Antioxidant activity and inhibition of α-glucosidase by hydroxyl-functionalized 2-arylbenzo[b]furans

Article abstract of DOI:10.1016/j.ejmech.2015.02.024

This study synthesized a series of hydroxyl-functionalized 2-arylbenzo[b]furans based on the structure of tournefolic acid A and evaluated them for antioxidant and a-glucosidase inhibitory activities. Compounds 5a, 5e, and 5n showed remarkable inhibition

Full text of DOI:10.1016/j.ejmech.2015.02.024

Accepted Manuscript
Antioxidant Activity and Inhibition of ?-Glucosidase by Hydroxyl-functionalized 2-
Arylbenzo[b]furans
Jung-Feng Hsieh, Wei-Jen Lin, Kai-Fa Huang, Jiahn-Haur Liao, Ming-Jaw Don,
Chien-Chang Shen, Young-Ji Shiao, Wen-Tai Li
PII:
S0223-5234(15)00120-8
10.1016/j.ejmech.2015.02.024
EJMECH 7706
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 October 2014
Revised Date: 5 February 2015
Accepted Date: 15 February 2015
Please cite this article as: J.-F. Hsieh, W.-J. Lin, K.-F. Huang, J.-H. Liao, M.-J. Don, C.-C. Shen, Y.-J.
Shiao, W.-T. Li, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.024.
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Graphical abstract

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Highlights
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A series of new hydroxyl-functionalized 2-arylbenzo[b]furans has synthesized.
Antioxidant and α-glucosidase inhibition activities.
Inhibition kinetics of new compounds were determined.
Molecular docking study predicted the binding of compounds to α-glucosidase.

