Synthesis and bioactivity of novel isoxazole chalcone derivatives on tyrosinase and melanin synthesis in murine B16 cells for the treatment of vitiligo

Article abstract of DOI:10.1016/j.bmc.2016.08.066

A new series of chalcone derivatives 1–18, bearing isoxazole moieties were designed and synthesized, and biologically evaluated for their activity on mushroom tyrosinase and melanin synthesis in murine B16 cells. The result indicated that most of prepared compounds 1–18 showed potent activating effect on tyrosinase, especially for 1–2, 4, 6–7, 9 and 15. Among them, compounds 2, 4 and 9 demonstrated the best activity with EC₅₀?=?1.3, 2.5 and 3.0?μmol·L^?1respectively, much better than the positive control 8-methoxypsoralan (8-MOP, EC₅₀?=?14.8?μmol·L^?1); In B16 cells, all the tested compounds exhibited a stronger activity on melanogenesis than 8-MOP (with the value of 115%). It was interesting that derivatives substituted with halogen (1, 2, 4, 5, 7, 9) were generally more potent. Compounds 2 (463%) and 18 (438%) with 3 and 4-fold potency compared with 8-MOP respectively, were recognized as the most promising candidate hits for further pharmacological study of anti-vitiligo.

Full text of DOI:10.1016/j.bmc.2016.08.066

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and bioactivity of novel isoxazole chalcone derivatives on
tyrosinase and melanin synthesis in murine B16 cells for the
treatment of vitiligo
Chao Niu a,b, Li Yin a, Li Fei Nie a,b, Jun Dou a, Jiang Yu Zhao a,b, Gen Li a,b, Haji Aker 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
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of chalcone derivatives 1–18, bearing isoxazole moieties were designed and synthesized,
and biologically evaluated for their activity on mushroom tyrosinase and melanin synthesis in murine
B16 cells. The result indicated that most of prepared compounds 1–18 showed potent activating effect
on tyrosinase, especially for 1–2, 4, 6–7, 9 and 15. Among them, compounds 2, 4 and 9 demonstrated
Received 11 July 2016
Revised 23 August 2016
Accepted 31 August 2016
Available online xxxx
the best activity with EC50 = 1.3, 2.5 and 3.0
8-methoxypsoralan (8-MOP, EC50 = 14.8
l
molꢀLꢁ1 respectively, much better than the positive control
l
molꢀLꢁ1); In B16 cells, all the tested compounds exhibited a
Keywords:
Chalcone
Tyrosinase
Melanin synthesis
SAR
stronger activity on melanogenesis than 8-MOP (with the value of 115%). It was interesting that deriva-
tives substituted with halogen (1, 2, 4, 5, 7, 9) were generally more potent. Compounds 2 (463%) and 18
(438%) with 3 and 4-fold potency compared with 8-MOP respectively, were recognized as the most
promising candidate hits for further pharmacological study of anti-vitiligo.
Ó 2016 Published by Elsevier Ltd.
Vitiligo
1. Introduction
symptom of severe psychological distress, autoimmune abnormal-
ity, and increased risk of psychiatric morbidity. So far, several the-
Melanin, derived from dopaquinone and synthesized in the
melanosomes of melanocytes, serves number of valuable physio-
logical functions with the most important being photoprotection
of the human skin from ultra-violet (UV) radiation.1 More than
100 distinct genes have been found being involved in the regula-
tion of melanogenesis.2 Melanin synthesis is regulated by enzy-
matic cascade, such as tyrosinase, tyrosinase-related protein 1
(TRP-1) and tyrosinase-related protein 2 (TRP-2).3,4 Among them,
tyrosinase is regarded as the rate-limiting enzyme of melanogene-
sis, which modulates this process by catalyzing the hydroxylation
of tyrosine into 3,4-dihydroxyphenylalanine (DOPA) and the fur-
ther oxidation of DOPA into dopaquinone.5–7
ories have been put forward to explain the pathogenesis of
vitiligo.9 However, none of them is generally accepted by acade-
mia, and the clinical treatment of this disease was largely absent
from the effective medicines. The Vernohia anthelmintica L.
(Fig. 1) is a kind of plant growing only in the high altitude localities
of southern Xinjiang and limited regions of Pakistan and India. Its
fruits extract is one of the most popular Uygur medicines used for
vitiligo and initially recorded in ‘‘Yao Yong Zong Ku” around
300 years ago.10–13 Some important flavonoid compounds are iso-
lated from the plant (Fig. 2),14–17 and it is suggested that they play
an important role in this treatment. Since these flavonoid com-
pounds may activate tyrosinase and improve the melanin
production.18,19
Unfortunately, few flavonoids as activator of tyrosinase had
been reported. Some derivatives of carbazole chalcone uera20 and
40-(phenylurenyl/thiourenyl)chalcone21 (Fig. 3), which bearing a
poor activator effect on banana tyrosinase were first synthesized
and investigated. In addition, Dubois et al.22 synthesized several
aurones (1a–1e) and discovered that 1c, 1d can behave as hyper-
bolic activators of mushroom tyrosinase (Fig. 4); Haudecoeur
et al.23 prepared a series of 24 aurones with different hydroxyla-
tion patterns on A, B rings, evaluated their abilities on tyrosinase
Vitiligo also named leukoderma, is an acquired, progressive,
depigmenting disorder characterized by the development of white
macules in the skin due to the chronic, selective loss of functional
melanocytes in the epidermis.8 Depigmentation may be the
Abbreviations: 8-MOP, 8-methoxypsoralan; UV, ultra-violet; TRP-1, tyrosinase-
related protein 1; TRP-2, tyrosinase-related protein 2; DOPA, 3,4-dihydroxypheny-
lalanine; EWG, electron-withdrawing group.
⇑
Corresponding author.
E-mail address: haji@ms.xjb.ac.cn (H.A. Aisa).
http://dx.doi.org/10.1016/j.bmc.2016.08.066
0968-0896/Ó 2016 Published by Elsevier Ltd.
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2
C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
O
O
R1
X
N
N
H
N
H
R2
R
Figure 3. The structure of the carbazole and phenylurenyl/thiourenyl chalcone
derivatives.
R1
4'
1a
R1=R2=R3=H
3'
R2
B
1b R1=OH; R2=R3=H
1c R1=R3=H; R2=OH
1
HO
6
O
1'
R3
1d
A
R1=R2=H; R3=OH
1e R1=R2=OH; R3=H
3
Figure 1. The plant of the Vernohia anthelmintica L.
O
Figure 4. The structure of the synthetic aurones.
from mushroom to bacterial respectively, and the result showed
that 6b–6c, 7b–7c, 8b–8c and 9b–9c can improve the activity of
mushroom tyrosinase (Fig. 5).
