Design and synthesis of novel xanthone-triazole derivatives as potential antidiabetic agents: α-Glucosidase inhibition and glucose uptake promotion

Article abstract of DOI:10.1016/j.ejmech.2019.05.045

Inhibiting the decomposition of carbohydrates into glucose or promoting glucose conversion is considered to be an effective treatment for type 2 diabetes. Herein, a series of novel xanthone-triazole derivatives were designed, synthesized, and their α-glucosidase inhibitory activities and glucose uptake in HepG2 cells were investigated. Most of the compounds showed better inhibitory activities than the parental compound a (1,3-dihydroxyxanthone, IC₅₀ = 160.8 μM)and 1-deoxynojirimycin (positive control, IC₅₀ = 59.5 μM)towards α-glucosidase. Compound 5e was the most potent inhibitor, with IC₅₀ value of 2.06 μM. The kinetics of enzyme inhibition showed that compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h were noncompetitive inhibitors, and molecular docking results were consistent with the noncompetitive property that these compounds bind to allosteric sites away from the active site (Asp214, Glu276 and Asp349). On the other hand, the glucose uptake assays exhibited that compounds 5e, 6a, 6c and 7g displayed high activities in promoting the glucose uptake. The cytotoxicity assays showed that most compounds were low-toxic to human normal hepatocyte cell line (LO2). These novel xanthone triazole derivatives exhibited dual therapeutic effects of α-glucosidase inhibition and glucose uptake promotion, thus they could be use as antidiabetic agents for developing novel drugs against type 2 diabetes.

Full text of DOI:10.1016/j.ejmech.2019.05.045

Accepted Manuscript
Design and synthesis of novel xanthone-triazole derivatives as potential antidiabetic
agents: α-Glucosidase inhibition and glucose uptake promotion
Gao-Jie Ye, Tian Lan, Zhi-Xin Huang, Xiao-Ning Cheng, Chao-Yun Cai, Sen-Miao
Ding, Min-Li Xie, Bo Wang
PII:
S0223-5234(19)30455-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.045
EJMECH 11355
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 February 2019
Revised Date: 15 May 2019
Accepted Date: 16 May 2019
Please cite this article as: G.-J. Ye, T. Lan, Z.-X. Huang, X.-N. Cheng, C.-Y. Cai, S.-M. Ding, M.-L. Xie,
B. Wang, Design and synthesis of novel xanthone-triazole derivatives as potential antidiabetic agents: α-
Glucosidase inhibition and glucose uptake promotion, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.05.045.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and synthesis of novel xanthone-triazole derivatives as potential
antidiabetic agents: α-glucosidase inhibition and glucose uptake promotion
a
a
a
b
c
Gao-Jie Ye , Tian Lan , Zhi-Xin Huang , Xiao-Ning Cheng , Chao-Yun Cai ,
a
a
a
Sen-Miao Ding , Min-Li Xie , Bo Wang *
a
School of Chemistry, Sun Yat-sen University, 135 Xingang West Road, Guangzhou, 510275, PR
China
b
Instrumental Analysis & Research Center, Sun Yat-sen University, 135 Xingang West Road,
Guangzhou, 510275, PR China
c
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s
University, 8000 Utopia Parkway, Queens, New York 11439, United States
Keywords: xanthone-triazole derivatives, α-glucosidase inhibitors, noncompetitive,
molecular docking, cytotoxicity, glucose uptake
Abstract:
Inhibiting the decomposition of carbohydrates into glucose or promoting glucose
conversion is considered to be an effective treatment for type 2 diabetes. Herein, a
series of novel xanthone-triazole derivatives were designed, synthesized, and their
α-glucosidase inhibitory activities and glucose uptake in HepG2 cells were
investigated. Most of the compounds showed better inhibitory activities than the
parental compound
-deoxynojirimycin (positive control, IC50 = 59.5 µM) towards α-glucosidase.
Compound 5e was the most potent inhibitor, with IC value of 2.06 µM. The kinetics
a
(1,3-dihydroxyxanthone, IC50
=
160.8 µM) and
1
5
0
of enzyme inhibition showed that compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h were
noncompetitive inhibitors, and molecular docking results were consistent with the
noncompetitive property that these compounds bind to allosteric sites away from the
active site (Asp214, Glu276 and Asp349). On the other hand, the glucose uptake
assays exhibited that compounds 5e, 6a, 6c and 7g displayed high activities in
1

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promoting the glucose uptake. The cytotoxicity assays showed that most compounds
were low-toxic to human normal hepatocyte cell line (LO2). These novel xanthone
triazole derivatives exhibited dual therapeutic effects of α-glucosidase inhibition and
glucose uptake promotion, thus they could be use as antidiabetic agents for
developing novel drugs against type 2 diabetes.
1
. Introduction
Diabetes mellitus is a metabolic disorder characterized by prolonged
hyperglycemia, which leads to an increased risk of cancer, stroke, cardiovascular
disease, retinopathy, nephropathy, and metabolic syndrome [1-4]. According to the
World Health Organization, more than 420 million people are suffering from diabetes,
and the number would increase to 642 million in 2040 [5, 6]. Type 2 diabetes
(Noninsulin-dependent diabetes mellitus) is the most common form of diabetes
mellitus, accounting for more than 90 % of all diabetes cases [7].
The main strategy for treating this disease is to control the high blood glucose
levels. The strategies to control blood glucose levels can be retarding, regulating
and/or inhibiting carbohydrate hydrolytic enzymes [8]. α-Glucosidase (EC.3.2.1.20) is
an important hydrolytic enzyme playing a vital role in digestion of carbohydrates and
biosynthesis of viral envelope glycoproteins [9]. It catalyzes the final step of
carbohydrates digestion in biological systems, and converts unabsorbed
oligosaccharides and disaccharides into monosaccharides, thus resulting in
hyperglycemia for diabetic patients [7, 10]. Hence, it is an effective therapeutic
approach for type 2 diabetes by suppressing the activity of α-glucosidase.
α-Glucosidase inhibitors can retard carbohydrates digestion through inhibiting the
activity of α-glucosidase, which in turn balance postprandial blood glucose levels.
The well-known α-glucosidase inhibitors, such as acarbose, miglitol and voglibose,
have been used for the treatments of type 2 diabetes. However, it is attractive and
meaningful to discover novel α-glucosidase inhibitors due to the adverse effects (for
2

