Study on synthesis and biological evaluation of 3-aryl substituted xanthone derivatives as novel and potent tyrosinase inhibitors

Article abstract of DOI:10.1248/cpb.c19-00572

Tyrosinase plays a key role in the melanin biosynthesis since it catalyzes the transformation of tyrosine into L-dopaquinone. A large number of studies have also shown that molecules to efficiently inhibit the activity of tyrosinase would be potentially used in treating many depigmentation-related disorders. In this study, we targeted a series of structure-based 3-aryl substituted xanthone derivatives in which diverse functional groups were respectively attached on 3-aromatic ring moiety as new tyrosinase inhibitors. The results demonstrated that all obtained compounds had potent tyrosinase inhibitory activities with IC₅₀ values at micromolar range. Especially, compound 4t was found to be the most active tyrosinase inhibitor with the IC₅₀ value of 11.3μM, uncovering that the introduction of the proper hydroxyl group in the 3-aromatic ring was beneficial for enhancing the inhibitory potency against tyrosinase. Moreover, the inhibition mechanism and inhibition kinetics studies revealed that compound 4t presented such inhibitory effect by acting as the reversible and competitive–uncompetitive mixed-II type inhibitor. Further molecular docking simulation showed that 3-aromatic ring of compound 4t was inserted into the narrow regions of binuclear copper-binding site at the bottom of the enzyme binding pocket, while the xanthone skeleton was positioned at the surface of tyrosinase. Taken together, these data suggested that such type of molecules might be utilized for the development of new and promising candidate for the treatment of depigmentation-related disorders.

Full text of DOI:10.1248/cpb.c19-00572

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c19-00572
Advance Publication
September 6, 2019
Chem. Pharm. Bull.
Regular Article
Study on synthesis and biological evaluation of 3-aryl substituted
xanthone derivatives as novel and potent tyrosinase inhibitors
Lihong Yu,a, † Liandi Chen,a,b,† Guolin Luo,a Licai Luo,a Wenqi Zhu,a Pengke Yan,b
Peiquan Zhang,a Chao Zhang,*,a and Wenhao Wu,*,a
a Key Laboratory of Molecular Target & Clinical Pharmacology，School of Pharmaceutical
Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou
b
511436, Guangdong, China: and The Third Affiliated Hospital of Guangzhou Medical
University, Guangzhou, 510150, Guangdong, China.
† These authors contributed equally to this work.
* Correspondence e-mail: wenhao_wu@gzhmu.edu.cn (W.W.); chao-zh@163.com (C.Z.);
Ⓒ 2019 The Pharmaceutical Society of Japan

Summary
Tyrosinase plays a key role in the melanin biosynthesis since it catalyzes the
transformation of tyrosine into L-dopaquinone. A large number of studies have also shown
that molecules to efficiently inhibit the activity of tyrosinase would be potentially used in
treating many depigmentation-related disorders. In this study, we targeted a series of
structure-based 3-aryl substituted xanthone derivatives in which diverse functional groups
were respectively attached on 3-aromatic ring moiety as new tyrosinase inhibitors. The
results demonstrated that all obtained compounds had potent tyrosinase inhibitory activities
with IC50 values at micromolar range. Especially, compound 4t was found to be the most
active tyrosinase inhibitor with the IC50 value of 11.3 μM, uncovering that the introduction
of the proper hydroxyl group in the 3-aromatic ring was beneficial for enhancing the
inhibitory potency against tyrosinase. Moreover, the inhibition mechanism and inhibition
kinetics studies revealed that compound 4t presented such inhibitory effect by acting as the
reversible and competitive-uncompetitive mixed-II type inhibitor. Further molecular
docking simulation showed that 3-aromatic ring of compound 4t was insert into the narrow
regions of binuclear copper-binding site at the bottom of the enzyme binding pocket, while
the xanthone skeleton was positioned at the surface of tyrosinase. Taken together, these
data suggested that such type of molecules might be utilized for the development of new
and promising candidate for the treatment of depigmentation-related disorders.
Keywords xanthone derivative; tyrosinase inhibitor; synthesis; inhibition mechanism
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Tyrosinase (EC 1.14.18.1), also known as polyphenoloxidase (PPO), is a multifunctional
enzyme and structurally belong to the type-3 copper-containing protein family.1) The
crystallographic structure of tyrosinase from three different species has revealed that the
active center of the dicopper ions coordinated by six histidine residues and a lipophilic
long-narrow gorge near to the active center composed the ligand-binding domain (LBD) of
tyrosinase.2) This enzyme is widely distributed in nature and mainly employed as the
rate-limiting step for the melanin biosynthesis through catalyzing two distinct reactions: the
hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) (called
cresolase/monophenolase activity) and the oxidation of DOPA into ortho-quinone
(dopaquinone) (called catecholase/diphenolase activity). Recently, the crystal structures and
active binding pocket of enzyme-substrates (tyrosine and DOPA) were also analyzed.3)
Furthermore, the produced dopaquinone is highly reactive and can further spontaneously
polymerize to generate brown pigments of high molecular weight, namely melanin,4-5)
which are responsible for the color of mammalian skin and hair and also plays an important
role in the protection of the skin against UV light induced damage.6-10) Despite these good
physiological functions demonstrated by this enzyme, the overproduction of melanin would
cause not only serious dermatological problems such as melasma, freckles, senile lentigines,
age spots, and sites of actinic damage in human beings.11) but also the undesired enzymatic
browning of fruits and vegetables.12) Moreover, excessive accumulation of melanin in the
mammalian brain caused by abnormal tyrosinase activity is linked to the neurodegenerative
diseases including Parkinson's disease.13) Obviously, the unregulated action of tyrosinase
has become an important factor in a number of human disease etiologies. Accordingly,
tyrosinase inhibitors have been ardently explored as an avenue for therapies to these
diseases and have become increasingly important in agriculture and food industry, as well
as in medicinal and cosmetic products. To date, a huge number of naturally occurring and
synthetic compounds acting as the tyrosinase inhibitors have already been reported,14-19) but
most of them are not potent enough to put into practical use due to their weak inhibitory
activities or safety concerns. Undoubtedly, it is still necessary to develop new tyrosinase
inhibitors with improved therapeutic profiles.
Xanthones are a class of oxygen-containing heterocycles with γ-pyran moiety condensed
Chemical and Pharmaceutical Bulletin Advance Publication