R4'
4'
Our group has been dedicated on the drug development of the
vitiligo for many years.24,25 In our previous research,26 three novel
benzothiazole chalcones and two amide chalcones with potent
activating effect on mushroom tyrosinase were exploited (Fig. 6).
In addition, 1,2,3-triazole was introduced to A ring of chalcone
instead of thiazole. Fortunately, it was found that six novel chal-
cone derivatives were able to activate the mushroom tyrosinase
(Fig. 7), especially for compounds 10a and 14a, which possessed
a better activity than the positive control 8-MOP.27
R3'
B
1
3'
6
R6
R5
O
2'
R2'
A
5
3
4
O
R4
6a-6f R4=OH; R5=R6=H 7a-7f R4=R6=H; R5=OH
8a-8f 9a-9f
R4=R5=H; R6=OH
R4=R6=OH; R5=H
a: R2'=R3'=R4'=H, b: R2'=OH; R3'=R4'=H c: R3'=OH; R2'=R4'=H
d
e
f
: R4'=OH; R2'=R3'=H
: R3'=R4'=OH; R2'=H : R3'=R4'=OH; R3'=H
The isoxazole was widely use in medicinal chemistry, can be
regarded as isostere of the thiazole or triazole and possessed an
extensive range of biological activities.28–34 Beside, it could actively
participate in hydrogen bonding and was very stable in most reac-
tion conditions. Inspired by above results, the active molecules
were further optimized by replacing the 1,2,3-triazole with isoxa-
zole with the aim of developing a better medication for the vitiligo.
In this paper, eighteen novel chalcone derivatives bearing substi-
tuted isoxazole on ring A were synthesized, and the activity of
them on mushroom tyrosinase in cell-free and melanin content
in murine B16 cells was studied and the SAR was summarized.
Figure 5. The structure of 24 hydroxyl aurones.
H
N
R
N
H
R
R'
A
R'
N
A
S
O
O
4d,4h
5d,7b,7d
Compound
4d
4h
5d
Substituent
R=Cl,R'=Bz
R=Cl,R'=Ts
R=Cl,R'=H
EC50(-M)
21.1
25.8
30.6
17.1
2. Result and discussion
2.1. Synthesis
7b
R=CH3,R'=Bz
7d
R=Cl,R'=Bz
9.6
14.8
The synthetic procedure of the target compounds was shown in
Scheme 1. Commercially available aromatic aldehydes were con-
densed with hydroxyl amine hydrochloride to obtain their
8-MOP(positive control)
-
Figure 6. The structure of the benzothiazole and amide chalcones.
OH
OH
OH
HO
OH
O
OH
HO
OH
OH
HO
OH
O
HO
HO
OH
O
OH
OH
O
OH
O
3,4,2',4',5'-pentahydroxychalcone
Isoliquiritigenin
3,4,2',4',5'-pentahydroxy-6'-methoxy-
2-methylchalcone
Butein
OH
OH
OH
OH
OH
OH
OH
HO
O
HO
O
O
HO
O
O
HO
O
OH
OH
OH
O
OH
OH
O
Quercetin
Myricetin
Liquiritigenin
Butin
Figure 2. The structure of the flavonoid compounds isolated from the Vernohia anthelmintica L.
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C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
molꢀLꢁ1) and 15 (EC50 = 11.4
l
molꢀLꢁ1) were better
N
N
N
(EC50 = 3.0
l
to the positive control 8-MOP, of which EC50 was 14.8
l
molꢀLꢁ1
.
R
A
B
The position, number and nature of the substituent on benzene
ring were varied in order to identify the most appropriate group.
The compounds with halogen (1–7, 9) and -CF3O (11) showed
higher activity compared with -N(CH3)2 (13), -OCH3 (8, 10) and
un-substituted (12), which suggested that the electron-withdraw-
ing group (EWG) may be favorable to enhance the activity. How-
ever, the activity dropped rapidly when substituted with -NO2
(14), indicating that substituents with too strong electron-with-
drawing ability must have no benefit to the activity.
O
Compound
3a
8a
Substituent
R=3-OCH3
R=3-Cl
R=4-Cl
R=3,4-Cl
R=3-F
EC50(-M)
37.4f2.2
11.9f0.9
23.9f2.6
1.71f0.23
5.6f0.5
9a
10a
14a
15a
14.6f1.4
14.80f1.5
R=4-F
-
8-MOP(positive control)
Among these halogenated compounds 1–7 and 9, the most
important factor in their efficacy was the location of the group.
The shift of -F substituent from the para (1, EC50 = 6.4
Figure 7. The structure of the chalcones with 1,2,3-triazole.
l
molꢀLꢁ1
)
molꢀLꢁ1), which had
corresponding oxime derivatives (a0). They were converted to their
corresponding hydroxymethylisoxazoles (b0) by intermolecular
cycloaddition with propynol in presence of N-chlorosuccinimide.
The intermediates b0 were finally brominated to give the desired
c0 by PBr3 in dichloromethane at 0 °C.35
into the meta position led to 5 (EC50 = 21.3
l
a 3.3-fold lower activity. The similar result was observed in com-
pounds 6 and 3 which were substituted with Cl group as well. It
is apparent that the presence of a halogen group in the meta or para
position of the phenyl ring was crucial for activity.
The 40-hydroxyl chalcone was prepared through the Claisen–
Schmidt condensation of the corresponding acetophenones and
benzaldehydes using NaOH as base,36,37 followed by deprotection
with HCl in ethanol. The 40-hydroxyl chalcone was then refluxed
with c, catalyzed by K2CO3 in acetone to yield the final compounds
1–18.
The replacement of the -F group with -Cl contributed no
enhancement to the activity. Nevertheless, the number of the halo-
gen atom on benzene made a great influence on activity. Introduc-
tion of a second -F or -Cl to the benzene strongly increased activity,
such as 2, 4, 7 and 9 compared with 1, 6, 3 and 5. The interesting
thing was that 3,5-disubstituted compound 7 was less potent than
All new compounds were characterized by 1H NMR, 13C NMR, IR
and HRMS (ESI). As shown in Figure 8, the single crystals of the
3,4-disubstituted compound 2 (EC50 = 1.3
l
molꢀLꢁ1), 4 (EC50 = 2.5 -
l
molꢀLꢁ1) and 9 (EC50 = 3.0
l
molꢀLꢁ1), which exhibited a most
compounds
1 and 5 (1: CCDC No. 1476858; 5: CCDC No.
promising activity than any other compounds.
1476859) used for X-ray diffraction analysis were obtained by slow
evaporation of acetone-ethanol (V/V = 1:3) mixed solution at room
temperature.