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instance, flatulence, diarrhoea, stomach ache and liver damage [11]) of the available
α-glucosidase inhibitors.
The imbalances of glucose homeostasis in type 2 diabetes results in reduced
glucose intake in peripheral tissues and increased hepatic glucose output [12]. The
Liver plays a major role in blood glucose control and utilization, providing glucose to
both insulin-sensitive (fat and skeletal muscle) and insulin-insensitive (nervous, skin,
red blood cells, smooth muscle, etc.) tissues. Therefore, promoting glucose uptake in
the liver is also an effective strategy for diabetic patients to control blood glucose
levels [13].
Xanthone and their derivatives are widely distributed in nature and they exhibit
various pharmacological activities, such as anti-inflammatory, anti-oxidant,
anti-bacterial, anti-viral, anti-tumor etc. [14-16]. In the previous studies, we have
shown that the synthetic polyhydroxyxanthones, benzoxanthones, noncoplanar
xanthones, 3-acyloxyxanthones, and oxazolxanthones, could be used as new classes
of α-glycosidase inhibitors [17-21]. The IC50 values of the most compounds were
ranging from 10-200 µM. To further increase the activity, we hope to introduce
appropriate groups at the suitable position of xanthone core. Therefore, we checked
all the xanthone derivatives we synthesized, we noticed that introducing oxygen or
nitrogen containing group and/or aromatic group into the C3 position of xanthone ring
through the ether or ester bond would increase the inhibitory activities [17, 21]
(
Figure 1: (A)). Compound b as the ester derivatives of xanthone, is a representative
compound, which exhibited much higher inhibitory activity than its parental
compound a (Figure 1: (A)). Accordingly, we hypothesized that potential
α-glycosidase inhibitors can be obtained by modifying the OH at the C3 position of
xanthone ring, and this modification can be easily to achieve. Considering the
ester-bearing compounds are thought to be susceptible to hydrolysis by intestinal
esterases and the present groups (such as the groups of compounds c, d and e)
introduced through the ether bond showed no so good inhibitory activities, thus,
3

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introducing the other appropriate group at C3 position of xanthone through the ether
bond could improve the stability and increase the inhibitory activity.
1
,2,3-Triazole is a heterocyclic scaffold significantly widespread in the medicinal
chemistry field. This scaffold is stable to acid/basic hydrolysis and
reductive/oxidative conditions, showing high aromatic stabilization and resistance to
metabolic degradation [22-24]. Previous studies showed that 1,2,3-triazole derivatives
have a variety of biological activities, such as anti-cancer, anti-tuberculosis,
anti-fungal, anti-bacterial, anti-HIV etc. [25]. Ferreira et al. synthesized a series of
4
-substituted 1,2,3-triazoles. These compounds showed high α-glucosidase inhibitory
activities and reduced the postprandial blood glucose levels in normal rats [23]. Wang
et al. synthesized two series of 2,4,5-triarylimidazole-1,2,3-triazole derivatives and
triazine-triazole derivatives. The biological activity test showed that all the triazole
derivatives had good inhibitory activities towards α-glucosidase [26, 27].
At the same time, 1,2,3-triazole, with strong dipole moment, could actively
participate in hydrogen bonding and π-stacking interactions [23]. In our previous
research, π-stacking, hydrogen bonding and hydrophobic effect play key roles in
promoting the α-glucosidase inhibitory activity [18, 19]. Considering the significant
increase of inhibitory activities after the modification of xanthone at C3 position and
the fact that 1,2,3-triazoles with potential metabolic stability and good α-glycosidase
inhibitory activities, we designed the skeleton of xanthone-triazole (Figure 1: (B)).
From prior docking studies, the skeleton compound exhibited a higher docking score
(-9.02 kcal/mol) than compound a (-6.65 kcal/mol). It was also found that the nitrogen
atom of the triazole and the oxygen atom of the amide bond formed hydrogen bonds
with the enzyme (Figure 1: (C)), which may further enhance the binding of
α-glucosidase and the compound, thus increasing the α-glucosidase inhibitory activity.
The docking results rationalized the idea of introducing triazole into the parental
compound a.
4

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Thus, in this study, a novel series of xanthone derivatives substituted with triazole
group at C3 position, and with OH, OMe or Br groups at C1, C6 or C7 position were
synthesized (Scheme 1), and their in vitro α-glucosidase activities and inhibitory
mechanism were evaluated. Moreover, molecular docking was performed to gain
insight into possible binding modes with α-glucosidase.
Furthermore, it was reported that flavonoids could increase glucose uptake in
HepG2 hepatocellular liver carcinoma cell line [28], and the xanthone-triazole
derivatives have the common structure with these compounds (Figure 2), which
enlightened us that the synthetic xanthone triazole derivatives may also promote
glucose uptake in the live cells. Therefore, in order to determine whether the
xanthone-triazole derivatives, which possessed high α-glucosidase inhibitory
activities, can be used as potential anti-diabetes agents, glucose uptake and
cytotoxicity assays were also carried out.
5

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Figure 1. Xanthones as α-glucosidase inhibitors and molecular docking of the
xanthone-triazole’s skeleton. (A) The synthetic xanthones as the α-glucosidase
inhibitors. (B) 1,3-dihydroxyxanthone and the skeleton of xanthone-triazole. (C)
Predicted interactions of the skeleton of xanthone-triazole with α-glucosidase. The
green dashed lines stand for hydrogen bonds.
6

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Figure 2. The structure of flavonoids and xanthone-triazole derivatives
2
2
. Result and discussion
.1 Chemistry
The xanthone-triazole derivatives 5a-5h, 6a-6h, 7a-7h were synthesized as showed
in Scheme 1. Compounds 1a-1d were synthesized from different substituted
hydroxybenzoic acids with phloroglucinol in the presence of Eaton’s reagent [29].
Compounds 1a-1d reacted with 3-bromopropyne in acetone at 60 ℃ for 5-14 h to
produce 2a-2d with the yields of 61-75% [30]. Different substituted anilines reacted
with chloroacetyl chloride to obtain compounds 3a-3b in 96 and 98 % yields,
respectively [31]. Compounds 3a-3b reacted with NaN in DMF at room temperature
3
for 4 h to generate the intermediates 4a-4b [31, 32]. Then, intermediates 2a-2d and
4
a-4b underwent copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) in the
presence of CuSO .5H O and sodium ascorbate, then products 5a-5h were obtained
4
2
with the yields of 71-89 % [33]. Demethylation of compounds 5a-5h in CH Cl with a
2
2
large excess BBr gave products 6a-6h in the yields of 22-45 % [34]. Finally,
3
xanthone-triazole derivatives 7a-7h were achieved with the yields of 81-93 % by the
reaction of compounds 5a-5h with Ac O under the catalysis of NaOAc at 90 °C [35].
2
7

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Scheme 1. Synthesis of xanthone-triazole derivatives. Reagents and conditions: (a)
Eaton's reagent, reflux; (b) 3-bromopropyne, K CO , acetone, reflux;(c) dry THF, N ,
2
3
2
0
℃ ; (d) NaN , DMF, 40 ℃ ; (e) CuSO ⋅5H O, sodium ascorbate, DMF, rt; (f) BBr ,
3 4 2 3
dry DCM, 0 ℃ ; (g) Ac O, NaOAc, 90 ℃ .
2
2
.2 α-Glucosidase inhibitory activities and structure-correlation analysis
8