with two benzene rings, which are widely distributed in nature.18) In recent years, xanthone
derivatives have been received considerable attention in the field of medicinal chemistry
because of their diversely biological functions,20-21) such as anti-oxidant,22)
anti-hypertensive,23)
anti-convulsant,24)
anti-cholinesterase,25)
anti-malarial,26)
antimicrobial,27) anti-inflammatory,28) and anti-cancer activities.29) Besides, they also
served as the effective inhibitors of several enzymes like α-glycosidase,30-31)
topoisomerase,32) proteinkinase,33) aromatase.34) Driven by both the biologically applied
powers and the favorable pharmacological properties, indeed, xanthone skeleton has been
defined as one of the privileged structural motifs for next stage of certain new drug
screening and discovery.
On the other hand, polyphenol derivatives have attracted considerable attention
compared with the other well-known tyrosinase inhibitors due to their favorable interaction
with the hydrophobic protein pocket surrounding the binuclear copper active center of
tyrosinase.35) As a result, several different kinds of polyphenol compounds as the tyrosinase
inhibitors have been reported, such as flavanones,36) chalcones,37) aurones,37) resveratrols,38)
benzoate ester derivatives,39) hydroxylated biphenyls,40) hydroxyl substituted
benzaldoximes.41) and thiosemicarbazone derivatives.42-44) On the contrary, despite most of
naturally occurring or synthetic xanthones possess a polyphenol moiety,44) their tyrosinase
inhibitory activity have not yet been studied. On the basis of the crystallographic structure
of tyrosinase and by analogy with the chemical structures of xanthones and other reported
tyrosinase inhibitors, we reasoned that a proper introduction of the substituent to the
xanthone core might deliver the acceptable inhibitory effect against tyrosinase through the
potent interaction with the LBD of tyrosinase.
Take advantage of the above information and as a part of our ongoing studies on the
synthetic and application of new biologically active compounds as the promising
candidates for new drug discovery. Herein, a series of 3-aryl substituted xanthones bearing
various functional groups (e.g. hydroxyl, methoxyl, formyl, carboxyl, methoxy carbonyl,
acetyl and thiosemicarbazide) were designed, synthesized and evaluated as novel tyrosinase
inhibitors. Moreover, the inhibition mechanism and inhibition kinetics of the selected
compounds were investigated. Besides, the molecular docking analysis was also performed
to elucidate the inhibition binding mode of the potent xanthones with the LBD of
Chemical and Pharmaceutical Bulletin Advance Publication

tyrosinase. We hope that the present findings can provide the scientific evidence to lead to
the discovery of alternative and highly efficient pharmacological agents for the treatment of
tyrosinase-related disorders.
Results and discussion
Chemistry Taking account into the specificity of hydroxylated flavone structure as the
tyrosinase inhibitor, we herein design 3-aryl substituted xanthone derivatives by
introducing hydroxylated and other functionalities to screen and then to disclose and the
key factors such as the efficient substituent and its position for determining the inhibitory
ability against tyrosinase. The synthetic route of target compounds was shown in Chart 1.
Briefly, the parent nucleus of xanthone 1 was firstly prepared via a condensation reaction of
salicylic acid and phloroglucinol in the presence of Eaton's reagent at 80 oC. 46) It was then
treated with triflic anhydride and trimethylamine in dichloromethane to give the triflated
intermediate 2 in a decent yield of 87%. Because of the existence of the hydrogen bond
between hydroxyl group at position-1 and carbonyl part at position-9, the triflate moiety
was regioselectively attached at 3-hydroxyl position of xanthone 1. Subsequently, treatment
of triflate 2 with appropriately substituted phenyl boronic acid 3 in the presence of K3PO4
proceeded smoothly via the classical Suzuki-Miyaura coupling to afford 3-aryl substituted
xanthone derivatives 4a-4n in good yields, in which Pd2(dba)3 and XPhos were employed
as the ligand and catalyst, respectively. Moreover, carboxyl substituted compounds 4o-4q
can be obtained from methoxycarbonyl xanthones 4d-4f by the convenient hydrolysis in
NaOH solution. Finally, demethylation of methoxyl xanthones 4h-4n with
o
boron tribromide in dichloromethane at 0 C was carried out to provide the corresponding
3-polyphenolsubstituted xanthone derivatives 4r-4x in excellent yields.
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1. Synthesis of 3-aryl substituted xanthone derivatives
Biological activity To explore the inhibitory effect of xanthone derivatives and define
the importance of 3-aryl moiety, all the newly synthesized compounds 4a-4x were screened
for the inhibitory activity on diphenolase of mushroom tyrosinase, where the well-known
tyrosinase inhibitor kojic acid was used as the standard reference. The structures and IC50
values of all the synthesized compounds were summarized as shown in Table 1.The result
showed that all the target compounds have favorable tyrosinase inhibitory activities
compared with the parent compound with different inhibition profiles (the ranges of IC50
values from 11.3 to 115.8 μM vs 1-hydroxyl xanthone IC50 > 150 μM), which was in
agreement with our structure-based design. Among them, compounds 4r-4t bearing a
hydroxyl group on the 3-aryl part demonstrated more potent tyrosinase inhibitory activities
than that of these bearing other hydrogen-bond donor/receptor, such as formyl (4a-4c),
ester (4d-4f) acetyl (4g), or carboxyl (4o-4q) groups. Especially, the compound 4t was
Chemical and Pharmaceutical Bulletin Advance Publication

found to be the most active tyrosinase inhibitor with the IC50 value of 11.3 μM, even more
active than kojic acid (IC50 = 17.3 μM). Taken together, the results revealed that: (1) the
introduction of the hydroxyl group on the 3-aryl part obviously facilitated tyrosinase
inhibitory activity, and (2) the position of the hydroxyl group played a vital role in
determining the tyrosinase inhibitory activity, and in the present investigation, the
para-position was optimal. It should be emphasized that their corresponding
3-mono-methoxyphenyl substituted derivatives 4h-4j exhibited a less efficiency for the
inhibition of tyrosinase. For example, compound 4j manifested a relatively low inhibitory
activity with the IC50 value of 36.5 μM, which was 3-fold less than that of 4t. Moreover,
further loss of the inhibitory activity against tyrosinase was detected from compounds 4h
and 4i since they gave the relatively high IC50 values (for 4h, IC50 = 82.2 μM; for 4i, IC50 =
109.1 μM), compared with 4t. Similarly, it was found that either ortho- or meta-methoxyl
substituted inhibitors were less active than para-substituted analogues, which further
validated the importance of the substituent attached at the para-position. Meanwhile, the
results also implied that the hydroxyl substituent might form the potential hydrogen-bond
interaction with the LBD of tyrosinase to contribute the inhibition of tyrosinase.
Compared with compounds 4r-4t, unfortunately, the results from Table 1 showed that
dihydroxylated compounds 4u-4w possessed relatively weak tyrosinase inhibitory potency,
revealing that the incorporation of an additional hydroxyl group onto the 3-aryl moiety led
to a reduction in potency. Of note, IC50 values of 4u-4w were less than their corresponding
methoxyl-substituted compounds 4k-4m. The results suggested that the nature, type and
number of the substituent played a key role in determining inhibitory effects, possibly due
to that the different interaction modes of the compound with the LBD of tyrosinase
depended by the structure and size of the ligand was involved in these cases.
Interestingly, trimethoxy substituted compound 4n only exhibited comparable inhibitory
activities compared with dimethoxy substituted analogues 4l-4m. A similar result can be
observed from trihydroxy substituted compounds 4x. The main reason might be that, the
introduction of the additional functional group led to the big steric hindrance, which
prevented the substrate to access the dicopper ions active center of tyrosinase via the
lipophilic long-narrow gorge near to the active center, thereby showing a relatively low
tyrosinase inhibitory activity.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 1. Structures and inhibitory activities of all synthesized compounds.
Compound
R1
CHO
H
R2
R3
R4
H
IC50 (µM) a
45.6±0.25
54.1±0.37
42.1±0.23
78.1±0.34
115.8±0.38
56.1±0.31
45.1±0.28
82.2±0.30
109.1±0.35
36.5±0.18
37.4±0.20
34.8±0.21
39.8±0.23
35.2±0.27
55.1±0.31
73.9±0.38
44.2±0.23
14.1±0.15
16.2±0.19
11.3±0.16
53.6±0.29
50.7±0.28
92.6±0.38
52.6±0.26
> 150
4a
H
H
4b
4c
CHO
H
H
H
H
CHO
H
H
4d
4e
COOMe
H
H
H
COOMe
H
H
H
4f
H
COOMe
COCH3
H
H
4g
H
H
H
4h
4i
OMe
H
H
H
OMe
H
H
H
4j
H
OMe
OMe
OMe
H
H
4k
4l
OMe
H
H
H
OMe
OMe
OMe
H
H
4m
4n
4o
H
OMe
OMe
H
H
OMe
H
COOH
H
4p
4q
4r
COOH
H
H
H
H
COOH
H
H
OH
H
H
H
4s
OH
H
H
H
4t
H
OH
OH
OH
H
H
4u
4v
OH
H
H
H
OH
OH
OH
H
4w
4x
H
OH
OH
H
OH
1
Kojic acid
17.3±0.15
a All compounds were examined in a set of experiments repeated three times. IC50 values of
compounds represent the concentration that caused 50% enzyme activity loss.
Chemical and Pharmaceutical Bulletin Advance Publication