However, the activity decreased dramatically when the benzene
on isoxazole was substituted by furan (15, 16), thiophen (17) and
1,4-benzodioxane (18), which suggested that benzene was funda-
mental for the activity.
2.2. Tyrosinase activity evaluation in cell-free system
Compared with the 1,2,3-triazole compounds in our previous
research21 (Fig. 3), the active isoxazole compounds in this work
(1, 2, 4, 6, 7, 9 and 15) generally showed a better activity, which
may be influenced by the shortened distance between benzene
and isoxazole, characteristics of the isoxazole as well.
The activities on tyrosinase of synthesized compounds 1–18
were performed according to a modified method,38 with 8-
MOP39–41 as positive control. The effects of the compounds were
summarized in Table 1. As shown in Table 1, most of prepared
compounds 1–18 showed good to excellent activating effect on
tyrosinase. The potencies of compound 1 (with the value of
2.3. Melanin synthesis evaluation in B16 cells
EC50 = 6.4
l
l
molꢀLꢁ1),
2
(EC50 = 1.3
l
l
molꢀLꢁ1),
4
(EC50 = 2.5
In order to avoid the possibility that inhibition of melanin syn-
thesis was due to cytotoxicity, we first performed CCK-8 assay to
molꢀLꢁ1) and 6 (EC50 = 8.1
molꢀLꢁ1), 7 (EC50 = 12.7
l
molꢀLꢁ1), 9
O
N
O
N
i
ii
iii
R
CHO
R
C
H
N OH
R
R
OH
Br
b'
c'
a'
MOMO
MOMO
HO
iv
v
H
O
O
O
O
N
O
R
HO
O
c
vi
O
O
1-18
1
R=4-fluorophenyl
2
R=3,4-difluorophenyl
5
R=3-fluorophenyl
3
6
9
R=3-chlorophenyl
4
7
R=3,4-dichlorophenyl
R=3,5-dichlorophenyl
R=4-chlorophenyl
8
R= 2-methoxyphenyl
R=4-chloro-3-fluorophenyl
12 R=phenyl
10 R=4-methoxyphenyl
11 R=3-trifluoromethoxyphenyl
13
14
15
R=4-dimethylaminophenyl
16 R=5-methylfuran-2-yl
R=4-nitrophenyl
17 R=thiophen-2-yl
R=5-iodofuran-2-yl
18 R=2,3-dihydrobenzo[b][1,4]dioxin-6-yl
Scheme 1. Synthetic route for the chalcones with isoxazole. (i) NH2OHꢀHCl, Na2CO3, 30% methanol aqueous solution, rt (ii) NCS, propynol, triethylamine, DCM, reflux (iii)
PBr3, DCM, 0 °C (iv) 10% NaOH, ethanol, rt (v) 3 M HCl, ethanol, rt (vi) K2CO3, acetone, reflux.
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C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
1
5
Figure 8. Chemical structure of the crystals of 1 and 5.
Table 1
determined whether these chalcone derivatives (1–18) were cyto-
toxic to B16 cells. The result showed that the cells treated with
compounds for 24 h caused mild cytotoxicity when compared with
Activator effect on tyrosinase of chalcone derivatives 1–18
a
Compd Substrate
R
EC50 (lmol/L)
the control at the dosage of 50 lM (Fig. 9); and the protein concen-
tration of B16 cells were examined as well in Figure 10.
1
4-Fluorophenyl
6.4 0.3
1.3 0.1
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
-Tyrosine
2
3,4-Difluorophenyl
3-Chlorophenyl
After that, all the compounds were further studied for their
activity on melanin synthesis, using a modified method.42 Accord-
ing to the screening data (Fig. 11), all the tested compounds from 1
to 18 exhibited a stronger activity than the control drug (8-MOP).
Among chalcones 1–14, compounds substituted with halogen (1, 2,
4, 5, 7, 9) and -CF3O (11) were generally more potent than that sub-
stituted with -OCH3 (8, 10) and -N(CH3)2 (13), It is apparent that
introduction of a EWG was accompanied by a noticeable increase
in activity, except -NO2 (14).
Different halogen were introduced into the different positions
of the benzene of isoxazole chalcone led to 1–7 and 9. Two mono
fluoro-substituted compounds 1, 5 and 3,4-dichloro substituted 2
possessed a better activity than monochloro-substituted 3, 6 and
4-chloro-3-fluoro substituted 9, especially for 2 (with the value
of 463%) and 5 (330%), which were separately 3 and 4-fold potent
than the control (8-MOP, 115%). Considering the meta-substituted
group (5) contributed more than the para one (1), it can be inferred
that the type and the position of the halogen was the most impor-
tant factor for their efficacy. The introduction of a second halogen
to benzene (2, 4, 7, 9) greatly improved the activity compared with
the mono. However, in the case of 7, a serious decrease (57%) was
3
23.8 1.0
2.5 0.2
21.3 0.8
8.1 0.4
12.7 0.3
65.3 3.4
3.0 0.3
47.2 2.3
25.5 0.7
78.1 4.7
28.9 0.8
>100
4
3,4-Dichlorophenyl
3-Fluorophenyl
5
6
4-Chlorophenyl
7
3,5-Dichlorophenyl
2-Methoxyphenyl
4-Chloro-3-fluorophenyl
4-Methoxyphenyl
3-Trifluoromethoxyphenyl
Phenyl
8
9
10
11
12
13
14
15
16
17
18
8-MOP
4-Dimethylaminophenyl
4-Nitrophenyl
5-Iodofuran-2-yl
5-Methylfuran-2-yl
Thiophen-2-yl
11.4 0.4
35.6 2.1
56.2 3.2
2,3-Dihydrobenzo[b][1,4]dioxin-6-yl >100
14.8
—
a
EC50 is the concentration of compounds that gave a 50% activator effect (when
the rate of activation = 50%). results are the mean SD from three independent
experiments.
Figure 9. Effect of chalcone derivatives on B16 cells viability. N means negative control; the B16 cells were incubated with 50 lM of different chalcone derivatives for 24 h
and the cell viability was assayed by adding CCK-8 solution. Values are expressed as the mean SD of three separate experiments. P > 0.05.
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C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 10. Effect of chalcone derivatives on protein concentration of B16 cells. N means negative control; 8-MOP as positive control. The B16 cells were incubated with 50
lM
of different chalcone derivatives for 48 h and the protein concentration was assayed by the method described below. Values are expressed as the mean SD of three separate
experiments. P > 0.05.
