells
The viability of 13a on B16 melanoma cells was examined using the MTT assay. The cells were treated with various concentrations
of 13a (6.25-200 μg/mL). As shown in Fig. 4, there was no significant difference between the control and treated group at
concentrations of 6.25-200 μg/mL. 13a showed very small cytotoxic effects on B16 cells at concentrations of 200 μg/mL.
The effect of 13a on tyrosinase in cells was measured by L-DOPA oxidation (Fig. 5). Compared with treatment with medium only
(untreated condition), treatment with 13a at 0-40μg/mL resulted in a dose-dependent increase in tyrosinase activity in B16 cells. In the
melanin content assay, to exclude the possibility that a rise in melanin content may be induced by cell-proliferating effect of 13a, the
absorbance of the same number of cells across 13a concentrations (0-40 μg/mL) was measured as well. We found that melanin levels
increased in a dose-dependent manner by 13a treatment in B16 cells (Fig. 6). At 40 μg/mL of 13a, the melanin content only slightly
increased, so 20 μg/mL was chosen as an effective concentration of 13a for further experiments.
3. Conclusion
This work showed that good activator effect can be obtained with small chalcone molecules and the SAR was summed up for the
first time as follows. (1) The hydroxyl on either benzene of chalcone may cause an inhibitory activity, especially for the B ring. (2).The
A ring of the chalcone can be substituted with other conjugate aromatic rings to retain the activator effect while the B ring cannot. It
was speculated that the planar molecule was essential for the activity. (3) The linker should not be prolonged for maintaining the
activity. In addition, 13a increased both activity of the tyrosinase and the contents of the melanin in a dose-dependent manner in
murine B16 cells.
4. Experimental
4.1 Chemistry
Reagents and solvents were purchased from Sigma, Sodipro or VMR, and used without further purification. Dimethylsulfoxide
(DMSO), mushroom tyrosinase, L-3, 4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylalanine and 3-(4,5-dimethylthiazol-2-
yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA).
4.2 General method for preparation of chalcone and analogues (1a-21a, 1b-2b, 1c-3c, 1d-4d)
The corresponding aldehyde (1.0 equiv) and ketone (1.0 equiv) were dissolved in ethanol at 0 °C and 10% NaOH (1.0 equiv.)
solution was slowly added. After that, the mixture was allowed to warm up to room temperature and stirred for 48 h. The resulting
mixture was evaporated to dryness and the residue was purified by chromatography on silica gel eluted with petroleum ether-ethyl
acetate to afford the final compound. The characterization data of the synthesized compounds are in the Supporting information.

4.3 Biological methods
Preparation of 13a: Compound 13a was obtained and then dissolved in DMSO. A stock solution of 13a (5 mg/mL) was prepared in
DMSO for further applications.
Cell culture: The murine B16 melanoma cell line was obtained from the CAS (Chinese Academy of Sciences, China). B16 cells
were grown in DMEM medium (Gibco, Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco),
100 U/mL penicillin and 100 μg/mL streptomycin (Hyclone, USA) in a humidified atmosphere with 5% CO2 at 37 °C.
Cell viability assay: Cell viability was determined using the MTT assay. B16 cells were plated in 96-well dishes at a density of 5 ×
103 cells per well. After 24 h, different concentrations of 13a were added and the cells were incubated for 48 h. Then, 10 μL of MTT (5
mg/mL in PBS) solution was added into each well and the cells were incubated at 37 °C for another 4 h. Following medium removal,
150 μL of DMSO was added to each well and plates were gently shaken for 10 min. Optical absorbance was determined at 570 nm
with a Spectra Max M5 (Molecular Devices, USA). Absorbance of cells without treatment was regarded as 100% cell survival. Each
treatment was performed in quintuplicate and each experiment was repeated three times.
4.3.1 Activator effect of tyrosinase in cell-free assay
The activities on tyrosinase of synthesized compounds were performed according to the modified method [31], with 8-MOP [32-35]
and Kojic acid [36-38] as positive control at the Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences.
Potassium phosphate buffer (0.06 mL, 50 mmol/L) at pH 6.5, 0.04 mL tyrosinase (250 U/mL) and 2 μL of the test compounds (0.5-300
μmol/L), dissolved in DMSO were inserted into 96-well plates. After 5 min incubation at 25-30 °C, 0.1 mL of L-tyrosine (2 mmol/L)
was added and incubated for additional 30 min. After that, the optical density of the systems at 490 nm was measured on ELASA and
the rate of activation (RA) was calculated according the formula: RA=[(C-D)-(A-B)]/(A-B)*l00, The A was optical density of the
system at 490 nm with only tyrosinase; B was the one with neither compounds nor tyrosinase; C was the one with both compounds and
tyrosinase; And the D was the one with only compounds. Finally, the activity of the compounds was expressed as the compounds
concentration that gave a 50% activator effect (when RA=50%) in the enzyme activity (EC50).
4.3.2 Tyrosinase activity and melanin content assay in B16 cells
Tyrosinase activity was estimated by measuring the rate of L-DOPA oxidation as previously reported. B16 cells were seeded in a 12-
well plate at a density of 2 × 105 cells per well and allowed to attach for 24 h. Then, cells were treated with 13a for 48 h, washed with
ice-cold PBS twice, trypsinized with 0.25% trypsin (Hyclone) and collected in an Ep tube. After being centrifuged at 3,000 rpm for 5
min the cells were washed once with PBS, and then 200 μL of Tris-0.1% Triton X-100 (pH 6.8) were added to each tube. All tubes
were incubated at -20 °C for 30 min, and then the lysates were centrifuged at 12,000 rpm for 15 min to obtain the supernatant for the
tyrosinase activity assay. Protein concentrations were determined by the Bradford method with bovine serum albumin (BSA) as a
standard. 100 mL of supernatant containing 10 μg total protein was added to each well in a 96-well plate and this was mixed with 100
μL of 0.1% L-DOPA in PBS (pH 6.8). After incubation at 37 °C for 1 h, the dopachrome was monitored by measuring the absorbance
at 475 nm. The total melanin in the cell pellet was dissolved in 100 mL of 1 mol/L NaOH/10% DMSO for 1h at 80 °C and solubilized
melanin was measured at 470 nm.
Acknowledgment
This work was supported by the Funds for the Xinjiang Key Research and Development Program (No. 2016B03038-3); Personalized
Medicines-Molecular Signature-based Drug Discovery and Development, Strategic Priority Research Program of the Chinese
Academy of Sciences (No. XDA12050301); West Light Foundation of the Chinese Academy of Science (No. XBBS201403).
Appendix A. Supplementary data
Supplementary data (experimental procedures, spectroscopic characterizations) associated with this article can be found.