Inhibition Mechanism Inspired by the above findings, next we were sought to explore
the inhibition mechanism of the selected compound 4t. As illustrated in Fig. 1, the
inhibition mechanism on mushroom tyrosinase by compound 4t for the oxidation of
L-DOPA was studied. The results showed that, the plots of the remaining tyrosinase activity
versus the concentrations of tyrosinase in the presence of different concentrations of 4t
gave a family of straight lines, which all passed through the origin point. Increasing the
concentration of 4t resulted in a decrease in the slope of the line, revealing that the
inhibition of compound 4t on tyrosinase was reversible.
Figure 1. Determination of the inhibitory effect of the selected compound 4t on mushroom
tyrosinase for the catalysis of L-DOPA. The concentrations of 4t for curves were 0, 10, 20
and 30 μM, respectively.
To further insight into the inhibition mechanism, we were interested to investigate the
inhibitory type of compound 4t determined by Lineweaver-Burk double reciprocal plots. In
the presence of 4t, the kinetics of the enzyme was shown in Fig. 2. The results displayed
that the plots of 1/V versus 1/[S] gave four straight lines with different slopes that
intersected one another in the third quadrant; with the increase of the concentration of 4t,
the values of Vm and Km all descended, suggesting that 4t was a competitive-uncompetitive
mixed-II type inhibitor.
Chemical and Pharmaceutical Bulletin Advance Publication

Figure 2. Determination of the inhibitory type of the selected compound 4t on mushroom
tyrosinase for the catalysis of L-DOPA. The concentrations of 4t for curves were 0, 10, 20,
and 40 μM, respectively.
Molecular Docking In order to further substantiate the interaction of the most active
compound 4t with tyrosinase, the molecular docking simulation was implemented using
SYBYL X-2.1 software. The crystal structure of Agaricus bisporus tyrosinase (PDB code:
2Y9X) was used for this study 47). As shown in Fig. 3A, it displayed that tyrosinase had a
very special binding pocket, which contained both spacious and narrow regions for the
binding of its active ligands. The two copper ions located at the bottom of binding pocket
were coordinated to the six histidine residues from the enzyme.48) According to the crystal
structure of tyrosinase, the inhibitor tropolone was small enough to be bound deeply in the
binuclear copper-binding site and its oxygen atom had a short distance of ~ 3.5 Å to either
copper ion. Interestingly, the relatively large inhibitor 4t had a different binding mode. As
the docking results demonstrated in Fig. 3A and 3B, compound 4t could be divided into
Chemical and Pharmaceutical Bulletin Advance Publication

two parts: the large part of xanthone skeleton and the small part of para-hydroxyphenyl
group at position-3. As a result, the small part of the para-hydroxyphenyl group could enter
narrow regions of ligand-bound pocket adopted a similar behavior as tropolone. In the
binuclear copper-binding site, the distance between oxygen atom of para-hydroxyphenyl
group and copper ion was 4.1 Å, which meant that the para-hydroxyphenyl group was not
bound as deeply as tropolone (4.1 Å vs 3.5 Å). Moreover, a same direction of the
para-hydroxyphenyl group in active site was observed compared with tropolone and it was
manifested that the benzene ring part was almost parallel with tropolone which ever existed
in the binding pocket. Nevertheless, the benzene ring of the para-hydroxyphenyl group
fitted into the hydrophobic residues of binding pocket formed by Met280, Gly281, Ser282,
Val283, and Ala286. On the other hand, the large part of xanthone skeleton was mostly
positioned at the surface of the enzyme binding pocket with hydrophobic residues
surrounded by Val248, Gly249, and Met257. In addition, the dihedral angle between the
xanthone scaffold and the para-hydroxyphenyl moiety was nearly 120 degree, which made
the xanthone skeleton block the substrate such as L-DOPA to access the binuclear
copper-binding active site at the bottom of the enzyme binding pocket. Taken together, the
inhibitor 4t presented a potent inhibition against tyrosinase by acting as a reversible and
competitive-uncompetitive mixed-II type inhibitor, which was consistent with the results
from the inhibitory activity evaluation and the inhibition mechanism and inhibition kinetics
studies. Finally, it should be noted that two or three hydroxy xanthones such as compounds
4v and 4x have a different binding mode compare with compound 4t according to their
docking results. The part that insert into narrow regions of ligand-bound pocket is xanthone
scaffold rather than 3-aryl moiety and xanthone scaffold doesn’t entre as deeply as 3-aryl of
compound 4t, it is proven by a relatively low total score. One possible reason could
attribute to steric hinder and this result can explain the inhibitory activity of compound 4t is
more potent.
Chemical and Pharmaceutical Bulletin Advance Publication

Figure 3. (A) Surface and binding pocket with compound 4t on tyrosinase. (B) Predicated
binding mode of compound 4t docked with tyrosinase at the active site. Copper ions are
displayed as green sphere. (Color figure can be accessed in the online version.)
Conclusion
In summary, we have defined for the first time a series of 3-aryl substituted xanthone
Chemical and Pharmaceutical Bulletin Advance Publication

derivatives with the corresponding diversely functional groups as novel and potent
tyrosinase inhibitors. The results demonstrated that all synthesized compounds had potent
tyrosinase inhibitory activities. Especially, the compound 4t exhibited the most potent
tyrosinase inhibitory activity with the IC50 value of 11.3 µM, which was more active than
the well-known tyrosinase inhibitor kojic acid. The structure-activity relationships (SARs)
analysis indicated that: (1) the introduction of the hydroxyl group on the 3-aryl part
obviously facilitated tyrosinase inhibitory activity, (2) the position and number of the
hydroxyl group played a vital role in determining the tyrosinase inhibitory activity.
Moreover, the inhibition mechanism and inhibition kinetics studies revealed that compound
4t exhibited such inhibitory effect by acting as the reversible and
competitive-uncompetitive mixed-II type tyrosinase inhibitor. The subsequent molecular
docking simulation further disclosed the feasible interaction mode between 4t with the
LBD of tyrosinase. Taken together, these results suggested that such type of 3-aryl
xanthone compounds could serve as a new class of promising candidates for future
investigation to treat the tyrosinase-related disorders.
Experimental
Chemistry The 1H NMR and 13C NMR spectra were recorded using TMS as the internal
standard on a Bruker Avnace III 400 spectrometer or JOEL ECA-400 spectrometers at 400
MHz and 101 MHz in either CDCl3, DMSO-d6 or Acetone-d6. Chemical shifts (δ) were
13
referenced to TMS as an internal standard (1H NMR, C NMR). Coupling constants are
given in Hz. High-resolution mass (HRMS) spectra were obtained with Shimazu
LCMS-IT-TOF mass spectrometer or Thermo-DFS mass spectrometer. Reaction progress
was monitored using analytical thin layer chromatography (TLC) on precoated silica gel
GF254 (Qingdao Haiyang Chemical Plant, Qingdao, China) plates and the spots were
detected under UV light (254 nm). Flash column chromatography was performed with
silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. All
commercially available compounds and solvents were used without further purification.
Mushroom tyrosinase (activity of the enzyme is ≥1000 unit/mg) was purchased from
Merck Life Science (Shanghai) Co. Ltd. All the purity of tested compounds was
1
determined by combining H NMR and HRMS to ensure the purity over 95%. Some
Chemical and Pharmaceutical Bulletin Advance Publication