Figure 11. Effect of chalcone derivatives on melanin content in B16 cells. N means negative control; P means positive control (8-MOP); The B16 cells were treated with 50 lM
of different chalcone derivatives for 48 h. After that, melanin content was measured directly. Values are expressed as the mean SD of three separate experiments. The
statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test for multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001 compared with
control.
observed compared with 4, which indicated that position of two
halogen substituent on benzene was essential for the activity as
well.
effect on tyrosinase of mushroom, especially for the derivatives
with two halogen atoms on phenyl (2, 4 and 9); In cell system,
compounds from 1 to 18 exhibited a stronger activity on melano-
genesis than 8-MOP (positive control). Among them, 2 and 18 were
3 and 4-fold potent than the 8-MOP, respectively. Additional
experiments are also needed on human tyrosinase or melanocytes
to support the potential of these compounds for human-directed
applications. Currently, further studies on action mechanism of
these chalcone derivatives and animal experiment on vitiligo
transgenic mouse were under way. This research may provide
some new suggestion for the design of the novel activator of tyrosi-
nase based on chalcone.
When the benzene ring was substituted with furan, thiophen
and 1,4-benzodioxane (15–18), a remarkable increase was noticed,
which suggested that the benzene ring was not required for main-
taining the activity. The introduction of -I (15) also enhanced the
melanogenesis as same as we discovered above. It was impressed
that compound 18 (438%) with [1,4]dioxine demonstrated the sec-
ond most promising activity of all chalcone derivatives, which was
nearly 4-fold potent than the control 8-MOP.
3. Conclusion
4. Experimental section
4.1. Chemistry
In summary, eighteen chalcone compounds as novel activator of
tyrosinase was developed and biologically evaluated, It was found
that the introduction of substituted-phenyl isoxazole was an effec-
tive way to improve the activity of tyrosinase and the melanogen-
esis of murine B16 cells: Most of compounds (1–18) had potent
Reagents and solvents were purchased from Sigma, and used
without further purification. Thin-layer chromatography (TLC)
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C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
was carried out on glass plates coated with silica gel (Qingdao
Haiyang Chemical Co., G60F-254) and visualized by UV light
(254 nm). The products were purified by column chromatography
over silica gel (Qingdao Haiyang Chemical Co., 200–300 mesh).
Melting points were determined on a Buchi B-540 apparatus and
uncorrected. All the NMR spectra were recorded with a Vari-
an400 MHz NMR spectrometer in CDCl3, using TMS as an internal
standard. High-resolution mass spectra (HRMS) were recorded on
AB SCIEX QSTAR Elite quadrupole time-of-flight mass spectrome-
try. The IR data were recorded on a Thermo Fisher Scientific Nilolet
6700 FT-IR infrared spectrometer (KBr).
4.1.3.4.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(bro-
7.31 (d,
momethyl)isoxazole. 1H NMR (400 MHz, CDCl3)
d
J = 2.0 Hz, 1H), 7.29–7.26 (m, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.53 (s,
1H), 4.49 (s, 2H), 4.30 (s, 4H).
4.1.4. Preparation of 40-hydroxy chalcone
To the mixture of 40-methoxymethoxy acetophenone (0.18 g,
1.0 mmol) and benzaldehyde (1.06 g, 1.0 mmol) in ethanol
(20 mL) was added dropwise a solution of KOH (11.2 mg mmol,
0.2 mmol) in ethanol (2 mL) at 0–5 °C. The resulting mixture was
stirred at room temperature for 24 h. The mixture was concen-
trated under reduced pressure, and the residue was diluted with
water (35 mL). The aqueous mixture was neutralized by the addi-
tion of aqueous 10% HCl solution and extracted with ethyl acetate
(2 ꢂ 30 mL). The organic phase was washed with aqueous satu-
rated NH4Cl solution and brine. The organic layer was separated
and dried over anhydrous MgSO4, filtered, and concentrated under
reduced pressure to give the crude product, which was purified by
silica gel chromatography to produce the pure compound 40-meth-
oxymethoxy chalcone.
4.1.1. General procedure of preparation of intermediate a0
To a mixture of aldehyde (10.0 mmol) in 30% methanol aqueous
solution, NH2OHꢀHCl (0.695 g, 10.0 mmol) was added slowly. After
the NH2OHꢀHCl was fully dissolved, Na2CO3 (0.53 g 5.0 mmol) was
added and then the resulting mixture was stirred at room temper-
ature for 2 h. The reaction mixture was diluted with water and
extracted with CH2Cl2. The organic phase was dried to afford inter-
mediate a0 as white solids.
A
stirred mixture of 40-methoxymethoxychalcone (0.27 g,
1.0 mmol) in 20 mL 3 M HCl ethanol solution was refluxed for
2 h. After cooling, water was added, the resulted mixture was neu-
tralized with 10% NaOH and extracted with chloroform
(2 ꢂ 30 mL). The organic phase was washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated under reduced pres-
sure to give the crude product, which was purified by silica gel
chromatography with petroleum ether/ethyl acetate to give 40-
hydroxychalcone.
4.1.2. General procedure of preparation of intermediate b0
A mixture of a0 (10.0 mmol) in CH2Cl2 was stirred at room tem-
perature, NCS (1.60 g, 12.0 mmol) was then added. The resulted
mixture was stirred at reflux and propynol (0.56 g, 10.0 mmol)
was added dropwise. After 0.5 h, triethylamine (1.01 g, 10 mmol)
was added dropwise and refluxed for another 4 h. After cooling,
the reaction mixture was washed with water and evaporated
under reduced pressure. The obtained residue was purified by sil-
ica gel chromatography with petroleum ether/ethyl acetate as elu-
ent to give intermediate b0.
4.1.4.1. 40-Hydroxy chalcone. Yield 41%, white solid, mp 178–
181 °C, 1H NMR (400 MHz, CD3OD) d 8.01 (d, J = 8.4 Hz, 2H), 7.81
(d, J = 15.7 Hz, 1H), 7.72–7.60 (m, 2H), 7.54 (d, J = 15.7 Hz, 1H),
7.47–7.37 (m, 3H), 6.94 (d, J = 8.4 Hz, 2H). 13C NMR (101 MHz, CD3-
OD) d 188.82, 159.84, 144.23, 131.29, 131.16, 130.40, 128.93,
128.38, 121.79, 115.44, 110.00; ESI-MS (m/z, %): 223 ([MꢁH]ꢁ,
100).
4.1.2.1. 3-(4-Dimethylaminophenyl)-5-(hydroxymethyl)isoxa-
zole. 1H NMR (400 MHz, CDCl3) d 7.67 (d, J = 8.9 Hz, 2H), 6.76 (d,
J = 8.3 Hz, 2H), 6.48 (s, 1H), 4.79 (s, 2H), 3.02 (s, 6H).