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The α-glucosidase inhibitory activities of all the synthetic compounds were assayed
and the IC values were listed in Table 1.
5
0
As shown in the Table 1, all the synthetic compounds 5a-5h, 6a-6h and 7a-7h
showed higher inhibitory activities than the parental compound a and almost half of
the compounds showed greater inhibition than compound b (Figure 1: (A)), which
demonstrated that the introduction of triazole structure at the C3-OH position can
increase the inhibitory activity as expected. More exciting results were that almost of
the compounds showed higher inhibitory activities than 1-deoxynojirimycin (positive
control) except 5b, 7a and 7b, and the IC value of compound 5e reached 2.06 µM,
5
0
nearly 29 folds lower than 1-deoxynojirimycin, and 78 folds lower than the parental
compound a.
Compounds 7a-7h showed lower inhibition compared with 5a-5h, indicating that
the inhibitory activities decreased when OH was changed to OAc at C1 position.
These facts suggested that this OH was vital for the compound binding to the enzyme,
probably involved in hydrogen bonding or other electrostatic interaction with the
enzyme.
Comparing the IC values of the pair of compounds a/c, b/d, e/g and f/h among all
5
0
the series, it was found that the inhibitory activity was enhanced when the OH or
OCH group at the C6 or C7 of xanthone ring were replaced by Br except 5g. In our
3
previous QASR studies, it revealed that the chemical softness of the groups was a key
factor influencing the inhibitory activity [36]. Based on this, we hypothesized that the
chemical softness of Br might be the key factors in promoting the inhibitory activity.
Comparing the IC50 values of compounds 5a-5h bearing OCH at different
3
positions of the aromatic rings and 6a-6h with OCH reduced to OH, respectively.
3
The results showed that 6a-6d exhibited higher inhibitory activities with the IC50
values ranging from 3.17 µM to 7.06 µM, nearly 2~14 folds increased compared to
5
a-5d. On the contrary, compounds 5e-5f were about 2~8 folds more active than 6e-6f,
with the IC values ranging from 2.06 µM to 8.31 µM. These facts clearly indicated
5
0
9

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that when the C3’and C4’ positions were substituted by OH and the C2’and C4’
positions were substituted by OCH , the inhibitory activity can be increased.
3
Compounds 6a, 6e with OH at C6 position had similar IC50 values with the
compounds 6b, 6f which had OH at C7 position. And compounds 5c, 5g, 6c, 6g, 7c,
7
g bearing Br at the C6 position also had similar IC values with compounds 5d, 5h,
50
6
d, 6h, 7d, 7h which Br at the C7 position except compound 5c. These results
suggested that the positions of OH and Br have no significant effect on the inhibitory
activity. Interestingly, compounds 5a, 5e, 7a, 7e, which had OCH at the C6 position,
3
showed higher activities than the corresponding isomers 5b, 5f, 7b, 7f with OCH at
3
the C7 position, suggesting that OCH at the C6 position could improve the inhibitory
3
activity.
Table 1. In vitro α-glucosidase inhibitory activity of compounds 5a-5h, 6a-6h,7a-7h.
1
0

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a
Compounds R
R1
OCH
H
Br
H
R2
H
OCH
H
Br
H
OCH
H
Br
H
OH
H
Br
H
OH
H
Br
H
OCH
H
Br
H
OCH
H
R3
R4
R5
IC (µM)
50
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
a
b
c
d
e
f
g
h
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
3
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OH
OH
OH
OH
OH
3
3
3
3
3
3
3
3
OCH
OCH
OCH
OCH
H
H
H
H
OH
OH
OH
OH
H
H
H
H
OCH
OCH
OCH
OCH
H
H
H
H
3
3
3
3
H
H
H
H
OCH
OCH
OCH
OCH
H
H
H
H
OH
OH
OH
OH
H
H
H
H
OCH
OCH
OCH
OCH
15.90±0.91
b
>100
3
11.82±0.82
29.84±3.47
2.06±0.16
8.31±0.88
2.78±0.22
3.07±0.56
6.13±0.09
7.06±0.89
5.23±0.53
3.17±0.61
17.61±1.68
15.62±1.13
5.87±0.76
5.88±0.32
98.63±4.12
OCH
H
3
3
3
3
3
3
Br
H
OH
H
Br
H
OH
H
OH
OH
OH
Br
H
OCH
H
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
3
3
3
3
3
3
3
b
>100
3
Br
H
26.11±2.95
32.33±0.82
33.16±2.51
42.60±0.09
12.78±0.01
12.13±1.84
OCH
H
3
3
3
3
3
3
g
h
Br
H
7
a
Br
IC value: concentration that inhibits the activity of α-glucosidase by 50% (mean
5
0
±
SD). The IC value of positive control (1-deoxynojirimycin, PG) is 59.50 ± 4.7
50
[17]
µM. The IC value of parental compound a is 160.8 µM , and compound b is
5
0
[21]
1
0.6 µM
.
b
The compounds precipitated when the concentration was higher than 100 µM, but
the inhibition is lower than 50%. Therefore, the IC value of compounds 5b and 7b
50
can not be obtained.
2
.3 Inhibition kinetics of α-glucosidase
1
1

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To further explore the interaction mechanism of xanthone-triazole derivatives with
α-glucosidase, the inhibition types of potential compounds 5e, 5g, 5h, 6c, 6d, 6g and
6
h were tested using Lineweaver-Burk plot analysis [37, 38]. As shown in Figure 3,
the double reciprocal plots showed straight lines with the same Michaelis constant
km), indicating that compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h are noncompetitive
inhibitors of α-glucosidase.
(
1
2

ACCEPTED MANUSCRIPT
Figure3. Lineweaver-Burk plots (1/V vs 1/[S]) of α-glucosidase inhibition of
compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h.
1
3

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2
.4 Molecular docking
To reveal the binding mode of these compounds towards α-glucosidase, molecular
docking studies were performed. Compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h with high
enzyme inhibition, were evaluated for their possible interactions with the enzyme,
particularly the interactions between the triazole moiety and the enzyme. The
homology model of α-glucosidase provided by SWISS-MODEL Repository was used
[
39], with the model quality estimation performed, due to the unavailability of the
crystal structure of α-glucosidase from Saccharomyces cerevisiae.
From the results of the docking studies, it can be found that all of the examined
compounds bind to allosteric sites away from the active site (Asp214, Glu276 and
Asp349) of the enzyme (Figure 4), which was consistent with their noncompetitive
property validated in the enzyme kinetic assay.
Various interactions could be found between these compounds and the allosteric
sites of the enzyme. For example, hydrogen bonds could be formed between the OH
and carbonyl oxygen atoms of the compounds and enzyme, and the aromatic xanthone
core and the side benzene ring could also form π-π or π-cation interactions with
residues of the enzyme (Figure 5: (A), (B)). Besides, the promoting effects of the
triazole ring could be illustrated in that it could not only form a hydrogen bond but
also participate in π-π interactions (Figure 5: (C), (D)).
1
4

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Figure 4. Binding positions of compounds 5e, 5g, 5h, 6c, 6d, 6g and 6h. All the
examined compounds bind to allosteric sites away from the active site (Asp214,
Glu276 and Asp349) of α-glucosidase.
Figure 5. Predicted interactions between α-glucosidase and compounds 6c, 5e, 5h and
6
g. The green dashed lines stand for hydrogen bonds, yellow columns stand for π-π
interactions, and yellow cones stand for π-cation interactions. (A) Compound 6c:
Hydrogen bonds, π-π interactions, π-cation interactions. (B) Compound 5e: Hydrogen
bonds, π-π interactions, π-cation interactions. (C) Compound 5h: Hydrogen bonds,
π-π interactions. (D) Compound 6g: Hydrogen bonds, π-π interactions.
2
2
.5 Cytotoxicity and glucose uptake assays
.5.1 In vitro cytotoxicity
1
5