selected compounds such 4t, its purity was further examined by HPLC.
1-Hydroxy-9-oxo-9H-xanthen-3-yl trifluoromethanesulfonate 2 Under nitrogen,
xanthone 1 (0.69 g, 3 mmol) was dissolved in 60 mL of dry DCM in a 250 mL Schlenk
flask. Analytical-grade Et3N (15 mL) was added and the solution was cooled to 0 oC. Then
triflic anhydride (1.1 mL, 6.6 mmol) was added dropwise via syringe over 5 min and the
o
mixture was stirred at 0 C for 30 min. The reaction solution was following allowed to
o
warm up to 50 C and kept stirred for another 6 h. At the conclusion of the reaction, the
mixture was quenched with saturated NaCl solution and extracted by CH2Cl2 three times.
The combined extract was then washed successively with aqueous HCl (10%), saturated
NaHCO3 and brine. The organic layer was dried over Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography gave the
1
desired intermediate 2 as a yellow solid, yield 87%. H NMR (400 MHz, DMSO-d6) δ
12.90 (s, 1H), 8.22 - 8.19 (dd, J = 8.0, 1.5 Hz, 1H), 7.99 - 7.95 (ddd, J = 8.7, 7.2, 1.7 Hz,
1H), 7.71 - 7.69 (d, J = 8.5, 1H), 7.58 - 7.54 (ddd, J = 8.1, 7.2, 1.0 Hz, 1H), 7.38 (d, J = 2.3
Hz, 1H), 7.05 (d, J = 2.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 181.67, 162.97,
156.92, 156.10, 154.11, 137.40, 126.02, 125.72, 120.43, 120.24 (JC-F = 160 Hz, 1C), 118.54
(JC-F = 160 Hz, 1C), 117.05 (JC-F = 160 Hz, 1C), 108.92, 104.43, 101.84. ESI-HRMS Calcd.
for C14H7O6F3S [M+H]+: 360.9949; found: 360.9982.
General Procedure for the Preparation of 4a-4n An oven dried round bottom flask
was charged with compound 2 (1 mmol, 1.0 equiv), Pd2(dba)3 (5 mol%), XPhos (6 mol%),
K3PO4 (3 mmol, 3.0 equiv) and phenylboronic acid 3 (2 mmol, 2.0 equiv.). The flask was
capped with a rubber septum and twice evacuated and backfilled with N2. Toluene (30 mL)
was injected sequentially and the reaction was stirred at 110 oC for 24 h. After completion,
the reaction mixture was passed through small pad of celite. The filtrate was diluted with
EtOAc and water, the combined organic layer was dried over Na2SO4 and concentrated in
vacuum. The obtained crude product was purified by flash chromatography on silica gel
gave the corresponding target compounds 4a-4n.
2-(1-Hydroxy-9-oxo-9H-xanthen-3-yl)benzaldehyde (4a)
Compound 2 was treated with (2-formylphenyl)boronic acid (3a) according to general
1
procedure to give the desired product 4a as yellow solid. Yield 73%, H NMR (400 MHz,
DMSO-d6) δ 12.67 (s, 1H), 9.99 (s, 1H), 8.25 – 8.22 (dd, J = 8.0, 1.6 Hz, 1H), 7.99 – 7.94
Chemical and Pharmaceutical Bulletin Advance Publication

(m, 2H), 7.83 – 7.79 (td, J = 7.5, 1.4 Hz, 1H), 7.71 – 7.66 (m, 2H), 7.62 (d, J = 7.7 Hz, 1H),
7.56 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 1.4 Hz, 1H), 6.93 (d, J = 1.4 Hz, 1H). 13C NMR (101
MHz, DMSO-d6) δ 191.95, 182.04, 161.07, 156.25, 155.88, 147.35, 143.45, 137.15, 134.45,
133.75, 131.03, 129.55, 128.43, 125.98, 125.33, 120.52, 118.60, 112.35, 109.54, 108.17.
ESI-HRMS Calcd. for C20H12O4 [M+H]+: 317.0769; found: 317.0803.
3-(1-Hydroxy-9-oxo-9H-xanthen-3-yl)benzaldehyde (4b)
Compound 2 was treated with (3-formylphenyl)boronic acid (3b) according to general
1
procedure to give the desired product 4b as yellow solid. Yield 70%, H NMR (400 MHz,
CDCl3) δ 12.73 (s, 1H), 10.14 (s, 1H), 8.33 (d, J = 7.9 Hz, 1H), 8.21 (s, 1H), 7.98 (t, J = 7.9
Hz, 2H), 7.81 (t, J = 7.8 Hz, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.46 (t,
13
J = 7.5 Hz, 1H), 7.25 (d, J = 1.4 Hz, 1H), 7.11 (d, J = 1.3 Hz, 1H). C NMR (126 MHz,
DMSO-d6) δ 193.54, 181.88, 161.84, 156.73, 156.32, 148.02, 139.36, 137.07, 133.63,
130.52, 129.84, 129.54, 127.92, 125.98, 125.26, 120.58, 118.56, 114.36, 108.95, 106.05.
ESI-HRMS Calcd. for C20H12O4 [M+H]+: 317.0769; found: 317.0804.
4-(1-Hydroxy-9-oxo-9H-xanthen-3-yl)benzaldehyde (4c)
Compound 2 was treated with (4-formylphenyl)boronic acid (3c) according to general
1
procedure to give the desired product 4c as yellow solid. Yield 70%, H NMR (400 MHz,
DMSO-d6) δ 12.60 (s, 1H), 10.09 (s, 1H), 8.21 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 8.1 Hz, 2H),
8.03 (d, J = 8.3 Hz, 2H), 7.95 (t, J = 7.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.54 (t, J = 7.6
Hz, 1H), 7.51 (s, 1H), 7.24 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 193.27, 181.85,
161.72, 156.57, 156.27, 147.81, 143.98, 137.06, 136.72, 130.55, 128.59, 125.94, 125.24,
120.53, 118.53, 109.21, 108.38, 106.37. ESI-HRMS Calcd. for C20H12O4 [M+H]+:
317.0769; found: 317.0805.
Methyl 2-(1-hydroxy-9-oxo-9H-xanthen-3-yl)benzoate (4d)
Compound 2 was treated with (2-(methoxycarbonyl)phenyl)boronic acid (3d) according
1
to general procedure to give the desired product 4d as yellow solid. Yield 73%, H NMR
(400 MHz, DMSO-d6) δ 12.66 (s, 1H), 8.30 – 8.28 (dd, J = 8.0, 1.7 Hz, 1H), 7.91 – 7.88
(dd, J = 7.8, 1.2 Hz, 1H), 7.77 – 7.73 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.59 – 7.55 (td, J = 7.5,
1.5 Hz, 1H), 7.50 – 7.46 (td, J = 7.5, 1.3 Hz, 2H), 7.42- 7.38 (ddd, J = 8.2, 7.2, 1.1 Hz, 2H),
6.89 (d, J = 1.5 Hz, 1H), 6.75 (d, J = 1.5 Hz, 1H), 3.70 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 182.21, 168.26, 161.48, 156.42, 155.89, 150.85, 141.27, 135.67, 131.71, 130.47,
Chemical and Pharmaceutical Bulletin Advance Publication