4.1.2.2. 3-(4-Methoxyphenyl)-5-(hydroxymethyl)isoxazole. 1H
NMR (400 MHz, CDCl3)
J = 8.9 Hz, 2H), 6.49 (s, 1H), 4.78 (s, 2H), 3.84 (s, 3H).
d 7.71 (d, J = 8.9 Hz, 2H), 6.95 (d,
4.1.5. General procedure of preparation of 1–18
40-Hydroxychalcone (0.224 g, 1.0 mmol),
c (1.0 mmol) and
K2CO3 (0.69 g, 5 mmol) were dissolved in 20 mL of acetone. This
solution was refluxed for 5 h, the progress of the reaction was
monitored by TLC. When the reaction was complete, 100 mL of
water was poured into the solution, the reaction mixture was
extracted with CHCl3 for three times. The organic layer was sepa-
rated and washed with distilled water and dried over anhydrous
MgSO4. The solvent was removed at reduced pressure, and the resi-
due was purified by a silica gel column eluted with petroleum
ether/ethyl acetate to give the corresponding c.
4.1.2.3. 3-(3,5-Dichlorophenyl)-5-(hydroxymethyl)isoxazole. 1H
NMR (400 MHz, cdcl3) d 7.69 (d, J = 1.9 Hz, 2H), 7.44 (t, J = 1.9 Hz,
1H), 6.55 (s, 1H), 4.84 (s, 2H).
4.1.3. General procedure of preparation of intermediate c0
A mixture of b0 (10 mmol) in anhydrous CH2Cl2 (25 mL) was
cooled to 0 °C, a solution of PBr3 (0.95 mL, 10 mmol) in CH2Cl2
(2 mL) was added dropwise and stirred in ice-bath until the raw
material consumed. When warmed up to room temperature, the
reaction mixture was neutralized with 10% NaOH and extracted
with CH2Cl2 two times, The organic phase was evaporated under
reduced pressure and purified by silica gel chromatography with
petroleum ether/ethyl acetate to give c0.
4.1.5.1.
1-(4-((3-(4-Fluorophenyl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (1). Yield 89%, light yellow solid,
mp 163–166 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.7 Hz,
2H), 7.84–7.77 (m, 3H), 7.67–7.62 (m, 2H), 7.53 (d, J = 15.6 Hz,
1H), 7.45–7.39 (m, 3H), 7.15 (t, J = 8.6 Hz, 2H), 7.08 (d, J = 8.7 Hz,
2H), 6.64 (s, 1H), 5.29 (s, 2H). 13C NMR (101 MHz, CDCl3) d
188.78, 167.98, 165.30, 162.81, 161.44, 144.54, 135.09, 132.36,
131.05, 130.61, 129.09, 128.90, 128.54, 121.84, 116.36, 116.14,
114.71, 101.69, 61.47; IR (KBr) v: 2920, 2359, 1726, 1657, 1604,
1527, 1343, 1226, 839 cmꢁ1; HRMS (ESI) calcd for C25H19 FNO3
[M+H]+ 400.1349, found 400.1338.
4.1.3.1.
NMR (400 MHz, CDCl3)
3-(4-Methoxyphenyl)-5-(bromomethyl)isoxazole. 1H
7.73 (d, J = 8.7 Hz, 2H), 6.97 (d,
d
J = 8.6 Hz, 2H), 6.57 (s, 1H), 4.50 (s, 2H), 3.86 (s, 3H).
4.1.3.2. 3-(Thiophen-2-yl)-5-(bromomethyl)isoxazole. 1H NMR
(400 MHz, CDCl3) d 7.43 (ddd, J = 6.1, 4.3, 1.0 Hz, 2H), 7.11 (dd,
J = 5.0, 3.7 Hz, 1H), 6.50 (s, 1H), 4.80 (s, 2H).
4.1.5.2.
1-(4-((3-(3,4-Difluorophenyl)isoxazol-5-yl)methoxy)
4.1.3.3. 3-(5-Methylfuran-2-yl)-5-(bromomethyl)isoxazole. 1H
NMR (400 MHz, CDCl3) d 6.73 (d, J = 3.3 Hz, 1H), 6.43 (s, 1H),
6.09–6.06 (m, 1H), 4.75 (s, 2H), 2.34 (s, 3H).
phenyl)-3-phenylprop-2-en-1-one (2). Yield 85%, white solid,
mp 160–162 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.7 Hz,
2H), 7.82 (d, J = 15.7 Hz, 1H), 7.70–7.62 (m, 3H), 7.58–7.49 (m,
Please cite this article in press as: Niu, C.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.066

C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
2H), 7.45–7.40 (m, 3H), 7.08 (d, J = 8.6 Hz, 2H), 6.64 (s, 1H), 5.30 (s,
2H). 13C NMR (101 MHz, CDCl3) d 168.44, 161.39, 161.06, 144.62,
135.09, 131.11, 130.65, 129.11, 128.57, 123.45, 118.26, 118.08,
116.32, 116.13, 114.71, 101.62, 61.46; IR (KBr) v: 2921, 2357,
1760, 1640, 1600, 1545, 1488, 1323, 1215 cmꢁ1; HRMS (ESI) calcd
for C25H18 F2NO3 [M+H]+ 418.1255, found 418.1270.
1304, 1171, 975 cmꢁ1; HRMS (ESI) calcd for C25H18Cl2NO3 [M
+H]+ 450.0664, found 450.0669.
4.1.5.8. 1-(4-((3-(2-Methoxyphenyl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (8). Yield 80%, white solid, mp
132–135 °C; 1H NMR (400 MHz, CDCl3) d 8.06 (d, J = 8.8 Hz, 2H),
7.89 (dd, J = 7.7, 1.7 Hz, 1H), 7.81 (d, J = 15.7 Hz, 1H), 7.67–7.61
(m, 2H), 7.54 (d, J = 15.7 Hz, 1H), 7.45–7.38 (m, 4H), 7.09 (d,
J = 8.8 Hz, 2H), 7.04 (td, J = 7.6, 0.7 Hz, 1H), 7.00 (d, J = 8.3 Hz,
1H), 6.89 (s, 1H), 5.28 (s, 2H), 3.89 (s, 3H). 13C NMR (101 MHz,
CDCl3) d 188.77, 166.22, 161.66, 160.36, 157.32, 144.38, 135.09,
132.12, 131.54, 130.97, 130.53, 129.59, 129.05, 128.50, 121.87,
121.08, 114.73, 111.57, 105.51, 61.41, 55.68; IR (KBr) v: 2925,
2360, 1711, 1641, 1606, 1444, 1347, 1309, 824 cmꢁ1; HRMS (ESI)
calcd for C26H22NO4 [M+H]+ 412.1549, found 412.1559.