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Most of the drugs are metabolized in the liver, which in turn have impacts on liver
function. If the drugs are toxic to the liver, it would cause liver damage. In order to
determine the toxicities of the synthetic xanthone-triazole derivatives, we conducted
MTT assay on human hepatocyte cell line (LO2).
As shown in Table 2, the IC values of all the synthesized compounds exhibited
5
0
more than 100 µM in the LO2 cells except 5e, 5f, 6g, 7b, 7c and 7h, showing that
most of the compounds have low toxicity to liver in the range of tested dose.
Table 2. LO2 cytotoxicity of compounds 5a-5h, 6a-6h,7a-7h
a
Compounds IC (µM)
Compounds IC (µM)
Compounds IC (µM)
50
5
0
50
5
5
5
5
5
5
5
5
a
b
c
d
e
f
>100
>100
>100
>100
6a
6b
6c
6d
>100
>100
>100
>100
>100
>100
7a
7b
7c
7d
7e
7f
>100
24.92±2.38
25.95±3.10
>100
89.06±4.89 6e
65.43±3.51 6f
>100
>100
g
h
>100
>100
6g
6h
73.95±6.73 7g
>100
7h
>100
80.72±1.63
a
IC value: concentration that inhibits cells survival by 50% (means ± SD).
5
0
Furthermore, the effects of compounds a, 5e, 5f, 5g, 5h, 6a, 6b, 6c, 6d, 6g, 6h, 7g
and 7h at the low concentrations on HepG2 cells growth were showed in the Figure 6.
It indicated that the low concentrations of compounds had no effect on cells survival,
which revealed the glucose uptake assay on HepG2 cells could be performed at these
concentrations.
1
6

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Figure 6. Effects of different concentrations of compounds a, 5e, 5f, 5g, 5h, 6a, 6b,
6
c, 6d, 6g, 6h, 7g and 7h on HepG2 cells growth.
2
.5.2 Glucose uptake in HepG2 cells
HepG2 cells maintain many functions of the human live and have the same
glycometabolic function as human hepatocytes, meanwhile, HepG2 cells are easy to
cultivate and reproduce rapidly, thus it is widely used as glucose uptake model in vitro
[
12, 13, 38].
To investigate whether the xanthone-triazole derivatives had another treatment
approach to type 2 diabetes, the potent α-glycosidase inhibitors were chosen to
evaluate the glucose uptake in HepG2 cells at 0.625, 1.25 and 2.5 µM. The results
were shown in Figure 7. Rosiglitazone was used as positive control and parental
compound a was also used as a reference. Rosiglitazone exerted improvement of an
approximately 50 % glucose uptake in HepG2 cells compared with the control group
and there is no significant difference when the concentration changed at the range of
0
.625, 1.25 and 2.5 µM. Inspiringly, the seven compounds 5e, 5g, 6a, 6c, 6d, 6h and
7
g significantly enhanced the glucose uptake in HepG2 cells, but the parental
1
7

ACCEPTED MANUSCRIPT
compound a had little effect on promoting the glucose uptake. Among them, 5g, 6d
had similar effects to rosiglitazone, and 5e, 6a, 6c and 7g displayed higher activities
than rosiglitazone with concentration dependence. In particular, at 0.625, 1.25 and 2.5
µM, the most active compound 7g increased the activity by 61 %, 90 % and 163 %,
respectively, compared with the control. Besides, comparing the glucose uptake of the
pair of compounds 5e/5f, 5g/5h, 6a/6b, 6c/6d and 7g/7h, it showed that bearing a
substituent such as OCH , OH or Br at C6 position of the xanthone ring presented
3
more positive effects than the substituent at C7 position.
Figure 7. Effects of compounds a, 5e, 5f, 5g, 5h, 6a, 6b, 6c, 6d, 6g, 6h, 7g and 7h on
glucose uptake of HepG2 cells.
3
. Conclusion
In summary, twenty-four novel xanthone-triazole derivatives have been designed,
synthesized, and their inhibitory activities of α-glucosidase, inhibition kinetics,
molecular docking, cytotoxicity and glucose uptake were evaluated.
As expected, the introduction of aromatic ring substituted triazole to the C3-OH
position of the parental xanthone ring enhanced the α-glucosidase inhibitory activities,
1
8

ACCEPTED MANUSCRIPT
and the IC values of the most synthetic xanthone-triazole derivatives ranging from
5
0
2
.06~17.61 µM, showed higher activities than 1-deoxynojirimycin. Especially, the
IC50 value of compounds 5e, 5g, 5h and 6d were 2.06, 2.78, 3.07 and 3.17 µM,
exhibiting greater inhibitory activities than the most potent reported synthetic
xanthone derivatives (the IC value was 5.8 µM [18]). Docking studies reveal that the
5
0
increase of the activities may due to the hydrogen bond and π-π or π-cation interaction
of aromatic ring substituted triazole moiety with the enzyme.
The structure-activity relationship analysis suggests that OH at the C1 position of
xanthone ring also has great influence on improving the inhibitory activity, and the
different substituents at the different positions may influence the inhibition towards
α-glucosidase. It may due to the chemical softness of the substituents, the
hydrophobic interaction or hydrogen bonding between the compounds and the
α-glucosidase.
Kinetic analysis reveals that the compounds exhibit potent inhibitory activities
toward yeast’s α-glucosidase via a noncompetitive mechanism, and molecular
docking results consist with the results of the kinetic study that these compounds did
not interact with the active site of the enzyme.
It is worth to highlight that compounds 5e, 6a, 6c and 7g showed good inhibitory
effects toward α-glucosidase and high activities in promoting the glucose uptake in
HepG2 cells, suggesting that these four compounds may have dual effects in the
glucose levels control. Additionally, the cytotoxicity assays confirmed that most of the
synthetic xanthone-triazole derivatives have low toxicity. Accordingly, these
compounds may be potentially developed as antidiabetic agents for the type 2 diabetes
in the future.
4
. Experimental designs and methods
4
.1 General
1
9

