Synthesis and evaluation of xanthine oxidase inhibitory and antioxidant activities of 2-arylbenzo[b]furan derivatives based on salvianolic acid C

Article abstract of DOI:10.1016/j.ejmech.2016.08.019

Xanthine oxidase (XO) is the key enzyme in humans which is related to a variety of diseases such as gout, hyperuricemia and cardiovascular diseases. In this work, a series of 2-arylbenzo[b]furan derivatives were synthesized based on salvianolic acid C, and they were evaluated for xanthine oxidase inhibitory and antioxidant activities. Compounds 5b, 6a, 6e and 6f showed potent xanthine oxidase inhibitory activities with IC₅₀values ranging from 3.99 to 6.36?μM, which were comparable with that of allopurinol. Lineweaver-Burk plots analysis revealed that the representative derivative 6e could bind to either xanthine oxidase or the xanthine oxidase-xanthine complex, which exhibited a mixed-type competitive mechanism. A DPPH radical scavenging assay showed most of the hydroxyl-functionalized 2-arylbenzo[b]furan derivatives possessed the potent antioxidant activity, which was further validated on LPS-stimulated RAW 264.7 macrophages model. The structure-activity relationships were preliminary analyzed and indicated that the structural skeleton of 2-arylbenzo[b]furan and phenolic hydroxyl groups played an important role in maintaining xanthine oxidase inhibitory effect and antioxidant property for the series of derivatives. Meanwhile, molecular docking studies were performed to further confirm the structure-activity relationships and investigate the proposed binding mechanisms of compounds 5d, 6d and 10d binding to the protein.

Full text of DOI:10.1016/j.ejmech.2016.08.019

Accepted Manuscript
Synthesis and evaluation of xanthine oxidase inhibitory and antioxidant activities of 2-
arylbenzo[b]furan derivatives based on salvianolic acid C
Hong-Jin Tang, Xiao-Wei Zhang, Lin Yang, Wei Li, Jia-Huang Li, Jin-Xin Wang, Jun
Chen
PII:
S0223-5234(16)30662-6
10.1016/j.ejmech.2016.08.019
EJMECH 8815
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 April 2016
Revised Date: 9 August 2016
Accepted Date: 10 August 2016
Please cite this article as: H.-J. Tang, X.-W. Zhang, L. Yang, W. Li, J.-H. Li, J.-X. Wang, J. Chen,
Synthesis and evaluation of xanthine oxidase inhibitory and antioxidant activities of 2-arylbenzo[b]furan
derivatives based on salvianolic acid C, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.08.019.
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ACCEPTED MANUSCRIPT
XO inhibitory assay
DPPH assay
Structural
modification
SAR analysis
The binding mechanisms
Salvianolic acid C
non-purine XO inhibitor
Molecular modeling
2-arylbenzo[b]furan derivatives

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Synthesis and evaluation of xanthine oxidase inhibitory and
antioxidant activities of 2-arylbenzo[b]furan derivatives based on
salvianolic acid C
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Hong-Jin Tanga, Xiao-Wei Zhanga, Lin Yanga, Wei Lia, Jia-Huang Lib, Jin-Xin
Wangc, Jun Chena,*
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State Key Laboratory of Natural Medicines, China Pharmaceutical University,
7
Nanjing 210009, P. R. China
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State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
9
Nanjing 210023, P. R. China
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Jiangsu Key Laboratory of Drug Design & Optimization, Department of Medicinal
Chemistry, China Pharmaceutical University, Nanjing 210009, P. R. China
Abbreviations: XOR, xanthine oxidoreductase; ROS, reactive oxygen species; XO, xanthine
oxidase; XDH, xanthine dehydrogenase; SARs, structure-activity relationships; DPPH,
1,1-diphenyl-2-picrylhydrazyl; RMSD, root mean square deviation; Mo-Pt, molybdopterin.
* Corresponding author. Tel.: +86-25-8327-1382; fax: +86-25-8327-1379 (J. Chen);
E-mail address: chenj2002cpu@126.com (J. Chen).
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Abstract
Xanthine oxidase (XO) is the key enzyme in humans which is related to a variety
of diseases such as gout, hyperuricemia and cardiovascular diseases. In this work, a
series of 2-arylbenzo[b]furan derivatives were synthesized based on salvianolic acid C,
and they were evaluated for xanthine oxidase inhibitory and antioxidant activities.
Compounds 5b, 6a, 6e and 6f showed potent xanthine oxidase inhibitory activities
with IC50 values ranging from 3.99 to 6.36 ꢀM, which were comparable with that of
allopurinol. Lineweaver-Burk plots analysis revealed that the representative derivative
6e could bind to either xanthine oxidase or the xanthine oxidase-xanthine complex,
which exhibited a mixed-type competitive mechanism. A DPPH radical scavenging
assay showed most of the hydroxyl-functionalized 2-arylbenzo[b]furan derivatives
possessed the potent antioxidant activity, which was further validated on
LPS-stimulated RAW 264.7 macrophages model. The structure-activity relationships
were preliminary analyzed and indicated that the structural skeleton of
2-arylbenzo[b]furan and phenolic hydroxyl groups played an important role in
maintaining xanthine oxidase inhibitory effect and antioxidant property for the series
of derivatives. Meanwhile, molecular docking studies were performed to further
confirm the structure-activity relationships and investigate the proposed binding
mechanisms of compounds 5d, 6d and 10d binding to the protein.
Keywords: Salvianolic acid C, 2-arylbenzo[b]furan derivatives, Xanthine oxidase
inhibitor, Antioxidant, Molecular docking.

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1. Introduction
Reactive oxygen species (ROS) play an important role in the development of a
number of pathological events, such as cardiovascular diseases, metabolic disorders,
reperfusion damages and even carcinogenesis [1-3]. Superoxide anion radicals are the
important reactive oxygen involved in a variety of biological phenomena and also the
precursor to other ROS [4,5]. H2O2 is the more likely candidate to mediate signal
transduction or participate in oxidation reactions especially in the presence of
transition metals [6]. ROS can be constantly produced by a variety of enzymatic
reactions. XO is one of the critical enzymatic sources generating ROS [7] and it is a
more abundant source of H2O2 than superoxide [6]. XO can catalyze the conversion of
hypoxanthine and xanthine to uric acid accompanied by producing superoxide anions,
H2O2, and ROS during the catalytic process [8]. Excess ROS can cause the cellular
damage and involved in the initiation and progression of many diseases [3], and the
generation of excess uric acid is harmful to body and may lead to gout, hyperuricemia
and other symptoms of related diseases [9]. Hence, there is a need to discover novel
XO inhibitors for the treatment of XO-related diseases in clinical.
Natural products have been always the most important and productive sources of
lead compounds for the development of drugs and the treatment of many diseases
[10-12]. However, due to various reasons such as severe toxicity, poor solubility,
original natural products are not particularly excellent candidates and therefore there
is a need to improve their drug-like properties by structure modification, which has
been demonstrated by a variety of drugs used in clinical [13], just as etoposide [14],
polyene paclitaxel [15]. Hence, the structural modification of natural products with
specific bioactivity is an important and efficient approach to discover novel drugs.
Danshen, the dried root and rhizome of Salvia miltiorrhiza Bge., is one of the
most popular traditional Chinese medicine, and has been used clinically in the
treatment of cardiovascular diseases more than 20 centuries [16,17]. The herb
provides therapeutic relief from stoke, promotes blood circulation and exhibits
amounts of pharmacological activities such as antiviral, antioxidant, antitumor [18-21].
Benzofuran and its derivatives have attracted medicinal chemists and pharmacologists

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due to their wide range of biological activities and their potential applications as
agents [22]. The salvianolic acid C (Fig. 1), a hydrophilic and polyphenolic
benzofuran derivative isolated from Danshen, presented potent xanthine oxidase (XO)
inhibitory activity reported in our previous study [23]. Nevertheless, so far, few
attempts have been devoted to the research of salvianolic acid C except the
total-synthesis [24-26] and the pharmacokinetic study of rat plasma [27].
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Fig. 1.
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The basic scaffold of salvianolic acid C, especially the hydroxyl-functionalized
2-arylbenzo[b]furan ring may play a critical role in the XO inhibitory activity.
Therefore, to elucidate the structure-activity relationships (SARs) and further discover
more potent XO inhibitors based on the scaffold of benzofuran, we designed and
performed the synthetic experiments and the activity evaluation assay in vitro. In this
work, based on the natural product salvianolic acid C, further modifications were
carried out with substitution of C-9 ester bond by amide linkage to develop a series of
analogues which contained the planar structure of 2-arylbenzo[b]furan. The obtained
derivatives were evaluated for the inhibitory effect against XO and antioxidant
activity in vitro. Meanwhile, the structure-activity relationships (SARs) were
discussed here and the potential binding modes between the inhibitors and the protein
by molecular docking studies were investigated.
2. Results and discussions
2.1. Chemical synthesis
The synthesis of the target hydroxyl-functionalized 2-arylbenzo[b]furan
derivatives 5a-5f and 6a-6f was performed as outlined in Scheme 1. Salvianolic acid
C (1) was employed as the starting material. It was firstly converted to tournefolic