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Fig. 1. The plant of the Vernohia anthelmintica L.
Fig. 2. Structures of synthesized analogues
2c
13a
Fig. 3. Chemical structures of the crystals in 2c and 13a

Fig. 4. The Cell viability of 13a determined using the MTT assay with different concentrations in B16 cells.
Fig. 5. Effect of 11a-13a on tyrosinase in B16 cells (expressed as the percentage of untreated control).
Fig. 6. Effect of 11a-13a on melanin content in B16 cells (expressed as the percentage of untreated control).

Scheme 1. Synthesis of polyhydroxychalcone. Regents and condition: (i) ClCH2OCH3, K2CO3, Acetone, reflux (ii) 10% NaOH, ethanol, rt (iii) 3 mol/L HCl,
ethanol, reflux
Scheme 2. Synthesis of substituted chalcones. Regents and condition: (i) 10% NaOH, ethanol, rt

Table 1. The inhibitory effect of polyhydroxy chalcones on tyrosinase
a
IC50
Compound
R1
R2
R3
R4
R
Substrate
(μmol/L)
1a
2a
3a
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
L-tyrosine
L-tyrosine
L-tyrosine
NEb
35.4±2.02
NIc
OH
4a
5a
6a
7a
8a
H
H
OH
H
OH
H
H
H
H
H
H
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
25.1±1.57
NI
OH
OH
OH
H
OH
OH
OH
OH
H
OH
OH
OH
31.0±0.91
19.6±1.42
27.3±2.64
OH
OH
9a
OH
-
OH
OH
OH
OH
L-tyrosine
11.4±1.03
10.70
Kojic acidd
-
-
-
-
a 50% inhibitory concentration, result were the means±SD from three independent experiment; b not effect; c NI means slightly inhibited and IC50>200 μmol/L; d
Kojic acid as positive control
Table 2. The activator effect of substituted chalcones on tyrosinase
Compound
10a
R5
R6
H
Substrate
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
EC50a (μmol/L)
NAb
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
4-OCH3
2-OCH3
3-OCH3
4-NO2
11a
OH
NA
12a
CH3
58.4±6.54
17.1±2.18
26.3±1.67
20.6±3.58
126.0±8.21
NA
13a
N(CH3)2
Cl
14a
15a
F
16a
OCH3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
17a
18a
46.3±4.60
NA
19a
20a
4-NH2
35.5±0.87
NA
21a
8-MOPc
4-NHAc
14.6
a 50% effective concentration, result were the means±SD from three independent experiment; b NA means slightly activated and EC50>200 μmol/L; c 8-MOP as
positive control

Table 3. The activator effect of chalcone analogues on tyrosinase
Compound
Substrate
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
L-tyrosine
EC50a (μmol/L)
25.8±1.73
41.7±2.25
NAb
1b
2b
1c
2c
NA
3c
NA
1d
84.0±7.51
73.6±5.36
NAb
2d
3d
4d
8-MOPc
NA
14.6
a 50% effective concentration, result were the means±SD from three independent experiment; b NA means slightly activated and EC50>200 μmol/L; c 8-MOP as
positive control