ACCEPTED MANUSCRIPT
Melting points (Mp) were obtained on WRS-3 melting point instrument. Infrared
IR) spectra were collected by a Perkin-Elmer model Lambda 950-IR
(
spectrophotometer with KBr pellets. High resolution (HR) mass spectra were
measured using a Thermo LTQ Orbitrap Elite or TSQ Quantum XLS mass
1
13
spectrometer. H and C NMR spectra were recorded with Bruker Avance III 400
MHZ and Varian Inova 500 MHZ in DMSO-d6 or CDCl . UV spectra were
3
determined using a Shimadzu UV-3250 scanning spectrophotometer.
1
-Deoxynojirimycin, rosiglitazone, α-glucosidase (from Saccharomyces cerevisiae),
dimethylsulfoxide (DMSO) and p-nitrophenyl-α-D-glucopyranoside (PNP) were
purchased from Sigma (St. Louis, MO, USA). Analytical thin-layer chromatography
was performed with silica gel plates (Merck, TLC silica gel 60 F254). Column
chromatography was performed with 100-200 mesh silica gel (Qingdao Haiyang
Chemical Co., Ltd., China). Others commercial chemicals and solvents were of
analytical grade and used without further purification. All the final compounds had a
purity of > 95% determined by HPLC (Agilent 1260 LC system) on a Sapphire C18
column (Thermo Fisher Scientific, 4.6 mm × 150 mm, 5 µm) or Eclipse XDB C18
column (Agilent Technologies, 4.6 mm × 150 mm, 5 µm) with MeOH/H O (70/30 v/v)
2
at 1 mL/min flow rate and 254 nm detector wavelengths.
4
.1.1 Compounds 1a-1d were synthesized as previously reported [29].
.1.2 General methods for the synthesis of 2a-2d.
4
A mixture of compounds 1a-1d (1.0 mmol), 3-Bromopropyne (1.0 mmol) and
K CO (5.0 mmol) in acetone (15 mL) was stirred at 60 ℃ for 5-14 h. The reaction
2
3
mixture was filtered, and the residue was washed with CH OH (3 × 10 mL). The
3
filtrate was combined and concentrated, then the compounds 2a-2d were obtained by
flash chromatography.
4
.1.3 General methods for the synthesis of 3a and 3b.
2
0

ACCEPTED MANUSCRIPT
Chloroacetyl chloride (0.80 mL, 12.0 mmol) was added dropwise into the solution
of dry THF (10 mL) containing substituted aniline (10.0 mmol) with continuous
stirring to avoid the vigorous reaction at 0 °C under nitrogen atmosphere. After 1.5-2
h, the mixture was poured into 100 mL of ice water, and extracted with DCM (2 ×
3
00 mL). The collected organic layers were dried over Na SO and the solvent was
2 4
evaporated under reduced pressure to obtain the products 3a and 3b, which were pure
enough without further purification.
4
.1.4 General methods for the synthesis of 4a and 4b.
A solution of NaN (12.0 mmol) in DMF (20 mL) was added 3a or 3b (4.0 mmol),
3
and the reaction mixture was stirred at room temperature for 4 h. Water (200 mL) was
carefully added to the reaction mixture. Then, the aqueous layer was extracted with
CH Cl (2 × 200 mL). The collected organic layers were washed with water (500 mL)
2
2
and dried over Na SO The drying agent was filtered out and concentrated.
2
4.
Concentrated product was purified by flash chromatography.
4
.1.5 General methods for the synthesis of 5a-5h.
The corresponding xanthone derivatives 2a-2d (1.0 mmol) were reacted with 4a or
b (1.0 mmol) at room temperature overnight in the presence of CuSO .5H O (0.1
4
4
2
mmol), sodium ascorbate (0.2 mmol) using DMF (10 mL) as a solvent. Then the
solution was poured into ice water (100 mL), the obtained precipitate was filtered and
dried, purified by flash chromatography to obtain 5a-5h respectively.
4
.1.5.1.
N-(3,4-dimethoxyphenyl)-2-(4-(((1-hydroxy-6-methoxy-9-oxo-9H-xanthen-3-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (5a). White solid; Yield 84.9 %; Mp;
2
1
9
1
45-246 ℃ ; Purity 97.5 %; IR (KBr): 3446, 3250, 3144, 3079, 2938, 2833, 1663,
604, 1565, 1511, 1456, 1404, 1369, 1306, 1267, 1242, 1210, 1154, 1103, 1080, 1018,
-1 1
73, 940, 814, 671, 642, 556 cm ; H NMR (400 MHz, DMSO-d ) δ 12.95 (s, 1H),
6
0.37 (s, 1H), 8.33 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.30 (s, 1H), 7.16 - 6.97 (m, 3H),
2
1

ACCEPTED MANUSCRIPT
6
6
1
1
5
5
.90 (d, J = 8.7 Hz, 1H), 6.71 (s, 1H), 6.51 (s, 1H), 5.33 (s, 4H), 3.92 (s, 3H), 3.71 (s,
1
3
H); C NMR (100 MHz, DMSO-d ) δ 179.35, 165.30, 164.82, 163.65, 162.61,
6
57.51, 157.15, 148.57, 145.15, 141.54, 131.91, 126.81, 126.74, 113.66, 113.33,
12.04, 111.10, 104.25, 102.82, 100.48, 97.58, 93.46, 61.82, 56.22, 55.65, 55.30,
–
2.21; HRMS (ESI) calcd for C H O N [M-H] : m/z = 531.15213, found
2
7
23
8
4
31.15223.
.1.5.2.
4
N-(3,4-dimethoxyphenyl)-2-(4-(((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (5b). Yellow solid; Yield 81.3 %; Mp
2
1
1
1
2
04-205 ℃ . Purity 99.8 %; IR (KBr): 3564, 3261, 3141, 3073, 2964, 2933, 2833, 1667,
603, 1576, 1513, 1489, 1436, 1404, 1368, 1339, 1297, 1265, 1237, 1215, 1160, 1129,
-1
1
082, 1029, 970, 837, 814, 768, 692, 559 cm ; H NMR (400 MHz, DMSO-d ) δ
6
2.81 (s, 1H), 10.37 (s, 1H), 8.33 (s, 1H), 7.55 (d, J = 8.6 Hz, 1H), 7.51 - 7.40 (m,
H), 7.30 (s, 1H), 7.05 (d, J = 8.7 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 6.76 (s, 1H), 6.50
1
3
(
s, 1H), 5.34 (s, 4H), 3.86 (s, 3H), 3.71 (s, 6H); C NMR (100 MHz, DMSO-d ) δ
6
1
79.82, 165.14, 163.65, 162.42, 157.20, 155.75, 150.13, 148.56, 145.15, 141.51,
31.91, 126.75, 124.82, 120.16, 119.27, 112.03, 111.10, 105.18, 104.25, 102.96, 97.58,
1
–
9
3.24, 61.84, 55.75, 55.65, 55.30, 52.21; HRMS (ESI) calcd for C H O N [M-H] :
2
7
23
8
4
m/z = 531.15213, found 531.15217.
4
2
.1.5.3.
-(4-(((6-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)-N-(3,4-dimethoxyphenyl)acetamide (5c). Yellow solid; Yield 76.3 %; Mp
2
1
7
8
54-256 ℃ ; Purity 97.2 %; IR (KBr): 3465, 3285, 3069, 2941, 2836, 1657, 1598,
556, 1515, 1447, 1424, 1372, 1312, 1287, 1229, 1164, 1061, 1024, 924, 882, 818,
-1 1
96, 663, 553 cm ; H NMR (400 MHz, DMSO-d ) δ 12.62 (s, 1H), 10.37 (s, 1H),
6
.33 (s, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 1.7 Hz, 1H), 7.64 (dd, J = 8.5, 1.7
Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 6.90 (d, J = 8.6 Hz,
H), 6.76 (d, J = 2.3 Hz, 1H), 6.55 (d, J = 2.3 Hz, 1H), 5.34 (d, J = 5.2 Hz, 4H), 3.71
1
2
2