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acid A (2) by hydrolyzing the ester bond with potassium hydroxide in high yield.
Amino reagents 3a-3f were reacted with thionyl chloride in methanol to protect the
free carboxyl groups and products 4a-4f were efficiently obtained. An amidation
reaction between the intermediate 2 and compounds 4a-4f was carried out and then
afforded the target products 5a-5f, which was performed by using the combined
reagents EDCI/HoBt in the presence of triethylamine. The reaction conditions are
mild and all reactants are mixed in solvents DMF/CH2Cl2 at room temperature.
Finally, the title derivatives 6a-6f derived from compounds 5a-5f were synthesized
with sodium hydroxide under reflux.
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Scheme 1. Synthesis of 2-arylbenzo[b]furan derivatives 5a-5f, 6a-6f. Reagents and conditions: (a)
KOH, MeOH-H2O, reflux; (b) SOCl2, MeOH, 0 °C-R.T.; (c) R-NH2·HCl, EDCI·HCl, HoBt, TEA,
DMF, CH2Cl2; (d) NaOH, MeOH-H2O, reflux.
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Meanwhile, the methoxyl-substituted 2-arylbenzo[b]furan derivatives including
9a-9f and 10a-10f were synthesized as Scheme 2. Firstly, the substrate salvianolic
acid C was alkylated with dimethyl sulfate in acetone in the presence of potassium
carbonate to provide methyl (+)-pentamethylsalvianolic acid C (7). Hydrolysis of
compound 7 with potassium hydroxide followed by acidification afforded the
intermediate 8 which was then treated with compounds 4a-4f to give the title
derivatives 9a-9f. Similar to compounds 6a-6f, the target products 10a-10f were
obtained by deprotection of the carboxyl groups under alkaline condition.

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Compared to the derivatives 9c-9f, the ester bonds of compounds 9a and 9b
could not be efficiently hydrolyzed by sodium hydroxide under the same reaction
conditions such as reflux for 8 h, which may be due to the strong electron-donating
effect of 3,4-dihydroxyl group. Just as the reported literature, replacing the ester with
the amide led to a striking stabilization of compound both chemically and
enzymatically [28]. However, during the reaction (step d), it could be observed that
the newly generated amide bond was partly hydrolyzed simultaneously accompanied
by breaking the ester bond, which indicated that the optimization of the hydrolysis
conditions was important to effectively afford these target derivatives.
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Scheme 2. Synthesis of 2-arylbenzo[b]furan derivatives 9a-9f, 10a-10f. Reagents and conditions:
(a) Me2SO4, K2CO3, Me2CO, reflux, 14 h; (b) KOH, MeOH-H2O, reflux, 10 h; (c) R-NH2·HCl,
EDCI·HCl, HoBt, TEA, DMF, CH2Cl2; (d) NaOH/KOH, THF-MeOH-H2O, reflux.
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2.2. Evaluation of XO inhibitory activity in vitro
All of the synthesized 2-arylbenzo[b]furan derivatives were evaluated for XO
inhibitory activity according to the developed procedures [23]. For the accuracy and
reliability of the experimental analysis, the known XO inhibitor, allopurinol was
selected as a positive control. The IC50 values, indicating the strength of XO inhibition
by salvianolic acid C analogues were calculated and displayed in Table 1. All the
hydroxyl-functionalized 2-arylbenzo[b]furan derivatives exhibited the potent XO

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inhibitory activity with IC50 values below 32 ꢀM. Among them, compounds 5b, 6a
and 6c-6f were shown to be potent XO inhibitors with IC50 values ranging from 3.99
to 6.36 ꢀM, which presented stronger inhibition potency than salvianolic acid C (IC50
= 8.26 ꢀM) and were close to that of allopurinol. Other hydroxyl-functionalized
2-arylbenzo[b]furans including compounds 5a, 5c-5f and 6b, possessed modest
activity with IC50 from 9.88 to 31.72 ꢀM. However, the methoxyl-substituted
2-arylbenzo[b]furan derivatives 9a-9f and 10a-10f were inactive at the highest tested
concentration (IC50 > 60 ꢀM), which suggested that the phenolic hydroxyl groups on
2-arylbenzo[b]furan ring were essential to the XO inhibitory effect. When the
phenolic hydroxyl groups on the core scaffold were substituted by methoxyl groups, it
would lead to a distinct decrease of the activity and was demonstrated by compounds
5a-6f and 9a-10f. Meanwhile, it should be pointed out that the number of phenolic
hydroxyl group outside of 2-arylbenzo[b]furan ring seemed to have no direct
relationship with the strength of the activity, which was summarized from compounds
5a-5f and 6a-6f., Compared to salvianolic acid C, the dramatic decrease of XO
inhibitory activity of salvianolic acid A and isosalvianolic acid C (structures seen in
Fig. 1) was determined in our previous study [23]. Combined with the fact, the
obtained results revealed that the structural skeleton of 2-arylbenzo[b]furan for the
series of derivatives was indispensable to retain the potent XO inhibitory effect. In
addition, the free carboxyl group of these derivatives may be helpful to slightly
enhance the activity, which could be inferred relying on the related compounds such
as 5a and 6a, 5d and 6d, 5f and 6f.
Table 1 Xanthine oxidase inhibitory activity of 2-arylbenzo[b]furan derivatives.
Compound
IC50 (µM) a
3.61
Compound
IC50 (µM)
8.26
Allopurinol
Salvianolic acid C (1)
5a
5b
5c
5d
5e
5f
9.88
6a
6b
6c
4.81
3.99
12.12
6.36
10.8
14.91
13.48
31.72
> 60
6d
6e
6.04
4.51
6f
4.98
9a
10a
> 60

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9b
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9d
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> 60
> 60
> 60
> 60
> 60
10b
10c
10d
10e
10f
> 60
> 60
> 60
> 60
> 60
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a IC50 values represent as mean of four determinations.
2.3. Evaluation of antioxidant activity
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay is a widely-used
method to determine the antioxidant activity by measuring the hydrogen donating
ability of the studied compound. Herein, the assay was carried out to evaluate the
antioxidant activity of the synthesized 2-arylbenzo[b]furan derivatives which was
shown as IC50 values representing the potency of scavenging free radical. The
reported compound quercetin was selected as a positive control for the experiment. As
displayed in Table 2, most of the hydroxyl-functionalized 2-arylbenzo[b]furan
analogues exhibited the potent antioxidant activity in the low micromolar level. Even
compounds 5b, 6a-6d and 6f with IC50 values ranging from 3.27 ꢀM to 5.61 ꢀM were
stronger than that of quercetin. However, a majority of methoxyl-substituted
2-arylbenzo[b]furans did not present any DPPH radical scavenging activity due to the
IC50 values exceeding 100 ꢀM, whereas compounds 9a, 9b, 10a and 10b were
exceptional. They possessed potential free radical scavenging activity with IC50 of
7.26-9.52 ꢀM, which indicated that the presence of ortho phenolic hydroxyl group
rather than phenolic hydroxyl group was most essential to these derivatives
maintaining the antioxidant property. Meanwhile, the obtained results suggested that
the substituted position of ortho phenolic hydroxyl group (on or outside of the
2-arylbenzo[b]furan ring) was not important to the antioxidant activity, which could
be demonstrated by several compounds such as 5d and 9a, 6c and 10b. Moreover, the
existence of the free carboxyl group of these derivatives may contribute to slightly
improving the antioxidant potency, especially for the hydroxyl-substituted
2-arylbenzo[b]furans, which was observed by several examples, just as compounds 5a
and 6a, 5e and 6e.