4.1.5.3.
1-(4-((3-(3-Chlorophenyl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (3). Yield 87%, white solid, mp
116–119 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz, 2H),
7.85–7.78 (m, 2H), 7.70 (d, J = 7.2 Hz, 1H), 7.67–7.62 (m, 2H),
7.53 (d, J = 15.7 Hz, 1H), 7.47–7.37 (m, 5H), 7.09 (d, J = 8.8 Hz,
2H), 6.67 (s, 1H), 5.31 (s, 2H). 13C NMR (101 MHz, CDCl3) d
188.81, 168.25, 161.68, 161.41, 159.24, 144.56, 135.18, 135.11,
132.41, 131.08, 130.62, 130.42, 129.10, 128.55, 127.13, 125.11,
121.86, 114.72, 101.75, 61.48; IR (KBr) v: 2922, 2360, 1726,
1658, 1600, 1467, 1257, 1189, 1081 cmꢁ1; HRMS (ESI) calcd for
4.1.5.9. 1-(4-((3-(4-Chloro-3-Fluoropheny)isoxazol-5-yl)meth-
oxy)phenyl)-3-phenylprop-2-en-1-one (9). Yield 90%, white solid,
mp 130–132 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz,
2H), 7.82 (d, J = 15.7 Hz, 1H), 7.67–7.59 (m, 3H), 7.57–7.47 (m,
3H), 7.45–7.40 (m, 3H), 7.08 (d, J = 8.8 Hz, 2H), 6.66 (s, 1H), 5.31
(s, 2H). 13C NMR (101 MHz, CDCl3) d 188.76, 168.52, 161.35,
161.04, 159.71, 157.23, 144.57, 135.07, 132.42, 131.42, 131.06,
130.62, 129.09, 128.54, 123.34, 121.81, 115.24, 115.01, 114.68,
101.63, 61.41; IR (KBr) v: 2924, 2359, 1726, 1657, 1604, 1579,
1450, 1256, 1176, 982 cmꢁ1; HRMS (ESI) calcd for C25H18ClFNO3
[M+H]+ 434.0959, found 434.0951.
C
25H19ClNO3 [M+H]+ 416.1053, found 416.1070.
4.1.5.4. 1-(4-((3-(3,4-Dichlorophenyl)isoxazol-5-yl)methoxy)
phenyl)-3-phenylprop-2-en-1-one (4). Yield 90%, white solid,
mp 161–163 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz,
2H), 7.91 (d, J = 1.9 Hz, 1H), 7.85–7.78 (m, 2H), 7.69–7.60 (m,
4H), 7.57–7.50 (m, 3H), 7.45–7.38 (m, 3H), 7.08 (d, J = 8.8 Hz,
2H), 6.66 (s, 1H), 5.31 (s, 2H). 13C NMR (101 MHz, CDCl3) d
188.80, 168.53, 161.30, 144.60, 139.18, 134.62, 132.42), 131.19,
131.08, 130.63, 130.56, 129.10, 128.84, 128.55, 128.10, 126.59,
126.08, 121.81, 114.69, 101.62, 61.43; IR (KBr) v: 2923, 2359,
1805, 1726, 1666, 1599, 1548, 1357, 1219 cmꢁ1; HRMS (ESI) calcd
for C25H18Cl2NO3 [M+H]+ 450.0664, found 450.0657.
4.1.5.10.
1-(4-((3-(4-Methoxyphenyl)isoxazol-5-yl)methoxy)
phenyl)-3-phenylprop-2-en-1-one (10). Yield 86%, white solid,
mp 145–147 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz,
2H), 7.81 (d, J = 15.7 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.68–7.62
(m, 2H), 7.54 (d, J = 15.7 Hz, 1H), 7.44–7.39 (m, 3H), 7.09 (d,
J = 8.9 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.62 (s, 1H), 5.29 (s, 2H),
3.86 (s, 3H). 13C NMR (101 MHz, CDCl3) d 188.63, 167.33, 162.19,
161.40, 161.15, 144.31, 134.96, 132.11, 130.89, 130.44, 128.94,
128.40, 128.25, 121.72, 121.07, 114.58, 114.36, 101.49, 61.35,
55.35; IR (KBr) v: 2924, 2359, 1726, 1678, 1620, 1573, 1483,
1378, 1256 824 cmꢁ1; HRMS (ESI) calcd for C26H22NO4 [M+H]+
412.1549, found 412.1563.
4.1.5.5.
1-(4-((3-(3-Fluorophenyl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (5). Yield 83%, light yellow solid,
mp 118–121 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz,
2H), 7.81 (d, J = 15.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.59 (d,
J = 7.7 Hz, 1H), 7.53 (d, J = 15.5 Hz, 2H), 7.47–7.39 (m, 4H), 7.15
(td, J = 8.3, 2.3 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 6.67 (s, 1H), 5.30
(s, 2H). 13C NMR (101 MHz, CDCl3) d 188.78, 168.19, 164.35,
161.41, 144.53, 135.08, 132.37, 131.05, 130.82, 130.60, 129.09,
128.54, 122.73, 121.83, 117.42, 117.21, 114.70, 114.12, 113.89,
101.81, 61.44; IR (KBr) v: 2922, 1834, 1726, 1659, 1604, 1573,
1370, 1286, 1187, 978 cmꢁ1; HRMS (ESI) calcd for C25H19FNO3 [M
+H]+ 400.1349, found 400.1361.
4.1.5.11.
1-(4-((3-(3-Trifluoromethoxyphenyl)isoxazol-5-yl)
methoxy)phenyl)-3-phenylpr-op-2-en-1-one (11). Yield 74%,
light yellow solid, mp 138–140 °C; 1H NMR (400 MHz, CDCl3) d
8.07 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 15.7 Hz, 1H), 7.75 (d, J = 7.8 Hz,
1H), 7.70–7.62 (m, 3H), 7.57–7.48 (m, 2H), 7.44–7.40 (m, 3H),
7.35–7.29 (m, 1H), 7.09 (d, J = 8.9 Hz, 2H), 6.69 (s, 1H), 5.32 (s,
2H). 13C NMR (101 MHz, CDCl3) d 188.81, 168.41, 161.60, 161.41,
144.58, 135.12, 132.45, 131.09, 130.65, 130.64, 129.11, 128.56,
125.36, 122.71, 121.86, 119.61, 114.72, 101.76, 61.48; IR (KBr) v:
4.1.5.6.