ACCEPTED MANUSCRIPT
1
3
(
s, 6H); C NMR (100 MHz, DMSO-d ) δ 179.39, 165.46, 163.57, 162.51, 156.96,
6
1
1
55.53, 148.57, 145.17, 141.43, 131.88, 128.98, 127.78, 126.96, 126.67, 120.50,
19.03, 112.10, 111.14, 104.36, 103.22, 97.97, 93.67, 61.93, 55.67, 55.31, 52.19;
–
HRMS (ESI) calcd for C H O N Br [M-H] : m/z = 579.05208, found 579.05223.
2
6
20
7
4
4
2
.1.5.4.
-(4-(((7-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)-N-(3,4-dimethoxyphenyl)acetamide (5d). Yellow solid; Yield 71.8 %; Mp
2
1
8
1
8
36-239 ℃ ; Purity 97.7 %; IR (KBr): 3460, 3264, 3088, 2924, 2836, 1658, 1601,
561, 1515, 1461, 1412, 1368, 1306, 1277, 1228, 1162, 1129, 1075, 1022, 969, 830,
-1 1
07, 713, 645, 553 cm ; H NMR (400 MHz, DMSO-d ) δ 12.54 (s, 1H), 10.37 (s,
6
H), 8.33 (s, 1H), 8.17 (d, J = 2.5 Hz, 1H), 8.01 (dd, J = 8.9, 2.5 Hz, 1H), 7.60 (d, J =
.9 Hz, 1H), 7.30 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.7, 2.3 Hz, 1H), 6.90 (d, J = 8.7
Hz, 1H), 6.81 (d, J = 2.2 Hz, 1H), 6.56 (d, J = 2.2 Hz, 1H), 5.34 (d, J = 7.4 Hz, 4H),
1
3
3
1
1
5
5
.71 (d, J = 2.9 Hz, 6H); C NMR (100 MHz, DMSO-d ) δ 178.87, 165.60, 163.63,
6
62.50, 157.12, 154.45, 148.55, 145.14, 141.41, 138.29, 131.91, 127.25, 126.80,
21.49, 120.39, 116.57, 112.03, 111.09, 104.24, 103.23, 97.99, 93.78, 61.95, 55.65,
–
5.30, 52.20; HRMS (ESI) calcd for C H O N Br [M-H] : m/z = 579.05208, found
2
6
20
7
4
79.05159.
.1.5.5.
4
N-(2,4-dimethoxyphenyl)-2-(4-(((1-hydroxy-6-methoxy-9-oxo-9H-xanthen-3-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (5e). White solid; Yield 86.2 %; Mp
2
1
7
9
53-260 ℃ ; Purity 95.9 %; IR (KBr): 3266, 3066, 3011, 2943, 2833, 1664, 1602,
561,1503, 1445, 1362, 1307, 1269, 1211, 1158, 1107, 1080, 1018, 968, 916, 827,
-1 1
99, 726, 672, 641, 561, 517 cm ; H NMR (400 MHz, DMSO-d ) δ 12.96 (s, 1H),
6
.64 (s, 1H), 8.30 (s, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.12 (d, J
=
2.3 Hz, 1H), 7.05 (dd, J = 8.9, 2.3 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 6.64 (d, J = 2.6
Hz, 1H), 6.52 (d, J = 2.2 Hz, 1H), 6.48 (dd, J = 8.7, 2.6 Hz, 1H), 5.41 (s, 2H), 5.33 (s,
1
3
2
H), 3.92 (s, 3H), 3.84 (s, 3H), 3.74 (s, 3H); C NMR (100 MHz, DMSO-d ) δ
6
2
3

ACCEPTED MANUSCRIPT
1
79.41, 165.36, 164.87, 164.07, 162.64, 157.58, 157.20, 157.05, 151.34, 141.51,
26.87, 126.75, 123.37, 119.55, 113.74, 113.37, 104.10, 102.86, 100.55, 98.90, 97.62,
1
–
9
3.51, 61.84, 56.28, 55.77, 55.31, 52.11; HRMS (ESI) calcd for C H O N [M-H] :
2
7
23
8
4
m/z = 531.15213, found 531.15216.
4
.1.5.6.
N-(2,4-dimethoxyphenyl)-2-(4-(((1-hydroxy-7-methoxy-9-oxo-9H-xanthen-3-yl)oxy)
methyl)-1H-1,2,3-triazol-1-yl)acetamide (5f). Yellow solid; Yield 83.8 %; Mp
2
1
1
1
1
6
35-237 ℃ ; Purity 96.4 %; IR(KBr): 3270, 3139, 3086, 3006, 2959, 2836, 1659, 1607,
577, 1545, 1485, 1466, 1434, 1406, 1367, 1308, 1284, 1237, 1208, 1152, 1125, 1081,
-1
1
030, 966, 937, 825, 784, 712, 590, 509 cm ; H NMR (400 MHz, DMSO-d ) δ
6
2.81 (s, 1H), 9.63 (s, 1H), 8.31 (s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.56 (d, J = 8.8 Hz,
H), 7.50 - 7.39 (m, 2H), 6.75 (d, J = 2.2 Hz, 1H), 6.63 (d, J = 2.6 Hz, 1H), 6.55 -
1
3
.40 (m, 2H), 5.42 (s, 2H), 5.33 (s, 2H), 3.85 (d, J = 8.0 Hz, 6H), 3.74 (s, 3H); C
NMR (100 MHz, DMSO-d ) δ 179.83, 165.15, 164.05, 162.42, 157.20, 157.03,
6
1
55.75, 151.31, 150.14, 141.49, 126.75, 124.83, 123.33, 120.16, 119.55, 119.29,
1
05.18, 104.08, 102.96, 98.88, 97.58, 93.25, 61.85, 55.76 (2C), 55.29, 52.11; HRMS
–
(
ESI) calcd for C H O N [M-H] : m/z = 531.15213, found 531.15233.
2
7
23
8
4
4
2
.1.5.7.
-(4-(((6-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)-N-(2,4-dimethoxyphenyl)acetamide (5g).Yellow solid; Yield 70.5 %; Mp
2
1
8
1
44-246 ℃ ; Purity 97.5 %; IR(KBr): 3293, 3063, 2935, 2833, 1656, 1597, 1543, 1504,
449, 1422, 1373, 1310, 1282, 1207, 1161, 1126, 1081, 1062, 1036, 966, 923, 890,
-1 1
21, 794, 662, 574, 511 cm ; H NMR (400 MHz, DMSO-d ) δ 12.63 (s, 1H), 9.62 (s,
6
H), 8.30 (s, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 1.9 Hz, 1H), 7.70 (d, J = 8.8
Hz, 1H), 7.65 (dd, J = 8.6, 1.9 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H), 6.63 (d, J = 2.7 Hz,
1
3
1
H), 6.55 (d, J = 2.3 Hz, 1H), 6.47 (dd, J = 8.8, 2.7 Hz, 1H), 5.41 (s, 2H), 5.35 (s, 2H),
13
.84 (s, 3H), 3.74 (s, 3H); C NMR (125 MHz, DMSO-d ) δ 179.66, 165.58, 164.10,
6
62.62, 157.16, 157.07, 155.76, 151.36, 141.44, 129.16, 127.98, 127.18, 126.86,
2
4