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Table 2 DPPH radical scavenging activity of 2-arylbenzo[b]furan derivatives.
Compound
IC50 (µM)a
6.0
Compound
IC50 (µM)
6.87
Quercetin
Salvianolic C (1)
5a
5b
5c
5d
5e
5f
8.79
6a
6b
4.52
3.9
5.61
11.95
8.66
6c
3.27
6d
4.2
47.25
12.37
7.26
6e
6.47
6f
3.86
9a
9b
9c
9d
9e
9f
10a
10b
10c
10d
10e
10f
8.60
9.52
8.11
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
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a IC50 values represent as mean of three determinations.
2.4. Evaluation of antioxidant activity on LPS-induced RAW 264.7 cell model
Macrophages, located in different tissues in body, have various functions and
play a key role in maintaining homeostasis of body, tissue repairing and immune
responses [29,30]. They are activated by lipopolysaccharide (LPS) to produce
inflammatory cytokines such as TNF-α and reactive oxygen species such as
•
superoxide anion (O2 ‾) or nitric oxide [31]. Hydroethidine (HE) as an intercellular
probe is frequently used to detect superoxide anion generated in cells and tissues [32].
In this assay, LPS-stimulated RAW 264.7 macrophages model combined with the
detection probe HE was used to evaluate the antioxidant activity of the synthesized
derivatives. As shown in Fig. 2, compared to model group, all of the test compounds
including 5a-5c, 6b-6e, 1 and vitamin E (VE) presented the potency of scavenging
superoxide anion, which was consistent with the results obtained by DPPH radical
scavenging assay. It could be observed that derivatives 5a-5c, 6b, 6d and 6e displayed
higher scavenging activity than that of VE. Moreover, the scavenging ratios
(Supplementary material Table S1) of analogues 6b, 6d and 6e were 2-fold more
potent than that of salvianolic acid C, which suggested that they possessed stronger
antioxidant activity. However, there was a slightly difference existed for the

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antioxidant activities of the test derivatives between the two methods including DPPH
radical scavenging assay and LPS-induced RAW 264.7 cell model assay, which may
be attributed to the differential potency of permeating cell membrane into cell for
these analogues.
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Fig. 2.
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2.5. Structure-activity relationship
Combined with the determined results of activity including XO inhibitory and
antioxidant effects, we could achieve some useful information of the key structure
features influenced the activities for the synthesized 2-arylbenzo[b]furan derivatives.
Some notions about SARs could be summarized as follows: (a) the core structure of
2-arylbenzo[b]furan was essential for the series of derivatives to possess the potent
XO inhibitory activity (compare salvianolic acid C with salvianolic acid A and
isosalvianolic acid C); (b) the presence of phenolic hydroxyl groups on the
2-arylbenzo[b]furan ring played a key role in maintaining XO inhibitory effect
(compare 5a-5f and 6a-6f with 9a-9f and 10a-10f); (c) phenolic hydroxyl groups
outside of 2-arylbenzo[b]furan ring had no distinct relationship with the inhibition of
XO (compare 5a, 5b, 5e, 6a, 6b and 6e with 5c, 5d, 5f, 6c, 6d and 6f, respectively); (d)
in general, ortho phenolic hydroxyl group could be helpful to the antioxidant activity
for these derivatives (compare 5a-5f, 6a-6f, 9a, 9b, 10a and 10b with 9c-9f and
10c-10f, respectively), whereas (e) the substituted position of ortho phenolic hydroxyl

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group (on or outside of 2-arylbenzo[b]furan ring) made no contribution to the
antioxidant activity (compare 5a, 5b, 5c, 6a, 6b, 6c with 9a, 9b, 10a and 10b); (f) the
free carboxyl group of these derivatives seemed to benefit to increase the antioxidant
and XO inhibitory potency (compare 5a and 5c-5f with 6a and 6c-6f) except in few
cases (compare 5b with 6b). The obtained information will help us to reasonably
design novel inhibitors containing the planar structure of 2-arylbenzo[b]furan with
enhanced XO inhibitory potency and antioxidant property, and the further structural
optimizations based on the SARs are under way.
2.6. Mode of XO inhibition by 2-arylbenzo[b]furan derivatives
Inhibition kinetics study of 2-arylbenzo[b]furan derivatives were performed by
conducting a Lineweaver-Burk plot analysis with compound 6e. As shown in Fig. 3,
the X-axis presented a variety of concentrations of the substrate xanthine, and the 1/V
obtained from the absorbance rate/min (ꢁA/min) was displayed along the Y-axis. The
results suggested that compound 6e belonged to the mixed-type competitive mode
inhibitor with respect to xanthine for binding to XO, which could be inferred by the
fact that the plots did not intersect either the X or Y-axis and cross to the left of Y-axis
but above the 1/[xanthine]-axis. Likewise, salvianolic acid C was determined to
exhibit the similar kinetic features as compound 6e in our previous study [23]. With
this mode, the derivative 6e could bind to not only the protein XO but also the
xanthine-XO complex. Moreover, the competitive and uncompetitive inhibition
constant Ki and Kis were directly calculated based on the Lineweaver-Burk plot. As a
result, the Ki and Kis values of the inhibitor 6e were 3.43 ꢀM and 15.21 ꢀM,
respectively. The Ki value was smaller than the Kis value, which suggested that the
binding
affinity
of
the
inhibitor-enzyme
exceeded
that
of
the
inhibitor-enzyme-substrate complex.

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Fig. 3.
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2.7. Molecular modeling
Molecular docking study is a widely used method to predict the potential binding
mechanisms between the bioactive molecule and the target. Xanthine oxidoreductase
(XOR) includes two enzymatic forms: XO and xanthine dehydrogenase (XDH) which
can convert mutually under specific conditions [33,34]. The Molybdenum (Mo-Pt) is
the function domain where the oxidation of xanthine takes place [35] and the Mo-Pt
sites of both XO and XDH are structurally equivalent [36].
In this work, docking simulations of compounds 5d, 6d and 10d into the binding
pocket of the bovine milk XDH/Febuxostat complex (PDB code: 1N5X) were
performed. The original ligand Febuxostat was re-docked into XDH, which yielded a
root mean square deviation (RMSD) of 0.56 Å (Supplementary material Fig. S1). The
interaction modes of the molecules binding to the receptor XDH were showed in Fig.
4. As shown in Fig. 4A and 4B, the inhibitors 5d and 6d may exhibit similar binding
modes with XDH due to several same key residues such as Glu802, Arg880, Phe914,
Phe1009, Thr1010, Leu1014 and Ala1079. From Fig. 4D, it could be observed that
2-arylbenzo[b]furan rings of 5d and 6d both stretched into the innermost binding sites
of the active pocket, just as the original ligand Febuxostat. The phenolic hydroxyl on
the 2-arylbenzo[b]furan ring of compound 5d formed one hydrogen bond with residue
Thr1010. Similarly, one hydrogen bond was observed between the inhibitor 6d and
residue Arg880. Meanwhile, pi-stacking interaction (face to face with Phe914) and
other hydrophobic interactions found for Febuxostat were maintained by the inhibitors

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5d and 6d (Fig. 4A and 4B), which may be responsible for their potent inhibitory
activities in vitro. However, as displayed in Fig. 4C, the derivative 10d, which may
have bumped with the protein residues due to the presence of methoxyl groups,
interacted in a distinct way with XDH and a slightly deviation from the binding
pocket of the protein could be seen, especially the conformation of the
2-arylbenzo[b]furan ring. The molecule did not generate any hydrogen bonds with
Arg880, Thr1010 or other residues and lacked pi-stacking interactions with Phe914.
Interactions between compound 10d and XDH may be too weak compared to those of
5d and 6d to produce any apparent inhibitory effect in the experimental test. As
exemplified by 10d, other compounds in the methoxyl-substituted series may present
similar binding properties with the protein, which could reasonably explain their
inferior potencies compared with those of the hydroxyl-functionalized
2-arylbenzo[b]furan derivatives.
Hence, the docking results indicated that the structure skeleton of
2-arylbenzo[b]furan ring and the hydroxyl groups on the ring were critical for the
series of derivatives binding to the protein to show potent XO inhibitory activity,
which was consistent with the SAR analyses. The results described above indirectly
demonstrate that hydrogen bonds, pi-stacking and other hydrophobic interactions
played an important role in maintaining the stability of the ligand-receptor docking
conformation.

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Fig. 4.
3. Conclusions
In this work, a series of novel 2-arylbenzo[b]furan derivatives have been
synthesized based on the core structure of salvianolic acid C. The yields ranged from
poor to excellent relying on the structure of reactants. The obtained
2-arylbenzo[b]furans were evaluated for their XO inhibitory and antioxidant activities.
The results suggested that several target derivatives presented enhanced inhibition of
XO compared with the parent compound, and the inhibitory effects of compounds 5b,
6a and 6e were even close to that of allopurinol. A DPPH radical scavenging assay
revealed that most hydroxyl-functionalized 2-arylbenzo[b]furan derivatives exhibited
remarkable antioxidant effects, which was confirmed by LPS-induced RAW 264.7
macrophages model. Further investigation of binding kinetics indicated that the
mechanism of XO inhibition by compound 6e was mixed-competitive, which was

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similar to salvianolic acid C. Moreover, the SARs were presented combined with the
activity values of the inhibition against XO and antioxidant. Molecular docking
studies were carried out to further validate the SAR analyses and explore the proposed
binding modes between the inhibitors 5d, 6d and XDH. The further modifications
relying on the obtained SAR results are under way, and it is expected that the current
results can be helpful to develop novel XO inhibitors with improved activity.
4. Experimental Section
4.1. Chemical synthesis
Most chemicals and solvents were purchased from commercial sources (Aladdin
and Sigma-Aldrich). Further purification and drying by standard methods were
employed when necessary. NMR spectra were recorded with a Burker AV-300 or 500
(Burker biospin, Swiss) spectrometer in the indicated solvents (TMS as the internal
standard): the values of the chemical shifts are expressed in δ values (ppm), and the
coupling constants J in Hz. Pre-coated thin-layer chromatography plates (Institute of
Yantai Chemical Industry, China ) were used for TLC. The compounds analyzed on
the TLC plates were visualized using a UV detector, I2 vapor or 5% concentrated
sulfuric acid-vanillin with subsequent heating. The silica gel (particle size of 300-400
mesh) for column chromatography purification were purchased from Qingdao Marine
Chemicals Inc., China. Chromatographic grade methanol and acetonitrile were bought
from Tedia Inc., USA. High-resolution mass spectra were recorded on the Agilent
6520 Series Q-TOF-MS system (Agilent Technologies, Germany).
4.1.1. Tournefolic acid A (2)
To a solution of salvianolic acid C (245 mg, 0.498 mmol) in MeOH/H2O (10 mL,
4:1 v/v) was added potassium hydroxide (335 mg, 5.576 mmol). The mixture was
stirred with reflux until the material spot disappeared by TLC under nitrogen
atmosphere. After completing, the solution was concentrated to remove methanol.
Then the residue was poured into 10% HCl solution (15 mL) in an ice bath, and
extracted with ethyl acetate (15 mL × 3). The organic layer was washed with brine,