1-(4-((3-(4-Chlorophenyl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (6). Yield 88%, white solid, mp
135–139 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz, 2H),
7.81 (d, J = 15.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.67–7.62 (m,
2H), 7.53 (d, J = 15.6 Hz, 1H), 7.46–7.40 (m, 5H), 7.08 (d,
J = 8.8 Hz, 2H), 6.66 (s, 1H), 5.30 (s, 2H). 13C NMR (101 MHz, CDCl3)
d 188.80, 168.14, 161.81, 161.43, 144.57, 136.46, 135.10, 132.40,
131.07, 130.62, 129.42, 129.10, 128.55, 128.27, 127.23, 121.85,
114.71, 101.68, 61.48; IR (KBr) v: 2924, 1726, 1657, 1605, 1530,
2923, 1657, 1607, 1468, 1450, 1290, 1222, 1174, 983 cmꢁ1
;
HRMS (ESI) calcd for C26H19F3NO4 [M+H]+ 466.1266, found
466.1275.
1431, 1345, 1256, 1173, 1094, 826 cmꢁ1; HRMS (ESI) calcd for C25
-
H
19ClNO3 [M+H]+ 416.1053, found 416.1038.
4.1.5.12.
1-(4-((3-Phenylisoxazol-5-yl)methoxy)phenyl)-3-
phenylprop-2-en-1-one (12). Yield 85%, white solid, mp 131–
133 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz, 2H), 7.85–
7.78 (m, 3H), 7.67–7.63 (m, 2H), 7.53 (d, J = 15.7 Hz, 1H), 7.48–
7.40 (m, 6H), 7.09 (d, J = 8.8 Hz, 2H), 6.68 (s, 1H), 5.30 (s, 2H). 13C
NMR (101 MHz, CDCl3) d 188.78, 167.74, 162.72, 161.49, 144.47,
135.08, 132.28, 131.02, 130.56, 130.34, 129.07, 129.08, 128.51,
126.96, 121.85, 114.71, 101.84, 61.47; IR (KBr) v: 2923, 2360,
1678, 1603, 1450, 1343, 1253, 1194, 977 cmꢁ1; HRMS (ESI) calcd
for C26H19F3NO4 [M+H]+ 382.1443, found 382.1452.
4.1.5.7.
1-(4-((3-(3,5-Dichlorophenyl)isoxazol-5-yl)methoxy)
phenyl)-3-phenylprop-2-en-1-one (7). Yield 92%, light yellow
solid, mp 159–161 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d,
J = 8.8 Hz, 2H), 7.82 (d, J = 15.6 Hz, 1H), 7.72–7.62 (m, 4H), 7.53
(d, J = 15.6 Hz, 1H), 7.46–7.39 (m, 4H), 7.08 (d, J = 8.8 Hz, 2H),
6.66 (s, 1H), 5.31 (s, 2H). 13C NMR (101 MHz, CDCl3) d 188.64,
168.43, 161.11, 144.45, 139.04, 135.73, 130.92, 130.40, 130.09,
128.93, 128.38, 127.94, 126.42, 125.23, 121.68, 114.49, 101.48,
61.17; IR (KBr) v: 2923, 2360, 1726, 1630, 1599, 1547, 1367,
Please cite this article in press as: Niu, C.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.066

8
C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.5.13. 1-(4-((3-(4-Dimethylaminophenyl)isoxazol-5-yl)meth-
oxy)phenyl)-3-phenylprop-2-en-1-one (13). Yield 87%, yellow
solid, mp 106–110 °C; 1H NMR (400 MHz, CDCl3) d 8.06 (d,
J = 8.9 Hz, 2H), 7.81 (d, J = 15.7 Hz, 1H), 7.72–7.62 (m, 5H), 7.53
(d, J = 15.7 Hz, 1H), 7.44–7.39 (m, 3H), 7.09 (d, J = 8.9 Hz, 2H),
6.60 (s, 1H), 5.27 (s, 2H), 3.03 (s, 6H). 13C NMR (101 MHz, CDCl3)
d 188.84, 167.00, 162.61, 161.61, 144.43, 135.10, 132.18, 131.01,
130.55, 129.06, 128.52, 128.02, 121.89, 115.65, 114.73, 101.43,
77.48, 77.16, 76.84, 61.55, 40.64; IR (KBr) v: 2924, 1725, 1599,
1547, 1483, 1334, 1217, 975 cmꢁ1; HRMS (ESI) calcd for C27H25F3-
N2O3 [M+H]+ 425.1865, found 425.1852.
J = 2.0 Hz, 1H), 7.29 (dd, J = 8.4, 2.0 Hz, 1H), 7.08 (d, J = 8.9 Hz,
2H), 6.93 (d, J = 8.4 Hz, 1H), 6.58 (s, 1H), 5.27 (s, 2H), 4.29 (s, 4H).
13C NMR (101 MHz, CDCl3) d 188.81, 167.48, 162.23, 161.52,
145.51, 144.48, 143.98, 135.11, 132.27, 131.04, 130.58, 129.08,
128.54, 121.89, 122.06, 120.37, 117.95, 116.03, 114.72, 101.72,
64.64, 64.42, 61.49; IR (KBr) v: 2924, 1655, 1420, 1384, 1260,
1172, 1023, 890 cmꢁ1; HRMS (ESI) calcd for C27H22NO5 [M+H]+
440.1498, found 440.1488.
4.2. Biological activity
4.2.1. Tyrosinase activity assay
4.1.5.14.
1-(4-((3-(4-Nitrophenyl)isoxazol-5-yl)methoxy)phe-
Mushroom tyrosinase purchased from Sigma was stored in
refrigerator below ꢁ20 °C before use. Potassium phosphate buffer
(0.06 mL, 50 mM) at pH 6.5, 0.04 mL tyrosinase (250 U/mL) and
nyl)-3-phenylprop-2-en-1-one (14). Yield 70%, yellow solid, mp
121–123 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.8 Hz, 2H),
7.82 (d, J = 15.7 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.70–7.62 (m,
3H), 7.57–7.47 (m, 2H), 7.44–7.40 (m, 3H), 7.34–7.29 (m, 1H),
7.09 (d, J = 8.8 Hz, 2H), 6.69 (s, 1H), 5.31 (s, 2H). 13C NMR
(101 MHz, CDCl3) d188.74, 168.37, 161.46, 159.42, 149.77,
144.51, 135.06, 132.36, 131.03, 130.60, 129.06, 128.51, 125.33,
122.65, 121.81, 119.54, 114.68, 101.75, 61.40; IR (KBr) v: 2923,
1654, 1608, 1467, 1449, 1289, 1260, 1174, 995 cmꢁ1; HRMS (ESI)
calcd for C25H19N2O5 [M+H]+ 427.1294, found 427.1303.