130.34, 130.27, 128.39, 126.11, 124.24, 120.83, 118.00, 110.92, 107.98, 107.31, 52.35.
EI-HRMS Calcd. for C21H14O5 [M+H]+: 347.0875; found: 347.0906.
Methyl 3-(1-hydroxy-9-oxo-9H-xanthen-3-yl)benzoate (4e)
Compound 2 was treated with (3-(methoxycarbonyl)phenyl)boronic acid (3e) according
1
to general procedure to give the desired product 4e as yellow solid. Yield 71%, H NMR
(400 MHz, CDCl3) δ 12.65 (s, 1H), 8.34 (t, J = 1.6 Hz, 1H), 8.29 – 8.26 (dd, J = 8.0, 1.7 Hz,
1H), 8.11 – 8.08 (dt, J = 7.8, 1.4 Hz, 1H), 7.85 – 7.83 (ddd, J = 7.8, 2.0, 1.2 Hz, 1H), 7.78 –
7.73 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.57 – 7.53 (td, J = 7.8, 0.5 Hz, 1H), 7.49 – 7.47 (dd, J
= 8.5, 1.1 Hz, 1H), 7.40 – 7.38 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 7.19 (d, J = 1.6 Hz, 1H),
13
7.06 (d, J = 1.6 Hz, 1H), 3.96 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 182.03, 166.76,
162.22, 156.65, 156.43, 148.72, 139.69, 135.74, 131.71, 131.09, 130.05, 129.26, 128.59,
126.12, 124.32, 120.84, 117.99, 109.25, 108.21, 105.74, 52.43. EI-HRMS Calcd. for
C21H14O5 [M+H]+: 347.0875; found: 347.0907.
Methyl 4-(1-hydroxy-9-oxo-9H-xanthen-3-yl)benzoate (4f)
Compound 2 was treated with (4-(methoxycarbonyl)phenyl)boronic acid (3f) according
1
to general procedure to give the desired product 4f as yellow solid. Yield 76%, H NMR
(400 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.22 (dd, J = 8.0, 1.6 Hz, 1H), 8.08 (d, J = 8.5 Hz,
2H), 8.02 (d, J = 8.5 Hz, 2H), 7.96 (td, J = 7.8, 1.7 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.55
13
(t, J = 7.6 Hz, 1H), 7.51 (d, J = 1.5 Hz, 1H), 7.24 (d, J = 1.5 Hz, 1H), 3.90 (s, 3H). C
NMR (101 MHz, CDCl3) δ 181.93, 166.63, 162.14, 156.51, 156.35, 148.49, 143.64, 135.66,
130.46, 130.25, 127.34, 126.05, 124.25, 120.77, 117.88, 109.31, 108.30, 105.80, 52.24.
ESI-HRMS Calcd. for C21H14O5 [M+H]+: 347.0875; found: 347.0904.
1-Hydroxy-3-(4-acetylphenyl)- 9H-xanthen-9-one (4g)
Compound 2 was treated with (4-acetylphenyl)boronic acid (3g) according to general
1
procedure to give the desired product 4g as yellow solid. Yield 76%, H NMR (400 MHz,
DMSO) δ 12.67 (s, 1H), 8.30 – 8.27 (dd, J = 7.9, 1.7 Hz, 1H), 8.08 – 8.04 (m, 2H), 7.80 –
7.72 (m, 3H), 7.50 – 7.48 (dd, J = 8.4, 1.0 Hz, 1H), 7.43 – 7.49 (ddd, J = 8.1, 7.1, 1.0 Hz,
1H), 7.18 (d, J = 1.7 Hz, 1H), 7.05 (d, J = 1.6 Hz, 1H), 2.65 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 197.67, 182.06, 162.22, 156.61, 156.43, 148.44, 143.85, 137.21, 135.83, 129.13,
127.68, 126.15, 124.40, 120.83, 118.00, 109.40, 108.43, 105.94, 26.84. EI-HRMS Calcd.
for C21H14O4 [M+H]+: 331.0926; found: 331.0970.
Chemical and Pharmaceutical Bulletin Advance Publication

1-Hydroxy-3-(2-methoxyphenyl)-9H-xanthen-9-one (4h)
Compound 2 was treated with (2-methoxyphenyl)boronic acid (3h) according to general
1
procedure to give the desired product 4h as yellow solid. Yield 88%, H NMR (400 MHz,
DMSO-d6) δ 12.52 (s, 1H), 8.21 – 8.19 (dd, J = 8.0, 1.6 Hz, 1H), 7.95 – 7.90 (ddd, J = 8.7,
7.2, 1.7 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.54 – 7.50 (td, J = 7.5, 1.0 Hz, 1H), 7.47 – 7.42
(m, 2H), 7.20 – 7.17 (dd, J = 8.8, 1.1 Hz, 2H), 7.10 – 7.16 (td, J = 7.5, 1.0 Hz, 1H), 6.95 (d,
13
J = 1.4 Hz, 1H), 3.83 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 181.76, 160.78, 156.68,
156.24, 155.78, 147.89, 136.86, 130.92, 130.88, 128.24, 125.90, 125.06, 121.41, 120.48,
118.52, 112.50, 111.60, 108.46, 107.52, 56.10. ESI-HRMS Calcd. for C20H14O4 [M+H]+:
319.0926; found: 319.0963.
1-Hydroxy-3-(3-methoxyphenyl)-9H-xanthen-9-one (4i)
Compound 2 was treated with (3-methoxyphenyl)boronic acid (3i) according to general
1
procedure to give the desired product 4i as yellow solid. Yield 83%, H NMR (400 MHz,
DMSO-d6) δ 12.59 (s, 1H), 8.22 – 8.20 (dd, J = 8.0, 1.6 Hz, 1H), 7.97 – 7.92 (ddd, J = 8.7,
7.2, 1.7 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.56 – 7.52 (t, J = 7.5 Hz, 1H), 7.47 – 7.38 (m,
3H), 7.36 (d, J = 2.1 Hz, 1H), 7.18 (d, J = 1.5 Hz, 1H), 7.06 – 7.03 (ddd, J = 7.7, 2.4, 1.5
13
Hz, 1H), 3.87 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 181.81, 161.63, 160.28, 156.57,
156.30, 149.43, 140.08, 136.97, 130.67, 125.94, 125.17, 120.54, 120.04, 118.51, 115.72,
112.91, 108.97, 107.92, 105.95, 55.75. ESI-HRMS Calcd. for C20H14O4 [M+H]+: 319.0926;
found: 319.0965.
1-Hydroxy-3-(4-methoxyphenyl)-9H-xanthen-9-one (4j)
Compound 2 was treated with (4-methoxyphenyl)boronic acid (3j) according to general
1
procedure to give the desired product 4j as yellow solid. Yield 90%, H NMR (400 MHz,
DMSO-d6) δ 12.59 (s, 1H), 8.21 – 8.19 (dd, J = 8.0, 1.6 Hz, 1H), 7.95 – 7.91 (ddd, J = 8.7,
7.2, 1.7 Hz, 1H), 7.85 – 7.82 (d, J = 8.9 Hz, 2H),, 7.68 (d, J = 8.0 Hz, 1H), 7.56 – 7.49 (td,
J = 7.5, 1.0 Hz, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.13 (d, J = 1.6 Hz, 1H), 7.11 – 7.04 (d, J =
8.9 Hz, 2H), 3.83 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 181.61, 161.69, 160.84,
156.69, 156.27, 149.16, 136.86, 130.62, 129.14, 125.90, 125.12, 120.55, 118.48, 115.01,
108.05, 107.35, 104.92, 55.80. ESI-HRMS Calcd. for C20H14O4 [M+H]+: 319.0926; found:
319.0960.
3-(2,4-Dimethoxyphenyl)-1-hydroxy-9H-xanthen-9-one (4k)
Chemical and Pharmaceutical Bulletin Advance Publication