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dried with anhydrous Na2SO4, and concentrated by rotary evaporation under reduced
pressure. The crude product was purified by column chromatography
(chloroform/methanol/formic acid, 10/1/0.1 v/v/v) to afford tournefolic acid A (2)
(138 mg, yield 89%) as a yellow powder. 1H NMR (CD3OD, 300 MHz) δ 7.92 (d, 1H,
J = 15.9 Hz), 7.41-7.29 (m, 3H), 7.18 (s, 1H), 6.88 (d, 1H, J = 8.1 Hz), 6.73 (d, 1H, J
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= 8.1 Hz), 6.43 (d, 1H, J = 15.9 Hz); C NMR (CD3OD, 75 MHz) δ 171.3, 159.3,
148.0, 146.7, 145.7, 144.6, 144.3, 132.5, 126.1, 123.4, 119.7, 118.7, 116.7, 116.0,
113.3, 111.7, 99.2. HR-MS (ESI) m/z: calcd. for C17H11O6 [M-H]-: 311.0561; found:
311.0563.
4.1.2. General procedure for syntheses of compounds 4a-4f
To 4 mL of ice-bath cooled methanol was added drop-wise 1 mL of thionyl
chloride in 5 min under nitrogen, the resulting mixture was stirred for 0.5 h and amine
derivatives 3a-3f were added. The reaction mixture was stirred at room temperature
overnight until complete disappearance of the materials indicated by TLC. The
solvent and the surplus SOCl2 were removed in vacuum. The residue was dissolved in
20 mL of methanol and evaporated under reduced pressure. The procedure was
repeated for three times. Then 20 mL of ether was added to the residue and evaporated
in vacuum, which also took three times. Target compounds 4a-4f were obtained in
86%-95% yield and used without further purification for the following step.
4.1.2.1. Methyl (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoate (4a) White solid
powder, yield 92%; 1H NMR (D2O, 300 MHz) δ 6.78 (d, 1H, J = 8.2 Hz), 6.78 (d, 1H,
J = 1.8 Hz), 6.68 (dd, 1 H, J = 8.1, 1.8 Hz), 4.35 (t, 1 H, J = 6.6 Hz), 3.82 (s, 3H),
13
3.18 (dd, 1H, J = 14.6, 5.9 Hz), 3.06 (dd, 1 H, J = 14.6, 7.7 Hz); C NMR (D2O, 75
MHz) δ 170.6, 144.8, 144.2, 126.6, 122.3, 117.4, 117.1, 54.8, 54.0, 35.4. HR-MS (ESI)
m/z: calcd. for C10H14NO4 [M+H]+: 212.0917; found: 212.0920.
4.1.3. General procedure for syntheses of compounds 5a-5f
Tournefolic acid A (2) was dissolved in dry DMF/CH2Cl2 (4 mL, 4:1 v/v), and

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the solution was cooled to 0 °C in an ice-water bath. To the solution were added
N-Hydroxybenzotriazole (HoBt) (1.5 equiv mol) and 1-[3-(di-methylamino)propyl]-3-
ethyl carbodiimide hydrochloride (EDCI·HCl) (1.5 equiv mol). After stirring for 15
min, triethylamine (1.5 equiv mol) and derivatives 4a-4f were added and the resultant
mixture was stirred at room temperature under nitrogen. When completing by TLC,
the mixture was poured into 10% HCl solution, then 5% NaHCO3 and extracted with
CH2Cl2 (10 mL × 4). The organic layer was washed with brine, dried with anhydrous
Na2SO4 and concentrated under vacuum. The crude products were separated on silica
gel column chromatography (chroloform/methanol) to provide the title
2-arylbenzo[b]furan derivatives 5a-5f.
4.1.3.1. Methyl (S,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dihydroxyphenyl)-7-
1
hydroxybenzofuran-4-yl)acrylamido)propanoate (5a) Green oil, yield 71%; H NMR
(CD3OD, 300 MHz) δ 7.74 (d, 1H, J = 15.6 Hz), 7.39 (s, 1H), 7.34 (d, 1H, J = 8.7 Hz),
7.27 (d, 1H, J = 7.8 Hz), 7.26 (s, 1H), 6.87 (d, 1H, J = 8.1 Hz), 6.74-6.63 (m, 4H),
6.55 (d, 1H, J = 7.2 Hz), 4.76-4.72 (m, 1H), 3.72 (s, 3H), 3.11-3.00 (dd, 1H, J = 13.5,
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6.0 Hz), 2.99-2.89 (dd, 1H, J = 13.5, 7.5 Hz); C NMR (CD3OD, 75 MHz) δ 173.8,
169.3, 159.1, 147.9, 146.8, 146.3, 145.3, 145.3, 144.5, 140.7, 132.0, 129.5, 126.0,
123.5, 121.7, 120.2, 118.6, 118.4, 117.3, 116.7, 116,4, 113.4, 111.6, 99.5, 55.8, 52.6,
38.2. HR-MS (ESI) m/z: calcd. for C27H22NO9 [M-H]-: 504.1300; found: 504.1302.
4.1.3.2. Methyl (R,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dihydroxyphenyl)-7-
1
hydroxybenzofuran-4-yl)acrylamido)propanoate (5b) Green oil, yield 74%; H NMR
(CD3OD, 300 MHz) δ 7.74 (d, 1H, J = 15.6 Hz), 7.39 (s, 1H), 7.35 (d, 1H, J = 8.4 Hz),
7.28 (d, 1H, J = 6.9 Hz), 7.26 (s, 1H), 6.88 (d, 1H, J = 8.1 Hz), 6.74-6.65 (m, 4H),
6.55 (d, 1H, J = 8.4 Hz), 4.78-4.73 (m, 1H), 3.72 (s, 3H), 3.11-3.00 (dd, 1H, J = 13.5,
13
5.7 Hz), 2.99-2.89 (dd, 1H, J = 13.5, 7.5 Hz); C NMR (CD3OD, 75 MHz) δ 172.4,
165.6, 157.0, 146.9, 145.7, 145.0, 144.5, 143.9, 142.7, 138.0, 129.7, 127.8, 125.7,
121.1, 119.8, 118.7, 118.4, 117.0, 116.4, 116.1, 115,4, 112.3, 110.6, 96.9, 54.0, 51.8,