ACCEPTED MANUSCRIPT
1
5
5
23.40, 120.72, 119.55, 119.24, 104.11, 103.39, 98.91, 98.09, 93.83, 61.98, 55.80,
–
5.34, 52.13; HRMS (ESI) calcd for C H O N Br [M-H] : m/z = 579.05208, found
2
6
20
7
4
79.05188.
.1.5.8.
4
2
-(4-(((7-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)-N-(2,4-dimethoxyphenyl)acetamide (5h). White solid; Yield 88.6 %; Mp 245-248 ℃ ;
Purity 97.9 %; IR(KBr): 3250, 3214, 3072, 3002, 2961, 2936, 2833, 1658, 1602,
1
8
1
8
1
3
1
1
5
5
557, 1508, 1461, 1414, 1370, 1331, 1305, 1281, 1206, 1163, 1128, 1077, 1040, 968,
-1 1
19, 715, 649, 580, 517 cm ; H NMR (500 MHz, DMSO-d ) δ 12.54 (s, 1H), 9.63 (s,
6
H), 8.31 (s, 1H), 8.18 (d, J = 2.6 Hz, 1H), 8.01 (dd, J = 9.0, 2.6 Hz, 1H), 7.70 (d, J =
.9 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 6.82 (d, J = 2.3 Hz, 1H), 6.64 (d, J = 2.7 Hz,
H), 6.56 (d, J = 2.3 Hz, 1H), 6.47 (dd, J = 8.9, 2.7 Hz, 1H), 5.41 (s, 2H), 5.35 (s, 2H),
13
.84 (s, 3H), 3.74 (s, 3H); C NMR (125 MHz, DMSO-d ) δ 178.97, 165.63, 164.06,
6
62.53, 157.19, 157.05, 154.53, 151.34, 141.39, 138.35, 127.31, 126.80, 123.36,
21.58, 120.48, 119.53, 116.59, 104.10, 103.30, 98.90, 98.04, 93.84, 61.96, 55.77,
–
5.31, 52.10; HRMS (ESI) calcd for C H O N Br [M-H] : m/z = 579.05208, found
2
6
20
7
4
79.05194.
.1.6 General methods for the synthesis of 6a-6h.
A solution of 5a-5h (0.5 mmol) in dry CH Cl (30 mL) was added BBr (4.7 mL,
4
5
2
2
3
0 mmol) slowly through the septum with stirring at 0 °C. After 0.5 h, the solution
was stirred for 24-72 h at room temperature, the mixture was poured into ice water
300 mL) and stirred for 0.5 h. Solid was precipitated. After being filtered and dried,
the solid was purified by flash chromatography to obtain 6a-6h, respectively.
(
4
2
3
.1.6.1.
-(4-(((1,6-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(
,4-dihydroxyphenyl)acetamide (6a). White solid; Yield 29.5 %; Mp 294-296 ℃ ;
Purity 95.5 %; IR(KBr): 3496, 3415, 3255, 3139, 3084, 2705, 2583, 1665, 1608, 1578,
509, 1462, 1389, 1296, 1252, 1180, 1145, 1109, 1086, 965, 924, 823, 726, 668, 564,
1
2
5

ACCEPTED MANUSCRIPT
-1 1
5
30, 454 cm ; H NMR (400 MHz, DMSO-d ) δ 13.05 (s, 1H), 11.10 (s, 1H), 10.13
6
(s, 1H), 9.01 (s, 1H), 8.69 (s, 1H), 8.30 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.09 (d, J =
2
.4 Hz, 1H), 6.92 (dd, J = 8.8, 2.1 Hz, 1H), 6.87 - 6.78 (m, 2H), 6.74 (d, J = 2.3 Hz,
1
3
1
H), 6.65 (d, J = 8.5 Hz, 1H), 6.48 (d, J = 2.3 Hz, 1H), 5.30 (d, J = 11.8 Hz, 4H); C
NMR (100 MHz, DMSO-d ) δ 179.35, 164.71, 164.50, 163.19, 162.58, 157.58,
6
1
57.18, 145.07, 141.76, 141.54, 130.38, 127.30, 126.66, 115.37, 114.26, 112.35,
110.46, 107.90, 102.73, 102.05, 97.55, 93.41, 61.79, 52.23; HRMS (ESI) calcd for
–
C H O N [M-H] : m/z = 489.10519, found 489.10528.
24
17
8
4
4
2
3
.1.6.2.
-(4-(((1,7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(
,4-dihydroxyphenyl)acetamide (6b); White solid; Yield 36.7 %, Mp 226-229 ℃ ;
Purity 97.4 %; IR(KBr): 3271, 3146, 3083, 2945, 2734, 1652, 1611, 1582, 1513, 1478,
1
5
364, 1328, 1299, 1271, 1240, 1215, 1166, 1112, 1081, 981, 818, 798, 715, 644, 594,
-1 1
66, 446 cm ; H NMR (400 MHz, DMSO-d ) δ 12.88 (s, 1H), 10.14 (s, 1H), 10.05
6
(s, 1H), 9.03 (s, 1H), 8.71 (s, 1H), 8.31 (s, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.42 (d, J =
2
.9 Hz, 1H), 7.32 (dd, J = 9.0, 2.9 Hz, 1H), 7.09 (s, 1H), 6.84 - 6.75 (m, 2H), 6.65 (d,
13
J = 8.5 Hz, 1H), 6.49 (s, 1H), 5.31 (d, J = 17.7 Hz, 4H); C NMR (100 MHz,
DMSO-d ) δ 179.54, 165.53, 163.18, 162.57, 157.05, 155.64, 145.07, 141.76, 141.41,
6
1
1
4
30.38, 129.08, 127.88, 127.07, 126.73, 120.61, 119.13, 115.36, 110.44, 107.88,
–
03.30, 98.02, 93.74, 61.96, 52.23; HRMS (ESI) calcd for C H O N [M-H] : m/z =
2
4
17
8
4
89.10519, found 489.10522.
.1.6.3.
4
2
-(4-(((6-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)-N-(3,4-dihydroxyphenyl)acetamide (6c); White solid; Yield 21.7 %; Mp 280-281 ℃ ;
Purity 95.9 %; IR(KBr): 3270, 3070, 2927, 1658, 1598, 1559, 1516, 1428, 1373, 1310,
-1
1
1
286, 1226, 1203, 1166, 1109, 1062, 966, 926, 817, 793, 668, 577 cm ; H NMR
(
400 MHz, DMSO-d ) δ 12.63 (s, 1H), 10.13 (s, 1H), 9.02 (s, 1H), 8.70 (s, 1H), 8.31
6
(s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.65 (dd, J = 8.4, 1.8 Hz,
2
6