Compound 2 was treated with (2,4-dimethoxyphenyl)boronic acid (3k) according to
general procedure to give the desired product 4k as yellow solid. Yield 91%, 1H NMR (400
MHz, CDCl3) δ 12.58 (s, 1H), 8.30-8.28 (dd, J = 8.0, 1.6 Hz, 1H), 7.77-7.73 (ddd, J = 8.7,
7.1, 1.7 Hz, 1H), 7.49-7.47 (dd, J = 8.5, 1.0 Hz, 1H), 7.41-7.37 (ddd, J = 8.1, 7.1, 1.1 Hz,
1H), 7.35-7.33 (d, J = 8.2 Hz, 1H), 7.15-7.14 (d, J = 1.5 Hz, 1H), 7.00-6.99 (d, J = 1.5 Hz,
1H), 6.64 – 6.56 (m, 2H), 3.88 (s, 3H), 3.86 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 181.85,
161.42, 161.15, 157.72, 156.35, 155.86, 147.70, 135.35, 131.42, 125.97, 123.93, 121.72,
120.77, 117.86, 111.55, 108.00, 107.46, 104.94, 99.08, 55.64, 55.50. ESI-HRMS Calcd. for
C21H16O5 [M+H]+: 349.1031; found: 349.1071.
3-(3,4-Dimethoxyphenyl)-1-hydroxy-9H-xanthen-9-one (4l)
Compound 2 was treated with (3,4-dimethoxyphenyl)boronic acid (3k) according to
general procedure to give the desired product 4l as yellow solid. Yield 86%, 1H NMR (400
MHz, CDCl3) δ 12.60 (s, 1H), 8.23 – 8.21 (dd, J = 8.0, 1.6 Hz, 1H), 7.69 (ddd, J = 8.6, 7.3,
1.6 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.36 – 7.32 (t, J = 7.3 Hz, 1H), 7.21 – 7.18 (dd, J =
7.3, 2.1 Hz, 1H), 7.12 (d, J = 2.1 Hz, 1H), 7.08 (d, J = 1.4 Hz, 1H), 6.96 (d, J = 1.5 Hz, 1H),
6.91 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 3.88 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 180.77,
160.90, 155.50, 155.30, 148.97, 148.66, 148.27, 134.46, 130.98, 124.98, 123.09, 119.75,
118.98, 116.79, 110.37, 109.20, 107.61, 106.56, 103.98, 54.99, 28.68. ESI-HRMS Calcd.
for C21H16O5 [M+H]+: 349.1031; found: 349.1071.
3-(3,5-Dimethoxyphenyl)-1-hydroxy-9H-xanthen-9-one (4m)
Compound 2 was treated with (3,5-dimethoxyphenyl)boronic acid (3m) according to
1
general procedure to give the desired product 4m as yellow solid. Yield 82%, H NMR
(400 MHz, CDCl3) δ 12.66 (s, 1H), 8.31-8.29 (dd, J = 8.0, 1.6 Hz, 1H), 7.79-7.75 (ddd, J =
8.7, 7.1, 1.7 Hz, 1H), 7.51-7.49 (dd, J = 8.6, 1.0 Hz, 1H), 7.44-7.40 (ddd, J = 8.1, 7.2, 1.1
Hz, 1H), 7.17 (d, J = 1.6 Hz, 1H), 7.04 (d, J = 1.6 Hz, 1H), 6.80 (d, J = 2.3 Hz, 2H), 6.54 (t,
J = 2.2 Hz, 1H), 3.87 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 181.93, 161.90, 161.19,
156.41, 149.90, 141.50, 135.57, 126.02, 124.16, 120.75, 117.89, 109.26, 108.05, 105.71,
105.55, 100.95, 99.48, 55.52. ESI-HRMS Calcd. for C21H16O5 [M+H]+: 349.1031; found:
349.1071.
1-Hydroxy-3-(3,4,5-trimethoxyphenyl)-9H-xanthen-9-one (4n)
Compound 2 was treated with (3,4,5-trimethoxyphenyl)boronic acid (3n) according to
Chemical and Pharmaceutical Bulletin Advance Publication

general procedure to give the desired product 4n as yellow solid. Yield 82%, 1H NMR (400
MHz, CDCl3) δ 12.61 (s, 1H), 8.23 – 8.21 (dd, J = 8.0, 1.6 Hz, 1H), 7.72 – 7.68 (ddd, J =
8.7, 7.2, 1.7 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.36 – 7.32 (td, J = 7.6, 1.0 Hz, 1H), 7.07 (d,
J = 1.6 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 6.80 (s, 2H), 3.89 (s, 6H), 3.85 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 180.83, 160.88, 155.42, 155.29, 152.57, 148.88, 137.90, 134.56,
134.03, 125.00, 123.17, 119.72, 116.80, 107.98, 106.81, 104.38, 103.51, 59.98, 55.24.
ESI-HRMS Calcd. for C22H18O6 [M+H]+: 379.1137; found: 379.1175.
General Procedure for the Preparation of (4o-4q) To a solution of methoxycarbonyl
compound (4d-4f) (0.346 g, 1.0 mmol) in MeOH (30 mL) was added 2M aqueous NaOH
o
solution (6 mL) and the mixture was heated at 70 C for 6 h. After completion monitored
by TLC, the reaction was cooled to ambient temperature, 10% aqueous solution of HCl
were sequentially added until acidic pH. The mixture was extracted with EtOAc, the
combined organic layers were dried over Na2SO4 and the solvent was evaporated in
vacuum. The residue was purified by flash chromatography on silica gel gave the
corresponding carboxyl compounds 4o-4q.
2-(1-Hydroxy-9-oxo-9H-xanthen-3-yl)benzoic acid (4o)
Hydrolysis of compound 4d according to general procedure gave the desired product 4o
as yellow solid. Yield 92%, 1H NMR (400 MHz, DMSO-d6) δ 12.97 (br, 1H), 12.53 (s, 1H),
8.17 – 8.15 (dd, J = 7.9, 1.7 Hz, 1H), 7.91 – 7.86 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H), 7.79 –
7.77 (dd, J = 7.7, 1.4 Hz, 1H), 7.65 – 7.58 (m, 2H), 7.54 – 7.43 (m, 3H), 6.99 (d, J = 1.5 Hz,
13
1H), 6.73 (d, J = 1.5 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 181.85, 169.36, 161.03,
156.23, 155.75, 150.98, 140.30, 136.47, 132.27, 131.47, 130.43, 130.00, 128.68, 125.91,
124.77, 120.52, 118.27, 110.98, 107.69, 107.58. EI-HRMS Calcd. for C21H14O5 [M+H]+:
333.0718; found: 333.0748.
3-(1-Hydroxy-9-oxo-9H-xanthen-3-yl)benzoic acid (4p)
Hydrolysis of compound 4e according to general procedure gave the desired product 4p
as yellow solid. Yield 90%, 1H NMR (400 MHz, DMSO-d6) δ 13.21 (br, 1H), 12.57 (s, 1H),
8.25 (t, J = 1.8 Hz, 1H), 8.19 – 8.17 (dd, J = 8.0, 1.7 Hz, 1H), 8.07 – 8.04 (dt, J = 7.8, 1.5
Hz, 1H), 8.02 – 7.99 (dt, J = 7.8, 1.3 Hz, 1H), 7.91 (ddd, J = 8.7, 7.2, 1.5 Hz, 1H), 7.67 –
13
7.61 (m, 2H), 7.52 – 7.48 (m, 1H), 7.41 (d, J = 1.6 Hz, 1H), 7.14 (d, J = 1.6 Hz, 1H). C
NMR (101 MHz, DMSO-d6) δ 181.89, 167.54, 161.83, 156.71, 156.33, 148.56, 139.04,
Chemical and Pharmaceutical Bulletin Advance Publication