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36.7. HR-MS (ESI) m/z: calcd. for C27H22NO9 [M-H]-: 504.1300; found: 504.1311.
4.1.3.3. Methyl (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)
acryloyl)-L-alaninate (5c) Green oil, yield 64%; 1H NMR (CD3OD, 300 MHz) δ 7.77
(d, 1H, J = 15.9 Hz), 7.39 (s, 1H), 7.35 (d, 1H, J = 8.1 Hz), 7.32-7.25 (m, 2H), 6.87 (d,
1H, J = 8.1 Hz), 6.72 (d, 1H, J = 8.4 Hz), 6.68 (d, 1H, J = 15.9 Hz), 4.62-4.53 (q, 1H,
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J = 7.2 Hz), 3.75 (s, 3H), 1.46 (d, 3H, J = 7.2 Hz); C NMR (CD3OD, 75 MHz) δ
174.9, 169.2, 159.1, 148.0, 146.8, 145.4, 144.5, 140.7, 132.1, 125.9, 123.5, 120.2,
118.6, 118.4, 116.7, 113.4, 111.7, 99.5, 52.8, 49.7, 17.7. HR-MS (ESI) m/z: calcd. for
C21H18NO7 [M-H]-: 396.1089; found: 396.1103.
4.1.3.4. Methyl (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)
acryloyl)-L-phenylalaninate (5d) Green oil, yield 61%; 1H NMR (CD3OD, 300 MHz)
δ 7.73 (d, 1H, J = 15.9 Hz), 7.39 (s, 1H), 7.34 (d, 1H, J = 9.0 Hz), 7.30-7.17 (m, 7H),
6.87 (d, 1H, J = 8.1 Hz), 6.71 (d, 1H, J = 8.4 Hz), 6.65 (d, 1H, J = 15.9 Hz), 4.95-4.86
(m, 1H), 3.72 (s, 3H), 3.27-3.17 (dd, 1H, J = 13.8, 5.7 Hz), 3.12-3.01 (dd, 1H, J =
13.8, 8.4 Hz); 13C NMR (CD3OD, 75 MHz) δ 173.7, 169.3, 159.1, 148.0, 146.8, 145.3,
140.8, 138.2, 132.1, 130.2, 130.2, 129.5, 129.5, 127.9, 126.0, 123.5, 121.8, 120.2,
118.6, 118.3, 116.7, 113.4, 111.6, 99.5, 55.6, 52.7, 38.7. HR-MS (ESI) m/z: calcd. for
C27H22NO7 [M-H]-: 472.1402; found: 472.1414.
4.1.3.5. Methyl (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)
acryloyl)-L-tyrosinate (5e) Green oil, yield 58%; 1H NMR (CD3OD, 300 MHz) δ 7.73
(d, 1H, J = 16.2 Hz), 7.41-7.32 (m, 2H), 7.26 (s, 1H), 7.10-7.02 (d, 2H, J = 7.8 Hz),
6.92-6.84 (m, 2H), 6.74-6.64 (m, 4H), 4.69-4.58 (dd, 1H, J = 8.4, 6.0 Hz), 3.72 (s,
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3H), 3.16-3.07 (dd, 1H, J = 13.8, 6.0 Hz), 3.02-2.94 (dd, 1H, J = 13.8, 8.4 Hz); C
NMR (CD3OD, 75 MHz) δ 174.5, 169.2, 159.3, 155.7, 147.3, 146.6, 145.2, 143.9,
141.7, 130.2, 130.2, 129.2, 124.3, 123.0, 122.0, 121.6, 120.2, 119.8, 116.9, 115.8,
115.8, 114.2, 108.4, 99.8, 56.2, 52.0, 38.2. HR-MS (ESI) m/z: calcd. for C27H22NO8

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[M-H]-: 488.1351; found: 488.1353.
4.1.3.6. Methyl (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)
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acryloyl)-L-tryptophanate (5f) Green oil, yield 62%; H NMR (CD3OD, 300 MHz) δ
7.74 (d, 1H, J = 15.6 Hz), 7.56 (d, 1H, J = 7.5 Hz), 7.40-7.20 (m, 5H), 7.13-6.98 (m,
3H), 6.86 (d, 1H, J = 7.2 Hz), 6.70 (d, 1H, J = 6.0 Hz), 6.66 (d, 1H, J = 15.6 Hz),
4.95-4.89 (m, 1H), 3.69 (s, 3H), 3.43-3.35 (m, 1H), 3.29-3.20 (m, 1H); 13C NMR
(CD3OD, 75 MHz) δ 174.2, 169.4, 159.1, 148.2, 147.0, 145.9, 144.7, 140.8, 138.9,
138.1, 132.0, 128.8, 126.1, 124.4, 123.5, 122.5, 120.0, 119.9, 119.2, 118.6, 118.3,
117.3, 116.7, 113,4, 112.3, 111.8, 99.5, 55.2, 52.7, 28.8. HR-MS (ESI) m/z: calcd. for
C29H23N2O7 [M-H]-: 511.1511; found: 511.1508.
4.1.4. General procedure for syntheses of compounds 6a-6f
Compounds 5a-5f were dissolved in 3 mL of MeOH/H2O (4:1 v/v) and sodium
hydroxide (12 equiv mol) was added. The reaction mixture was heated to reflux under
nitrogen. After completing by TLC, methanol was removed under reduced pressure
and the syrup was diluted with H2O (10 mL). The mixture was acidified to pH = 2 by
adding 1N HCl solution in an ice-water bath, and then extracted with ethyl acetate
four times (15 mL × 4). Evaporating the solvent of the organic layer afforded the
crude products which were further purified by preparative HPLC (column: Agilent
zorbax, SB-C18, 5 ꢀm, 20 × 250 mm) using H2O containing 0.1% formic acid and
CH3CN as the eluents to yield the title compounds 6a-6f.
4.1.4.1. (S,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dihydroxyphenyl)
1
-7-hydroxybenzofuran-4-yl)acrylamido)propanoic acid (6a) Green oil, yield 56%; H
NMR (DMSO-d6, 300 MHz) δ 8.15 (br s, 4H), 7.54 (d, 1H, J = 15.9 Hz), 7.43 (s, 1H),
7.35 (s, 1H), 7.28 (d, 1H, J = 8.1 Hz), 7.23 (d, 1H, J = 8.4 Hz), 6.88 (d, 1H, J = 8.1
Hz), 6.79-6.72 (m, 2H), 6.65-6.60 (m, 2H), 6.50 (d, 1H, J = 8.1 Hz), 4.50 (dd, 1H, J =
8.4, 5.4 Hz), 3.00-2.91 (dd, 1H, J = 13.8, 5.4 Hz), 2.84-2.74 (dd, 1H, J = 13.8, 8.4 Hz);
13C NMR (DMSO-d6, 75 MHz) δ 173.3, 165.6, 156.9, 146.9, 145.7, 145.0, 143.9,

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143.8, 142.7, 137.8, 129.6, 128.3, 125.7, 121.1, 119.9, 119.2, 118.6, 117.0, 116.5,
116.1, 115.4, 112.4, 110.6, 99.0, 53.9, 36.7. HR-MS (ESI) m/z: calcd. for C26H22NO9
[M+H]+: 492.1289; found: 492.1296.
4.1.4.2. (R,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dihydroxyphenyl)
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-7-hydroxybenzofuran-4-yl)acrylamido)propanoic acid (6b) Green oil, yield 54%; H
NMR (DMSO-d6, 300 MHz) δ 8.21 (br s, 4H), 7.53 (d, 1H, J = 15.9 Hz), 7.43 (s, 1H),
7.35 (s, 1H), 7.23 (d, 1H, J = 8.1 Hz), 7.22 (d, 1H, J = 8.1 Hz), 6.87 (d, 1H, J = 8.4
Hz), 6.80-6.70 (m, 2H), 6.65-6.59 (m, 2H), 6.43 (d, 1H, J = 7.5 Hz), 4.50 (dd, 1H, J =
8.4, 5.1 Hz), 2.98-2.91 (dd, 1H, J = 13.8, 5.1 Hz), 2.81-2.73 (dd, 1H, J = 13.8, 8.4 Hz);
13C NMR (DMSO-d6, 75 MHz) δ 173.6, 165.4, 156.9, 146.9, 145.7, 144.9, 143.8,
143.8, 142.7, 137.6, 129.5, 128.5, 125.7, 121.0, 119.9, 119.3, 118.6, 116.9, 116.6,
116.1, 115.3, 112.4, 110.5, 99.0, 54.2, 36.8. HR-MS (ESI) m/z: calcd. for C26H22NO9
[M+H]+: 492.1289; found: 492.1293.
4.1.4.3. (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)acryloyl)
-L-alanine (6c) Green oil, yield 65%; 1H NMR (CD3OD, 300 MHz) δ 7.53 (d, 1H, J =
15.6 Hz), 7.44 (s, 1H), 7.36 (s, 1H), 7.32-7.20 (m, 2H), 6.88 (d, 1H, J = 7.8 Hz), 6.77
(d, 1H, J = 15.6 Hz), 6.75 (d, 1H, J = 6.6 Hz), 4.38 (q, 1H, J = 6.3 Hz), 1.35 (d, 3H, J
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= 6.6 Hz); C NMR (CD3OD, 75 MHz) δ 174.5, 165.5, 157.0, 147.0, 145.8, 143.9,
142.8, 137.8, 129.6, 125.8, 121.1, 119.3, 118.7, 117.0, 116.2, 112.4, 110.6, 99.0, 48.7,
17.8. HR-MS (ESI) m/z: calcd. for C20H16NO7 [M-H]-: 382.0932; found: 382.0941.
4.1.4.4. (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)acryloyl)
1
-L-phenylalanine (6d) Green oil, yield 57%; H NMR (DMSO-d6, 300 MHz) δ 8.20
(br s, 2H), 7.54 (d, 1H, J = 15.9 Hz), 7.42 (s, 1H), 7.35 (s, 1H), 7.30-7.20 (m, 7H),
6.88 (d, 1H, J = 7.8 Hz), 6.79-6.69 (m, 2H), 4.63 (m, 1H), 3.16-3.08 (m, 1H),
3.01-2.91 (m, 1H); 13C NMR (DMSO-d6, 75 MHz) δ 173.2, 165.5, 156.9, 146.9,
145.7, 143.8, 142.7, 137.73, 137.68, 129.6, 129.1, 129.1, 128.2, 128.2, 126.4, 125.6,
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calcd. for C26H20NO7 [M-H]-: 458.1245; found: 458.1252.
4.1.4.5. (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)acryloyl)
-L-tyrosine (6e) Green oil, yield 55%; 1H NMR (DMSO-d6, 300 MHz) δ 7.50 (d, 1H,
J = 16.2 Hz), 7.47 (s, 1H), 7.36 (s, 1H), 7.28 (d, 1H, J = 8.1 Hz), 7.20 (d, 1H, J = 8.1
Hz), 7.06-6.98 (d, 2H, J = 7.8 Hz), 6.87 (d, 1H, J = 8.1 Hz), 6.79 (d, 1H, J = 16.2 Hz),
6.74 (d, 1H, J = 8.1 Hz), 6.65-6.59 (d, 2H, J = 7.8 Hz), 4.66 (dd, 1H, J = 8.4, 4.8Hz),
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3.11-3.00 (dd, 1H, J = 13.8, 4.8 Hz), 2.90-2.80 (dd, 1H, J = 13.8, 8.4 Hz); C NMR
(DMSO-d6, 75 MHz) δ 174.3, 165.2, 156.9, 155.7, 147.0, 145.8, 143.8, 142.7, 137.3,
130.1, 130.1, 129.4, 128.4, 125.8, 121.0, 119.6, 118.6, 116.9, 116.2, 114.8, 114.8,
112.5, 112.5, 99.1, 54.8, 36.8. HR-MS (ESI) m/z: calcd. for C26H20NO8 [M-H]-:
474.1194; found: 474.1201.
4.1.4.6. (E)-(3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl)acryloyl)
1
-D-tryptophan (6f) Green oil, yield 48%; H NMR (DMSO-d6, 300 MHz) δ 10.85 (s,
1H), 8.17 (s, 1H), 7.59 (d, 1H, J = 6.0 Hz), 7.57 (d, 1H, J = 16.2 Hz), 7.44 (s, 1H),
7.3-7.21 (m, 4H), 7.18 (s, 1H), 7.10-6.95 (m, 2H), 6.89 (d, 1H, J = 8.1 Hz), 6.80 (d,
1H, J = 16.2 Hz), 6.75 (d, 1H, J = 9.3 Hz), 4.72 (dd, 1H, J = 7.8, 4.8 Hz), 3.69 (dd,
1H, J = 14.4, 4.8 Hz), 3.29-3.20 (dd, 1H, J = 14.4, 7.8 Hz); 13C NMR (DMSO-d6, 75
MHz) δ 173.7, 165.7, 157.0, 147.0, 145.8, 143.9, 142.8, 137.9, 136.2, 129.6, 127.4,
125.9, 123.6, 121.1, 121.0, 119.3, 118.7, 118.5, 118.4, 117.0, 116.2, 112.4, 111.5,
110.6, 110.0, 99.1, 53.2, 27.6. HR-MS (ESI) m/z: calcd. for C28H21N2O7 [M-H]-:
497.1354; found: 497.1360.
4.1.5. (2R)-3-(3,4-dimethoxyphenyl)-1-methoxy-1-oxopropan-2-yl (2E)-3-[2-(3 ,4-
dimethoxyphenyl)-7-methoxy-1-benzofuran-4-yl]prop-2-enoate (7)
Salvianolic acid C (206 mg, 0.419 mmol) was placed into a boiling flask and
dispersed in 15 mL of acetone containing anhydrous potassium carbonate (1.16 g,
8.374 mmol). To the suspension 714.5 ꢀL (7.542 mmol) of dimethyl sulfate was
added, and the reaction mixture was stirred for 18 h under reflux. After completing,