2
l
L of the test compounds (0.5–300
inserted into 96-well plates. After 5 min incubation at 25–30 °C,
0.1 mL of -tyrosine (2 mM) was added and incubated for addi-
lM), dissolved in DMSO were
L
tional 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).
4.1.5.15. 1-(4-((3-(5-Iodofuran-2-yl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (15). Yield 74%, yellow solid, mp
151–154 °C; 1H NMR (400 MHz, CDCl3) d 8.06 (d, J = 8.8 Hz, 2H),
7.81 (d, J = 15.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.53 (d, J = 15.7 Hz,
1H), 7.44–7.40 (m, 3H), 7.06 (d, J = 8.9 Hz, 2H), 6.84 (d, J = 3.4 Hz,
1H), 6.68 (d, J = 3.5 Hz, 1H), 6.64 (s, 1H), 5.28 (s, 2H). 13C NMR
4.2.2. Cell culture
(101 MHz, CDCl3)
d
188.72, 167.62, 161.22, 157.96, 154.14,
Murine B16 melanoma cell lines (B16F10) were obtained from
CAS (Chinese Academy of Sciences, China). The B16F10 cells were
grown in DMEM medium (GIBICO, USA) supplemented with 10%
heat-inactivated fetal bovine serum (GIBICO, USA), 100 U/mL peni-
cillin and 100 mg/mL streptomycin (GIBCO, USA) in a humidified
atmosphere with 5% CO2 at 37 °C.
149.00, 144.45, 134.94, 132.23, 130.93, 130.47, 128.95, 128.41,
122.45, 121.70, 114.57, 113.04, 101.15, 90.64, 61.19; IR (KBr) v:
2922, 2360, 1654, 1604, 1449, 1254, 1018, 978 cmꢁ1; HRMS (ESI)
calcd for C23H17INO4 [M+H]+ 498.0202, found 498.0216.
4.1.5.16. 1-(4-((3-(5-Methylfuran-2-yl)isoxazol-5-yl)methoxy)
phenyl)-3-phenylprop-2-en-1-one (16). Yield 73%, yellow solid,
mp 115–118 °C; 1H NMR (400 MHz, CDCl3) d 8.05 (d, J = 8.9 Hz,
2H), 7.81 (d, J = 15.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.53 (d,
J = 15.7 Hz, 1H), 7.44–7.39 (m, 3H), 7.06 (d, J = 8.9 Hz, 2H), 6.79
(d, J = 3.3 Hz, 1H), 6.57 (s, 1H), 6.11 (dd, J = 3.2, 0.8 Hz, 1H), 5.26
(s, 2H), 2.37 (s, 2H). 13C NMR (101 MHz, CDCl3) d 188.85, 167.20,
161.45, 155.08, 154.64, 144.53, 142.14, 135.09, 132.30, 131.04,
130.59, 129.08, 128.54, 121.86, 114.72, 112.06, 108.12, 101.13,
61.39, 13.85; IR (KBr) v: 2921, 1655, 1603, 1499, 1253, 1171,
4.2.3. Cell viability measurement
Cell viability was assayed by adding CCK-8 (TransGen Biotech,
Beijing, China) solution. B16 cells were plated in 96-well dishes
at a density of 5 ꢂ 103 cells per well. After 12 h incubation at
37 °C, the culture medium was removed and replaced with fresh
medium containing the candidate compounds in indicated concen-
trations. The cells were incubated for another 24 h, washed with
ice cold PBS, 10 ll of CCK-8 solution was added into each well
and cells were incubated at 37 °C for another 2 h. The absorbance
was determined at 450 nm. The cell proliferation of each group
was calculated as the absorbance of the treatment group relative
to the control.
1025, 890 cmꢁ1
;
HRMS (ESI) calcd for C24H20NO4 [M+H]+
386.1392, found 386.1380.
4.1.5.17.
1-(4-((3-(Thiophen-2-yl)isoxazol-5-yl)methoxy)phe-
nyl)-3-phenylprop-2-en-1-one (17). Yield 67%, yellow solid, mp
136–138 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.9 Hz, 2H),
7.81 (d, J = 15.7 Hz, 1H), 7.67–7.62 (m, 2H), 7.53 (d, J = 15.6 Hz,
1H), 7.47 (dd, J = 3.6, 1.1 Hz, 1H), 7.45–7.40 (m, 4H), 7.14–7.10
(m, 1H), 7.10–7.05 (m, 2H), 6.61 (s, 1H), 5.28 (s, 2H). 13C NMR
4.2.4. Protein concentration measurement
Cells were treated with different compounds at the indicated
concentrations for 48 h, after treatment according to the method
previously described, melanin amount was measured spectro-pho-
tometrically at 405 nm by a multi-plate reader, total protein con-
centration was measured by the Bradford (Biomed, Beijing,
China)assays, measured spectro-photometrically at 595 nm by a
multi-plate reader.
(101 MHz, CDCl3)
d 188.83, 167.79, 161.43, 157.97, 144.54,
135.12, 132.38, 131.06, 130.61, 129.10, 128.55, 128.07, 127.90,
127.84, 127.23, 126.92, 121.88, 114.72, 101.85, 61.44. IR (KBr) v:
2922, 1655, 1603, 1448, 1344, 1256, 1227, 1032, 977 cmꢁ1; HRMS
(ESI) calcd for C24H20NO4 [M+H]+ 388.1007, found 388.1021.
4.2.5. Melanin contents assay
Exponentially growing cells were seeded into 6-well plates at a
concentration of 5 ꢂ 105 cells per well. After 24 h incubation at
37 °C, the culture medium was removed and replaced with fresh
medium containing the candidate compounds in different concen-
trations. The cells were incubated for another 48 h, washed with
ice cold PBS, followed by lysis with RIPA buffer for 40 min on ice,
4.1.5.18. 1-(4-((3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)isoxa-
zol-5-yl)methoxy)phenyl)-3-phenylprop-2-en-1-one (18). Yield
63%, yellow solid, mp 133–135 °C; 1H NMR (400 MHz, CDCl3) d
8.06 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 15.7 Hz, 1H), 7.67–7.62 (m,
2H), 7.53 (d, J = 15.7 Hz, 1H), 7.44–7.40 (m, 3H), 7.33 (d,
Please cite this article in press as: Niu, C.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.08.066

C. Niu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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and the lysates were centrifuged at 10,000 g for 20 min. Super-
natants containing protein were subject to the protein assay and
the pellets with intracellular melanin were solubilized in 200 ll
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spectrophotometrically at 405 nm by a multi-plate reader. The
melanin amount was calculated by normalizing the total melanin
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Acknowledgements
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This work was supported by the Funds for the West Light Foun-
dation of The Chinese Academy of Science (No. XBBS201403).
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