137.09, 132.24, 130.47, 130.12, 129.21, 128.44, 126.00, 125.29, 120.59, 118.60, 108.99,
108.12, 106.08. EI-HRMS Calcd. for C20H12O5 [M+H]+: 333.0718; found: 333.0750.
4-(1-Hydroxy-9-oxo-9H-xanthen-3-yl)benzoic acid (4q)
Hydrolysis of compound 4f according to general procedure gave the desired product 4q
as yellow solid. Yield 93%, 1H NMR (400 MHz, DMSO-d6) δ 12.61 (s, 1H), 8.23-8.21 (dd,
J = 8.0, 1.7 Hz, 1H), 8.07-8.05 (d, J = 8.5 Hz, 2H), 7.99-7.97 (d, J = 8.5 Hz, 2H), 7.96 –
7.93 (m, 1H), 7.71-7.69 (dd, J = 8.5, 0.9 Hz, 1H), 7.56-7.52 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H),
7.49 (d, J = 1.6 Hz, 1H), 7.22 (d, J = 1.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ
181.85, 167.36, 161.73, 156.61, 156.29, 148.13, 142.55, 137.05, 131.64, 130.44, 128.05,
125.95, 125.24, 120.55, 118.54, 109.08, 108.27, 106.20. ESI-HRMS Calcd. for C20H12O5
[M+H]+: 333.0718; found: 333.0759.
General Procedure for the Preparation of (4r-4x) To a solution of methoxyl xanthones
4h-4n (1.0 mmol) in 20 mL dried dichloromethane was added boron tribromide (2.0 equiv
per hydroxyl group) dropwise at 0 oC. The mixture was stirred at 0 oC for 30 min and then
stirring reacted overnight at room temperature. When the reaction completed, the mixture
was quenched with ice water and extracted by CH2Cl2 twice. The combined extract was
then washed with brine and dried over Na2SO4. The solvent was removed under reduced
pressure and the crude product was purified by flash chromatography on silica gel gave the
corresponding desired C3-polyphenol substituted compounds 4r-4x.
1-Hydroxy-3-(2-hydroxyphenyl)-9H-xanthen-9-one (4r)
Demethylation of compound 4h according to general procedure gave the desired product
4r as yellow solid. Yield 91%, 1H NMR (400 MHz, CDCl3) δ 12.72 (s, 1H), 8.31 (d, J = 8.0
Hz, 1H), 7.82 – 7.78 (td, J = 7.8, 1.5 Hz, 1H), 7.51 (d, J = 8.5 Hz, 1H), 7.44 (t, J = 7.6 Hz,
1H), 7.35 (t, J = 7.5 Hz, 2H), 7.13 (d, J = 1.2 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 7.03 (d, J =
8.3 Hz, 1H), 6.98 (d, J = 1.2 Hz, 1H), 5.49 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ
181.67, 160.73, 156.23, 155.80, 155.20, 148.30, 136.80, 130.84, 130.62, 126.02, 125.86,
125.01, 120.47, 120.13, 118.50, 116.91, 111.28, 108.13, 107.30. ESI-HRMS Calcd. for
C19H12O4 [M+H]+: 305.0769; found: 305.0804.
1-Hydroxy-3-(3-hydroxyphenyl)-9H-xanthen-9-one (4s)
Demethylation of compound 4i according to general procedure gave the desired product
4s as yellow solid. Yield 88%, 1H NMR (400 MHz, DMSO-d6) δ 12.59 (s, 1H), 9.69 (s, 1H),
Chemical and Pharmaceutical Bulletin Advance Publication