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acetone was removed by a rotary evaporator. The residue was suspended in saturated
NH4Cl resolution (15 mL) and then extracted with ethyl acetate (20 mL × 4). The
organic layer was washed with brine, dried with anhydrous Na2SO4 and concentrated
in vacuum. The obtained residue was further separated by silica gel chromatography
using chroloform/methanol as the eluent to give pure compound 7 as a yellow foam
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(230 mg, yield 95%). H NMR (CDCl3, 300 MHz) δ 7.96 (d, 1H, J = 15.9 Hz), 7.53
(dd, 1H, J = 8.4, 2.1 Hz), 7.39 (m, 2H), 7.17 (s, 1H), 6.97 (d, 1H, J = 8.4 Hz), 6.82 (m,
4H), 6.50 (d, 1H, J = 15.9 Hz), 5.41 (m, 1H), 4.09 (s, 3H), 4.03 (s, 3H), 3.96 (s, 3H),
3.88 (s, 3H), 3.86 (s, 3H), 3.78 (s, 3H), 3.21-2.80 (m, 2H); 13C NMR (CDCl3, 75 MHz)
δ 170.5, 166.7, 157.6, 150.1, 149.2, 148.8, 148.8, 146.9, 144.1, 143.7, 130.7, 128.4,
125.8, 122.7, 121.4, 121.4, 118.5, 114.9, 112.5, 111.3, 111.2, 108.2, 106.6, 99.2, 73.1,
56.2, 56.2, 56.0, 55.8, 55.8, 52.4, 37.2. HR-MS (ESI) m/z: calcd. for C32H33O10
[M+H]+: 577.2079; found: 577.2068.
4.1.6. (2E)-3-[2-(3,4-dimethoxyphenyl)-7-methoxy-1-benzofuran-4-yl]prop-2-enoic
acid (8)
To a solution of the intermediate 7 in THF 1 mL, MeOH 3 mL and H2O 1 mL
was added potassium hydroxide (112 mg, 1.997 mmol), the resulting solution was
heated at reflux under nitrogen atmosphere. After 12 h, the reaction solution was
cooled down to room temperature and concentrated in vacuo to remove the solvents.
After dissolving the residue with water, 1N HCl solution was added drop-wise in an
ice bath. The resulting solid was collected by filtration, washed with brine and dried.
The crude product was subjected to silica gel chromatography with
chloroform/methanol/formic acid (25:1:0.1 v/v/v) as the eluent to afford compound 8
as a yellow solid (126 mg, yield 88%). 1H NMR (CDCl3, 300 MHz) δ 8.03 (d, 1H, J =
15.6 Hz), 7.53 (d, 1H, J = 8.1 Hz), 7.43 (d, 1H, J = 9.3 Hz), 7.41 (s, 1H), 7.20 (s, 1H),
6.96 (d, 1H, J = 8.4 Hz), 6.82 (d, 1H, J = 8.4 Hz), 6.50 (d, 1H, J = 15.6 Hz), 4.09 (s,
3H), 4.02 (s, 3H), 3.95 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 168.0, 156.8, 149.9,
149.1, 146.1, 142.8, 141.5, 130.2, 125.1, 122.1, 119.5, 117.8, 117.3, 111.9, 108.5,

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107.1, 99.9, 56.0, 55.7, 55.6. HR-MS (ESI) m/z: calcd. for C20H17O6 [M-H]-:
353.1108; found: 353.1105.
4.1.7. General procedure for syntheses of compounds 9a-9f
To a solution of compound 8 in dry DMF/CH2Cl2 (3 mL, 2:5 v/v) was added
successively HoBt (1.2 equiv mol) and EDCI·HCl (1.2 equiv mol). The mixture was
stirred for 15 min in an ice-water bath. After cooling, derivatives 4a-4f (1.2 equiv mol)
and triethylamine (2.5 equiv mol) were added to the solution. The reaction mixture
was stirred at ambient temperature under nitrogen with TLC detection. After
completely disappearing, the solvent was evaporated in a vacuum. The residue was
dissolved in ethyl acetate and successively washed with 10% HCl, saturated NaHCO3,
water and saturated brine. The organic layer was combined, dried over anhydrous
Na2SO4 and concentrated in vacuo. The crude products were further chromatographed
on silica gel using hexane/ethyl acetate as the eluent to provide target products 9a-9f.
4.1.7.1. Methyl (S,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dimethoxyphenyl)
-7-methoxybenzofuran-4-yl)acrylamido)propanoate (9a) Yellow-green powder, yield
1
67%; H NMR (CD3OD, 300 MHz) δ 7.71 (d, 1H, J = 15.6 Hz), 7.46 (d, 1H, J = 8.7
Hz), 7.42 (s, 1H), 7.32 (d, 1H, J = 8.4 Hz), 7.30 (s, 1H), 7.01 (d, 1H, J = 7.2 Hz), 6.83
(d, 1H, J = 8.1 Hz), 6.75-6.64 (m, 3H), 6.56 (d, 1H, J = 8.1 Hz), 4.76-4.60 (dd, 1H, J
= 7.8, 6.0 Hz), 4.02 (s, 3H), 3.92 (s, 3H), 3.87 (s, 3H), 3.72 (s, 3H), 3.13-3.01 (dd, 1H,
J = 13.8, 6.0 Hz), 2.99-2.91 (dd, 1H, J = 13.8, 7.8 Hz); 13C NMR (CD3OD, 75 MHz)
δ 173.8, 169.0, 158.5, 151.5, 150.8, 147.8, 146.3, 145.4, 140.5, 131.4, 129.5, 126.2,
124.4, 122.7, 121.7, 121.6, 119.54, 119.49, 117.3, 116.4, 113.2, 109.8, 107.9, 100.3,
56.71, 56.67, 56.5, 55.8, 52.7, 38.2. HR-MS (ESI) m/z: calcd. for C30H28NO9 [M-H]-:
546.1770; found: 546.1777.
4.1.7.2. Methyl (R,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dimethoxyphenyl)
-7-methoxybenzofuran-4-yl)acrylamido)propanoate (9b) Yellow-green powder, yield
1
62%; H NMR (CDCl3, 300 MHz) δ 7.61 (d, 1H, J = 15.3 Hz), 7.45-7.29 (m, 2H),