ACCEPTED MANUSCRIPT
1
H), 7.08 (d, J = 2.4 Hz, 1H), 6.81 (dd, J = 8.5, 2.4 Hz, 1H), 6.76 (d, J = 2.2 Hz, 1H),
1
3
6
.65 (d, J = 8.5 Hz, 1H), 6.55 (d, J = 2.2 Hz, 1H), 5.35 (s, 2H), 5.29 (s, 2H); C NMR
(
100 MHz, DMSO-d ) δ 180.08, 165.10, 163.19, 162.47, 157.34, 154.09, 149.13,
6
1
1
45.07, 141.76, 141.50, 130.38, 126.69, 124.82, 120.42, 119.07, 115.37, 110.45,
07.96, 107.89, 102.98, 97.44, 93.19, 61.83, 52.22; HRMS (ESI) calcd for
–
C H O N Br [M-H] : m/z =551.02078, found 551.02144.
24
16
7
4
4
2
.1.6.4.
-(4-(((7-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)
-N-(3,4-dihydroxyphenyl)acetamide (6d); White solid; Yield 24.5 %; Mp 265-269 ℃ ;
Purity 95.4 %; IR(KBr): 3499, 3270, 3235, 3091, 2925, 1659, 1600, 1562, 1530, 1463,
370, 1305, 1276, 1228, 1204, 1167, 1106, 1077, 1057, 957, 817, 714, 653, 563, 532
1
-1 1
cm ; H NMR (400 MHz, DMSO-d ) δ 12.52 (s, 1H), 10.14 (s, 1H), 9.02 (s, 1H),
6
8
.70 (s, 1H), 8.32 (s, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.99 (dd, J = 8.9, 2.5 Hz, 1H),
.58 (d, J = 8.9 Hz, 1H), 7.08 (d, J = 2.6 Hz, 1H), 6.87-6.75 (m, 2H), 6.65 (d, J = 8.5
7
1
3
Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H), 5.32 (d, J = 20.3 Hz, 4H); C NMR (100 MHz,
DMSO-d ) δ 178.83, 165.59, 163.18, 162.49, 157.09, 154.42, 145.06, 141.76, 141.39,
6
1
1
=
38.27, 130.38, 127.23, 126.74, 121.44, 120.37, 116.57, 115.36, 110.45, 107.89,
–
03.20, 97.97, 93.75, 61.96, 52.23; HRMS (ESI) calcd for C H O N Br [M-H] : m/z
2
4
16
7
4
551.02078, found 551.02131.
.1.6.5.
4
2
2
-(4-(((1,6-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(
,4-dihydroxyphenyl)acetamide (6e); White solid; Yield 35.2 %; Mp 290-292 ℃ ;
Purity 99.5 %; IR(KBr): 3441, 3284, 3138, 3086, 2927, 2703, 2584, 1663, 1608, 1580,
1
8
1
547, 1508, 1455, 1394, 1367, 1300, 1257, 1236, 1178, 1143, 1088, 1036, 977, 923,
-1 1
86, 841, 820, 725, 671, 597, 560, 534, 498 cm ; H NMR (400 MHz, DMSO-d ) δ
6
3.05 (s, 1H), 11.09 (s, 1H), 9.66 (s, 1H), 9.49 (s, 1H), 9.17 (s, 1H), 8.29 (s, 1H), 7.99
(d, J = 8.7 Hz, 1H), 7.43 (d, J = 8.6 Hz, 1H), 6.91 (dd, J = 8.7, 2.2 Hz, 1H), 6.84 (d, J
=
2.2 Hz, 1H), 6.74 (d, J = 2.3 Hz, 1H), 6.48 (d, J = 2.3 Hz, 1H), 6.35 (d, J = 2.6 Hz,
2
7

ACCEPTED MANUSCRIPT
1
3
1
H), 6.17 (dd, J = 8.6, 2.6 Hz, 1H), 5.37 (s, 2H), 5.31 (s, 2H); C NMR (100 MHz,
DMSO-d ) δ 179.34, 164.71, 164.48, 163.88, 162.58, 157.58, 157.18, 155.09, 149.69,
6
1
1
4
41.52, 127.29, 126.65, 124.02, 116.99, 114.25, 112.35, 105.64, 102.73, 102.68,
–
02.05, 97.54, 93.40, 61.79, 52.09; HRMS (ESI) calcd for C H O N [M-H] : m/z =
2
4
17
8
4
89.10519, found 489.10536.
.1.6.6.
4
2
2
-(4-(((1,7-dihydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(
,4-dihydroxyphenyl)acetamide (6f); White solid; Yield 30.2 %; Mp 266-267 ℃ ;
Purity 98.7 %; IR(KBr): 3286, 3133, 1656, 1607, 1577, 1479, 1413, 1389, 1328, 1300,
-1
1
232, 1155, 1109, 1082, 1058, 1026, 972, 951, 872, 825, 793, 713, 592, 546, 493 cm ;
1
H NMR (400 MHz, DMSO-d ) δ 12.87 (s, 1H), 10.06 (s, 1H), 9.69 (s, 1H), 9.51 (s,
6
1
H), 9.19 (s, 1H), 8.30 (s, 1H), 7.49 (d, J = 9.0 Hz, 1H), 7.45-7.39 (m, 2H), 7.31 (dd,
J = 9.0, 3.1 Hz, 1H), 6.75 (d, J = 2.3 Hz, 1H), 6.48 (d, J = 2.3 Hz, 1H), 6.36 (d, J =
13
2
.6 Hz, 1H), 6.16 (dd, J = 8.4, 2.6 Hz, 1H), 5.35 (d, J = 22.7 Hz, 4H); C NMR (100
MHz, DMSO-d ) δ 180.09, 165.10, 163.90, 162.48, 157.34, 155.11, 154.10, 149.71,
6
1
1
49.13, 141.50, 126.72, 124.82, 124.03, 120.42, 119.07, 117.01, 107.97, 105.63,
02.98, 102.70, 97.44, 93.18, 61.84, 52.11; HRMS (ESI) calcd for C H O N
4
24
17
8
–
[
M-H] : m/z = 489.10519, found 489.10493.
4
2
.1.6.7.
-(4-(((6-bromo-1-hydroxy-9-oxo-9H-xanthen-3-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl
)-N-(2,4-dihydroxyphenyl)acetamide (6g); White solid; Yield 27.2 %; Mp 264-267 ℃ ;
Purity 98.8 %; IR(KBr): 3268, 3141, 3071, 2956, 1658, 1598, 1555, 1498, 1458, 1434,
-1
1
403, 1301, 1201, 1150, 1112, 1064, 974, 922, 872, 845, 819, 671, 608, 557, 496 cm ;
1
H NMR (400 MHz, DMSO-d ) δ 12.61 (s, 1H), 9.67 (s, 1H), 9.49 (s, 1H), 9.17 (s,
6
1
H), 8.30 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.89 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.42
(d, J = 8.7 Hz, 1H), 6.74 (s, 1H), 6.53 (s, 1H), 6.35 (d, J = 2.7 Hz, 1H), 6.16 (dd, J =
1
3
8
1
.7, 2.7 Hz, 1H), 5.36 (d, J = 16.3 Hz, 4H); C NMR (100 MHz, DMSO-d ) δ 179.50,
6
65.52, 163.90, 162.57, 157.03, 155.62, 155.12, 149.72, 141.43, 129.09, 127.87,
2
8