8.22-8.20 (d, J = 7.9 Hz, 1H), 7.96-7.92 (t, J = 7.8 Hz, 1H), 7.71-7.68 (d, J = 8.5 Hz, 1H),
7.55-7.52 (t, J = 7.5 Hz, 1H), 7.35 – 7.29 (m, 2H), 7.25-7.23 (d, J = 7.7 Hz, 1H), 7.15-7.14
(t, J = 2.1 Hz, 1H), 7.06 (s, 1H), 6.90-6.88 (dd, J = 8.0, 2.3 Hz, 1H). 13C NMR (126 MHz,
DMSO-d6) δ 181.77, 161.61, 158.40, 156.56, 156.28, 149.72, 140.03, 136.93, 130.68,
129.16, 125.92, 125.16, 120.54, 118.50, 116.39, 114.39, 108.73, 105.68, 104.56.
ESI-HRMS Calcd. for C19H12O4 [M+H]+: 305.0769; found: 305.0808.
1-Hydroxy-3-(4-hydroxyphenyl)-9H-xanthen-9-one (4t)
Demethylation of compound 4j according to general procedure gave the desired product
4t as yellow solid. Yield 93%, 1H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 9.89 (s, 1H),
8.20-8.18 (dd, J = 7.9, 1.7 Hz, 1H), 7.94-7.90 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.72-7.70 (d,
J = 8.7 Hz, 2H), 7.67-7.65 (d, J = 8.2 Hz, 1H), 7.54-7.50 (t, J = 7.5 Hz, 1H), 7.32 (d, J =
1.6 Hz, 1H), 7.08 (d, J = 1.6 Hz, 1H), 6.91-6.88 (d, J = 8.7 Hz, 2H). 13C NMR (126 MHz,
DMSO-d6) δ 181.51, 161.65, 159.35, 156.66, 156.23, 149.58, 136.77, 129.16, 128.98,
125.86, 125.06, 120.54, 118.45, 116.38, 107.75, 107.12, 104.55. ESI-HRMS Calcd. for
C19H12O4 [M+H]+: 305.0769; found: 305.0810. Purity 97.2%.
3-(2,4-Dihydroxyphenyl)-1-hydroxy-9H-xanthen-9-one (4u)
Demethylation of compound 4k according to general procedure gave the desired product
1
4u as yellow solid. Yield 90%, H NMR (400 MHz, Acetone-d6) δ 12.58 (s, 1H), 8.74 (s,
1H), 8.60 (s, 1H), 8.28-8.25 (dd, J = 7.9, 1.7 Hz, 1H), 7.93-7.89 (ddd, J = 8.7, 7.1, 1.7 Hz,
1H), 7.63-7.61 (dd, J = 8.6, 1.0 Hz, 1H), 7.53-7.49 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H),
7.37-7.35 (d, J = 8.4 Hz, 1H), 7.31-7.30 (d, J = 1.5 Hz, 1H), 7.08-7.07 (d, J = 1.4 Hz, 1H),
13
6.59-6.58 (d, J = 2.3 Hz, 1H), 6.54-6.51 (dd, J = 8.4, 2.4 Hz, 1H). C NMR (126 MHz,
Acetone-d6) δ 181.55, 161.21, 159.40, 156.34, 155.92, 148.58, 135.84, 131.46, 125.53,
124.20, 120.56, 118.06, 117.92, 110.64, 107.92, 107.26, 106.70, 103.35. ESI-HRMS Calcd.
for C19H12O5 [M+H]+: 321.0718; found: 321.0752.
3-(3,4-Dihydroxyphenyl)-1-hydroxy-9H-xanthen-9-one (4v)
Demethylation of compound 4l according to general procedure gave the desired product
1
4v as yellow solid. Yield 86%, H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 9.47 (s,
1H), 9.19 (s, 1H), 8.21 – 8.18 (dd, J = 8.0, 1.6 Hz, 1H), 7.95 – 7.90 (ddd, J = 8.7, 7.2, 1.7
Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.56 – 7.49 (td, J = 7.5, 1.0 Hz, 1H), 7.24 (d, J = 1.6 Hz,
1H), 7.21 – 7.14 (m, 2H), 6.99 (d, J = 1.5 Hz, 1H), 6.87 (d, J = 8.1 Hz, 1H). 13C NMR (101
Chemical and Pharmaceutical Bulletin Advance Publication

MHz, DMSO-d6) δ 181.49, 161.58, 156.61, 156.24, 149.90, 147.62, 146.24, 136.75, 129.62,
125.86, 125.05, 120.54, 119.25, 118.47, 116.58, 114.81, 107.76, 107.13, 104.57.
ESI-HRMS Calcd. for C19H12O5 [M+H]+: 321.0718; found: 321.0751.
3-(3,5-Dihydroxyphenyl)-1-hydroxy-9H-xanthen-9-one (4w)
Demethylation of compound 4m according to general procedure gave the desired
1
product 4w as yellow solid. Yield 91%, H NMR (400 MHz, Acetone-d6) δ 12.68 (s, 1H),
8.52 (s, 2H), 8.29-8.27 (dd, J = 8.0, 1.6 Hz, 1H), 7.96-7.92 (ddd, J = 8.7, 7.2, 1.7 Hz, 1H),
7.65 (d, J = 8.4 Hz, 1H), 7.55-7.52 (t, J = 7.1 Hz, 1H), 7.22 (d, J = 1.5 Hz, 1H), 7.00 (d, J =
1.5 Hz, 1H), 6.76 (d, J = 2.2 Hz, 2H), 6.51-6.49 (t, J = 2.1 Hz, 1H). 13C NMR (126 MHz,
Acetone-d6) δ 181.79, 161.97, 159.16, 156.50, 156.37, 150.23, 141.22, 136.09, 125.59,
124.44, 120.53, 118.00, 108.51, 107.64, 105.88, 105.21, 103.37. ESI-HRMS Calcd. for
C19H12O5 [M+H]+: 321.0718; found: 321.0759.
3-(3,4,5-Trihydroxyphenyl)-1-hydroxy-9H-xanthen-9-one (4x)
Demethylation of compound 4n according to general procedure gave the desired product
1
4x as yellow solid. Yield 82%, H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 9.18 (s,
2H), 8.65 (s, 1H), 8.21 – 8.18 (dd, J = 7.9, 1.6 Hz, 1H), 7.93 (td, J = 7.8, 1.6 Hz, 1H), 7.68
(d, J = 8.3 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 1.4 Hz, 1H), 6.90 (d, J = 1.4 Hz,
1H), 6.73 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 181.49, 161.52, 156.56, 156.25,
150.25, 146.99, 136.76, 135.54, 128.79, 125.86, 125.06, 120.55, 118.51, 107.77, 107.18,
106.63, 104.58. ESI-HRMS Calcd. for C19H12O6 [M+H]+: 337.0667; found: 337.0703.
Assay of Tyrosinase Inhibitory Activities The inhibition of synthetic compounds on the
diphenolase activitiy of mushroom tyrosinase was investigated according to reported
procedure [12] with some slight modifications. Briefly, all the synthesized compounds were
screened for the diphenolase inhibitory activity of tyrosinase using L-DOPA as substrate.
Synthetic compounds and kojic acid (used as a control) were dissolved in DMSO and their
final concentration in DMSO was 5.0%. Phosphate buffer, pH 6.8, was used to dilute the
DMSO stock solution of test compounds. In a 96-well plate, 10 μL of mushroom tyrosinase
(30 U/ml) was mixed with 10 μL of samples in 170 μL of Phosphate buffer solution
o
(50 mM) and the mixture was first pre-incubated for 10 min at 25 C. After 10 μl of
L-DOPA (0.85 mM) was added, the solution was monitored for the formation of
dopachrome for 5 min, by measuring the change in absorbance at 475 nm. The
Chemical and Pharmaceutical Bulletin Advance Publication

measurement was completed in triplicate for each concentration and averaged before
further calculation.
IC50 value was determined by interpolation of the dose-response curves. The mode of
inhibition of the enzyme was performed using the Lineweaver-Burk plot. The assay was
performed varying the concentration of inhibitor and L-DOPA (0.1, 0.2, 0.3 and 0.4 μM).
Kinetics constants were determined by the second plots of the apparent Km/Vmax and/or
1/Vmax versus the inhibitor concentration.
Molecular Modeling For the docking studies, Surflex-dock in SYBYL X-2.1 was used.
The Crystal structure of Agaricus bisporus tyrosinase (PDB code: 2Y9X) was obtained
from the Protein Data Bank. The structures of inhibitors were built in Sybyl package with
standard bond lengths and angles and minimized using the Powell method. The termination
of Gradient, force field of Tripos and charge of Gasteiger-Hückel were applied for the
minimization process. For the protein, all the non-polar water molecules were removed
from the crystal structure and hydrogens were added, as well as the AMBER FF99 force
field were applied. The standard docking procedure was used with parameters set in default
and three rounds of computation were carried out. After docking, residues within 5 Å of the
inhibitors were identified and the conformations that appeared the most frequently and
possessed the lowest binding free energy were selected for further analysis.
Acknowledgements This research was funded by the Natural Science Foundation of
Guangdong Province of China (grant number: Q17056013), Foundation of Educational
Commission of Guangdong Province of China (grant number: 2016KQNCX129), and
Medical Scientific Research Foundation of Guangdong Province of China (A2017083,
A2018421, B2018272).
Conflict of Interest The authors declare no conflict of interest.
Supplementary Materials The online version of this article contains supplementary
materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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