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7.12 (d, 1H, J = 8.1 Hz), 6.93-6.58 (m, 6H), 6.28 (d, 1H, J = 15.3 Hz), 5.02-4.90 (dd,
1H, J = 5.4, 5.4 Hz), 3.97 (s, 3H), 3.91 (s, 3H), 3.74 (s, 3H), 3.49 (s, 3H), 3.24-3.11
(dd, 1H, J = 14.1, 5.4 Hz), 3.09-2.95 (dd, 1H, J = 14.1, 5.4 Hz); 13C NMR (CDCl3, 75
MHz) δ 172.5, 167.2, 157.3, 150.0, 149.3, 146.5, 144.4, 143.9, 140.1, 136.0, 130.9,
127.9, 124.1, 122.9, 121.2, 119.8, 118.6, 117.3, 116.3, 115.4, 111.4, 108.4, 106.6, 99.0,
56.3, 56.2, 56.1, 54.0, 52.6, 37.3. HR-MS (ESI) m/z: calcd. for C30H28NO9 [M-H]-:
546.1770; found: 546.1776.
4.1.7.3. Methyl (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)-
1
L-alaninate (9c) White solid powder, yield 73%; H NMR (CDCl3, 300 MHz) δ 7.89
(d, 1H, J = 15.6 Hz), 7.50 (dd, 1H, J = 8.4, 1.8 Hz), 7.40-7.34 (m, 2H), 7.17 (s, 1H),
6.95 (d, 1H, J = 8.4 Hz), 6.78 (d, 1H, J = 8.4 Hz), 6.46 (d, 1H, J = 15.6 Hz), 4.79 (q,
1H, J = 7.2 Hz), 4.06 (s, 3H), 4.01 (s, 3H), 3.94 (s, 3H), 3.79 (s, 3H), 1.50 (d, 3H, J =
7.2 Hz); 13C NMR (CDCl3, 75 MHz) δ 174.0, 165.9, 157.4, 150.2, 149.5, 146.5, 139.6,
135.0, 130.8, 124.2, 123.1, 120.4, 118.6, 118.5, 111.5, 108.5, 106.9, 99.4, 56.4, 56.3,
56.2, 52.7, 48.4, 18.9. HR-MS (ESI) m/z: calcd. for C24H26NO7 [M+H]+: 440.1704;
found: 440.1706.
4.1.7.4. Methyl (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)-
L-phenylalaninate (9d) Yellow-green powder, yield 60%; 1H NMR (CDCl3, 300 MHz)
δ 8.00 (d, 1H, J = 15.6 Hz), 7.61 (d, 1H, J = 8.4, 1.8 Hz), 7.57-7.28 (m, 7H), 7.23 (s,
1H), 7.05 (d, 1H, J = 8.4 Hz), 6.88 (d, 1H, J = 8.4 Hz), 6.53 (d, 1H, J = 15.6 Hz), 5.19
(m, 1H), 4.17 (s, 3H), 4.11 (s, 3H), 4.04 (s, 3H), 3.88 (s, 3H), 3.42-3.34 (dd, 1H, J =
13.8, 5.7 Hz), 3.34-3.27 (dd, 1H, J = 13.8, 5.7 Hz); 13C NMR (CDCl3, 75 MHz) δ
172.4, 166.0, 157.5, 150.1, 149.4, 146.5, 143.9, 139.7, 136.1, 130.9, 129.5, 128.7,
127.3, 124.2, 123.1, 120.3, 118.6, 118.3, 111.5, 108.5, 106.8, 99.4, 56.4, 56.3, 56.2,
53.6, 52.5, 38.1. HR-MS (ESI) m/z: calcd. for C30H30NO7 [M+H]+: 516.2017; found:
516.2014.
4.1.7.5. Methyl (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)

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-L-tyrosinate (9e) Yellow-green solid, yield 65%; H NMR (CDCl3, 500 MHz) δ 7.87
(d, 1H, J = 16.0 Hz), 7.48-7.45 (dd, 1H, J = 8.5, 2.0 Hz), 7.37 (d, 1H, J = 1.5 Hz),
7.31 (d, 1H, J = 8.5 Hz), 7.12 (s, 1H), 7.00-6.97 (m, 2H), 6.92 (d, 1H, J = 8.0 Hz),
6.78-6.73 (m, 3H), 6.41 (d, 1H, J = 16.0 Hz), 5.07-5.01 (m, 1H), 4.05 (s, 3H), 3.98 (s,
3H), 3.92 (s, 3H), 3.20-3.15 (dd, 1H, J = 14.0, 6.0 Hz), 3.12-3.06 (dd, 1H, J = 14.0,
6.0 Hz); 13C NMR (CDCl3, 125 MHz) δ 172.6, 166.3, 157.5, 155.4, 150.3, 149.5,
146.6, 143.9, 139.8, 135.4, 130.6, 127.7, 125.0, 124.2, 123.1, 120.3, 118.7, 115.7,
115.7, 111.6, 108.7, 106.9, 99.4, 56.4, 56.3, 56.2, 53.7, 52.5, 37.4. HR-MS (ESI) m/z:
calcd. for C30H30NO8 [M+H]+: 532.1966; found: 532.1953.
4.1.7.6. Methyl (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)
1
-D-tryptophanate (9f) Yellow-green solid, yield 57%; H NMR (CDCl3, 300 MHz) δ
8.17 (s, 1H), 7.88 (d, 1H, J = 15.6 Hz), 7.54 (d, 1H, J = 7.8 Hz), 7.49 (d, 1H, J = 8.4
Hz), 7.39-7.35 (m, 2H), 7.22-7.10 (m, 3H), 7.03 (s,1H), 7.00-6.87 (m, 2H), 6.77 (d,
1H, J = 8.1 Hz), 6.38 (d, 1H, J = 15.6 Hz), 5.20-5.11 (m, 1H), 4.06 (s, 3H), 4.00 (s,
3H), 3.94 (s, 3H), 3.74 (s, 3H), 3.51-3.35 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ
172.7, 166.0, 157.4, 150.2, 149.5, 146.5, 139.6, 136.3, 134.9, 130.8, 128.0, 125.0,
124.3, 123.1, 123.0, 122.4, 120.4, 119.9, 119.0, 118.7, 118.6, 111.5, 111.4, 108.5,
106.8, 99.5, 56.4, 56.3, 56.2, 53.4, 52.5, 28.0. HR-MS (ESI) m/z: calcd. for
C32H31N2O7 [M+H]+: 555.2126; found: 555.2110.
4.1.8. General procedure for syntheses of compounds 10a-10f
To a solution of compounds 9a-9f (1 equiv mol) in THF/MeOH/H2O (1:3:1 v/v/v)
was added potassium hydroxide (8 equiv mol for 9a and 9b) and sodium hydroxide (5
equiv mol for 9c-9f). The resulting solution was heated to reflux under nitrogen and
monitored by TLC. After completion of the reaction, the solvents were removed in a
vacuum and the residue was diluted with 1N HCl solution (15 mL). The aqueous layer
was extracted with ethyl acetate (20 mL × 4). Then the combined organic layer was
dried over anhydrous Na2SO4 and concentrated to afford the crude products. The title
derivatives 10a-10f were obtained after further separation by preparative HPLC

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using H2O containing 0.1% formic acid and CH3CN as the eluents.
4.1.8.1. (S,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dimethoxyphenyl)
-7-methoxybenzofuran-4-yl)acrylamido)propanoic acid (10a) Yellow-green powder,
yield 55%; 1H NMR (DMSO-d6, 300 MHz) δ 7.68 (s, 1H), 7.62 (d, 1H, J = 15.9 Hz),
7.54-7.39 (m, 3H), 7.11 (d, 1H, J = 8.1 Hz), 6.98 (d, 1H, J = 8.4 Hz), 6.82 (d, 1H, J =
15.9 Hz), 6.67-6.58 (m, 2H), 6.49 (d, 1H, J = 7.8 Hz), 4.58-4.46 (dd, 1H, J = 8.4, 4.8
Hz), 4.01 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H), 3.00-2.92 (dd, 1H, J = 14.1, 4.8 Hz),
13
2.83-2.75 (dd, 1H, J = 14.1, 8.4 Hz); C NMR (DMSO-d6, 75 MHz) δ 173.4, 165.2,
156.5, 149.9, 149.2, 145.7, 144.9, 143.9, 143.0, 136.8, 129.5, 128.4, 124.6, 122.2,
120.6, 120.3, 119.9, 117.9, 116.6, 115.3, 112.1, 108.4, 107.2, 100.0, 56.0, 55.8, 55.7,
54.2, 36.7. HR-MS (ESI) m/z: calcd. for C29H26NO9 [M-H]-: 532.1613; found:
532.1620.
4.1.8.2. (R,E)-3-(3,4-dihydroxyphenyl)-2-(3-(2-(3,4-dimethoxyphenyl)
-7-methoxybenzofuran-4-yl)acrylamido)propanoic acid (10b) Yellow-green solid,
yield 51%; 1H NMR (DMSO-d6, 300 MHz) δ 7.65 (d, 1H, J = 15.9 Hz), 7.52-7.37 (m,
4H), 7.10 (d, 1H, J = 7.8 Hz), 7.00-6.70 (m, 3H), 6.65 (d, 1H, J = 8.4 Hz), 6.58 (d, 1H,
J = 15.9 Hz), 4.54-4.33 (m, 1H), 4.00 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H), 2.96-2.89 (m,
1H), 2.80-2.71 (m, 1H); 13C NMR (DMSO-d6, 75 MHz) δ 173.5, 165.6, 156.5, 155.6,
149.9, 145.6, 144.9, 143.7, 136.6, 128.7, 125.5, 124.6, 122.5, 122.2, 120.7, 119.9,
117.9, 116.6, 115.3, 112.1, 108.5, 107.2, 99.8, 56.0, 55.8, 55.7, 54.4, 36.8. HR-MS
(ESI) m/z: calcd. for C29H26NO9 [M-H]-: 532.1613; found: 532.1626.
4.1.8.3. (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)
1
-L-alanine (10c) White solid powder, yield 62%; H NMR (DMSO-d6, 300 MHz) δ
7.75 (s, 1H), 7.65 (d, 1H, J = 15.9 Hz), 7.56-7.49 (m, 2H), 7.41 (d, 1H, J = 8.4 Hz),
7.10 (d, 1H, J = 9.0 Hz), 6.98 (d, 1H, J = 7.8 Hz), 6. 87 (d, 1H, J = 15.9 Hz), 4.39 (q,
1H, J = 7.2 Hz), 4.01 (s, 3H), 3.90 (s, 3H), 3.83 (s, 3H), 1.36 (d, 3H, J = 7.2 Hz); 13C
NMR (DMSO-d6, 75 MHz) δ 174.2, 165.1, 156.5, 149.9, 149.2, 145.7, 143.0, 136.8,

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129.4, 124.7, 122.2, 120.6, 120.3, 117.8, 112.1, 108.5, 107.2, 100.0, 56.0, 55.8, 55.6,
47.6, 17.5. HR-MS (ESI) m/z: calcd. for C23H22NO7 [M-H]-: 424.1402; found:
424.1407.
4.1.8.4. (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)
1
-L-phenylalanine (10d) Yellow-green powder, yield 45%; H NMR (DMSO-d6, 300
MHz) δ 7.71 (s, 1H), 7.61 (d, 1H, J = 15.9 Hz), 7.54-7.45 (t, 2H, J = 8.4 Hz), 7.40 (d,
1H, J = 8.4 Hz), 7.29-7.15 (m, 5H), 7.10 (d, 1H, J = 8.1 Hz), 6.97 (d, 1H, J = 8.7 Hz),
6.85 (d, 1H, J = 15.9 Hz), 4.65-4.54 (dd, 1H, J = 8.1, 5.7 Hz), 4.00 (s, 3H), 3.90 (s,
3H), 3.83 (s, 3H), 3.17-3.12 (dd, 1H, J = 12.9, 5.7 Hz), 3.20-2.94 (dd, 1H, J = 12.9,
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8.1 Hz); C NMR (DMSO-d6, 75 MHz) δ 173.4, 165.1, 156.5, 149.9, 145.6, 143.0,
138.0, 136.6, 129.4, 129.2, 129.2, 129.1, 128.1, 128.1, 126.2, 124.6, 122.2, 120.6,
120.3, 117.8, 112.1, 108.4, 107.1, 100.0, 56.0, 55.8, 55.6, 54.1, 37.2. HR-MS (ESI)
m/z: calcd. for C29H26NO7 [M-H]-: 500.1715; found: 500.1720.
4.1.8.5. (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)
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-L-tyrosine (10e) Yellow-green solid, yield 54%; H NMR (DMSO-d6, 300 MHz) δ
7.71 (s, 1H), 7.61 (d, 1H, J = 15.9 Hz), 7.53-7.39 (m, 3H), 7.10 (d, 1H, J = 8.1 Hz),
7.07-7.01 (m, 2H), 6.97 (d, 1H, J = 8.4 Hz), 6.85 (d, 1H, J = 15.9 Hz), 6.68-6.62 (d,
2H, J = 7.8 Hz), 4.56-4.48 (dd, 1H, J = 7.5, 4.5 Hz), 4.00 (s, 3H), 3.89 (s, 3H), 3.83 (s,
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3H), 3.09-2.99 (dd, 1H, J = 13.8, 4.5 Hz), 2.90-2.79 (dd, 1H, J = 13.8, 7.5 Hz); C
NMR (DMSO-d6, 75 MHz) δ 173.6, 165.1, 156.5, 155.8, 149.9, 149.2, 145.6, 143.0,
136.6, 130.1, 130.1, 129.4, 128.0, 124.6, 122.2, 120.7, 120.4, 117.9, 114.9, 114.9,
112.1, 108.4, 107.2, 100.0, 56.0, 55.8, 55.6, 54.5, 36.6. HR-MS (ESI) m/z: calcd. for
C29H26NO8 [M-H]-: 516.1664; found: 516.1666.
4.1.8.6. (E)-(3-(2-(3,4-dimethoxyphenyl)-7-methoxybenzofuran-4-yl)acryloyl)
-D-tryptophan (10f) Yellow-green solid, yield 57%; 1H NMR (DMSO-d6, 300 MHz) δ
10.82 (s, 1H), 7.71 (s, 1H), 7.63 (d, 1H, J = 16.2 Hz), 7.59-7.43 (m, 3H), 7.40 (d, 1H,
J = 8.4 Hz), 7.32 (d, 1H, J = 7.8 Hz), 7.17 (s, 1H), 7.10 (d, 1H, J = 8.4 Hz), 7.03 (d,

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1H, J = 7.8 Hz), 7.00-6.92 (m, 2H), 6.87 (d, 1H, J = 16.2 Hz), 4.73-4.55 (dd, 1H, J =
7.8, 4.5 Hz), 4.00 (s, 3H), 3.89 (s, 3H), 3.82 (s, 3H), 3.37-3.24 (dd, 1H, J = 14.7, 4.5
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Hz), 3.17-3.03 (dd, 1H, J = 14.7, 7.8 Hz); C NMR (DMSO-d6, 75 MHz) δ 173.9,
165.2, 156.5, 149.9, 149.1, 145.5, 143.0, 136.5, 136.1, 129.4, 127.5, 124.6, 123.5,
122.2, 120.9, 120.8, 120.4, 118.4, 118.2, 117.8, 112.1, 111.3, 110.3, 108.4, 107.1,
100.0, 55.9, 55.8, 55.1, 53.6, 27.7. HR-MS (ESI) m/z: calcd. for C31H27N2O7 [M-H]-:
539.1824; found: 539.1832.
4.2. Inhibition assay of XO activity
XO and xanthine were obtained from Sigma-Aldrich Chemical Co. (St. Louis
Missouri, USA), and allopurinol was purchased from Toronto (North York, Canada).
The XO inhibitory activity was determined based on the developed method [18].
Briefly, phosphate buffer (75 mM), XO and xanthine solution (the concentration of
stock solutions were 0.08 U/mL and 0.48 mmol/L respectively) were prepared before
using and stored on the ice-bath. The test compounds were diluted with PB at varying
concentrations (0-100 ꢀM). The blank control was prepared without test compounds
to determine the maximum uric acid formation of the reaction system, and allopurinol
was used as the positive control in this assay. Every concentration was set four
replicates. The reaction was performed in 96-well microplates with each containing
200 ꢀL of the final reaction mixture. 100 ꢀL of different test compound
concentrations and 50 ꢀL of 0.08 U/mL XO were equilibrated for 3 min, and then 50
ꢀL of 0.48 mmol/L xanthine solution was added to initiate the reaction. The
absorbance was read every 15 s for 7 min using a microplate spectrophotometer at
295 nm. Statistical analyses were dealt with Excel. The inhibition ratio (%) could be
calculated according to the change of the blank group and the sample group following
the formula: Inhibition ratio (%) = [(dA/dt)blank - (dA/dt)sample]/(dA/dt)blank × 100%.
Wherein, (dA/dt)blank and (dA/dt)sample represented the reaction rate of the blank and
samples, respectively. The IC50 value was calculated using GraphPad Prism 6.0.2 to
evaluate the XO inhibitory activity of the synthesized derivatives.