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Antioxidant Activity and Inhibition of α-Glucosidase by
Hydroxyl-functionalized 2-Arylbenzo[b]furans
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Jung-Feng Hsieh , Wei-Jen Lin , Kai-Fa Huang , Jiahn-Haur Liao , Ming-Jaw Don ,
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Chien-Chang Shen , Young-Ji Shiao , Wen-Tai Li *
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Department of Food Science, Fu Jen Catholic University
Taipei 24205, Taiwan
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National Research Institute of Chinese Medicine, Ministry of Health and Welfare
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Taipei 11221 Taiwan
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Institute of Biological Chemistry, Academia Sinica,
Taipei 11529, Taiwan
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* Correspondence to:
Wen-Tai Li, Ph.D.
National Research Institute of Chinese Medicine, Ministry of Health and Welfare
155-1, Sec. 2, Linong St., Beitou Distrct,
Taipei 11221, Taiwan, ROC
Tel: +886 2 2820 1999 ext. 8261;
Fax: +886 2 2825 0743;
E-mail: wtli@nricm.edu.tw
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Abstract
This study synthesized a series of hydroxyl-functionalized 2-arylbenzo[b]furans based
on the structure of tournefolic acid A and evaluated them for antioxidant and α-glucosidase
inhibitory activities. Compounds 5a, 5e, and 5n showed remarkable inhibition of
α-glucosidase (IC values of 1.9 to 3.0 µM), and they appear to be even more potent than
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quercetin. A kinetic binding study indicated that compounds 5a and 5n used a mechanism
of mixed-competition to inhibit α-glucosidase. This study also revealed that compounds 5a
and 5n bind to either the α-glucosidase or α-glucosidase-4-NPGP complex. Using the
crystal structure of the Saccharomyces cerevisiae α-glucosidase, the molecular docking
study has predicted the binding of compounds 5a and 5n to the active site of α-glucosidase
through both hydrophobic and hydrogen interactions. A DPPH radical scavenging assay
further showed that most hydroxyl-functionalized 2-arylbenzo[b]furans possess antioxidant
activity. The exception was compound 5p, which has only one hydroxyl group on the
2-phenyl ring of 2-arylbenzo[b]furan. Our results indicate that hydroxyl-functionalized
2-arylbenzo[b]furans possess both antidiabetic as well as antioxidant properties.
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1. Introduction
Diabetes mellitus is a metabolic disease characterized by hyperglycemia, an abnormal
postprandial increase of blood glucose [1,2]. α-Glucosidases are membrane-bound enzymes
that help to catalyze the reactions associated with carbohydrate digestion. These enzymes
are also required for the cleavage of the α-glycosidic linkage connecting two glucoses or
glycoconjugates, the reaction of which leads to the release of glucose [3]. Therefore, the
inhibition of α-glucosidases can cause the suppression of carbohydrate ingestion [4,5].
Indeed, for two decades, α-glucosidase inhibitors have been used to treat diabetic patients
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by lowering the blood glucose levels [6,7]. In addition, α-glucosidase inhibitors have the
potential to treat a broad-spectrum of viruses, cancers, and other degenerative diseases,
such as nojirimycin and castanospermine [8-11].
Oxidative damage and the increased production of free radicals have been implicated
in diabetic complications [12]. Therefore, considerable efforts have been made to develop
an anti-diabetic drug that possesses both hypoglycemic and antioxidant properties [13,14].
Catechin and quercetin are polyphenolic compounds found in a variety of plant-based foods
and beverages [15,16]. Both of these compounds have excellent antioxidant capacity and
are lead compounds in the design of anti-diabetic drugs. The antioxidant properties of
catechin and quercetin are due to phenolic structures, and it was found that the electron
donating effect of the hydroxyl group is essential [17]. Furthermore, the relatively planar
structures of polyphenols have a higher antioxidant capacity. For instance, the relatively
planar conformation of quercetin allows for the conjugated π-system of the AC-ring to
interact efficiently with the B-ring, which gives quercetin an antioxidant capacity that is
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higher than that of its nonplanar derivatives [18]. In addition, a number of polyphenols,
such as quercetin and epicatechin, have been found to possess both antioxidant properties
and inhibit α-glucosidase [19]. The relatively planar structures of catechin derivatives have
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more pronounced α-glucosidase inhibitory activity than catechin itself [20]. Therefore,
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planar polyphenols likely have stronger antioxidant and α-glucosidase inhibition activity
than non-planar polyphenols, suggesting that planar phenolic structures can improve the
therapeutic efficacy of antidiabetic drugs.
Tournefolic acid A, which is characterized by a planar phenolic structure, has been
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reported to inhibit Cu -induced low-density-lipoprotein (LDL) peroxidation, but it has
never been reported to inhibit α-glucosidase (Fig. 1) [21]. The planar scaffold of
tournefolic acid A, hydroxyl-functionalized 2-arylbenzo[b]furan conserves antioxidant
activity and may also possess the α-glucosidase inhibition potential. Therefore, we used
tournefolic acid A to develop novel antidiabetic agents that contain a planar scaffold of
hydroxyl-functionalized 2-arylbenzo[b]furan.
In this study, we demonstrate the efficient synthesis of hydroxyl-functionalized
2-arylbenzo[b]furan derivatives and report on the antioxidant and α-glucosidase inhibitory
effects of these derivatives. We also describe the potential docking model and mechanism
underlying enzymatic inhibition by hydroxyl-functionalized 2-arylbenzo[b]furans.
2. Results and Discussion
2.1. Synthesis
The synthetic strategies for new antioxidants and α-glucosidase inhibitors with the
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structural scaffold of 2-arylbenzo[b]furan are outlined in Schemes 1, 2 and 3. One-pot
palladium-catalyzed coupling of 2-iodophenol with alkynes was utilized to efficiently
construct the 2-arylbenzo[b]furan core structure (Scheme 1). First, phenylacetylene 2 was
synthesized via a two-step reaction, as follows. Substituted benzaldehyde was treated with
carbon tetrabromide to yield 1,1-dibromo-1-alkene 1. This interim compound was then
debrominated using n-butyl lithium, which generated phenylacetylene 2, as shown in
Scheme 1 [22]. Subsequently, one-pot palladium-catalyzed coupled phenylacetylene 2 with
2-iodo-6-methoxyphenol 3 using palladium catalysis, yielded 2-arylbenzo[b]furan 4a [23].
To synthesize hydroxyl-functionalized 2-arylbenzo[b]furan 5a, deprotection of three
methoxy groups on 2-arylbenzo[b]furan 4a was performed under strong acid conditions
using boron tribromide. However, attempts to carry out one-pot palladium-catalyzed
coupled reaction to obtain some halo-substituted 2-arylbenzo[b]furans were unsuccessful;
therefore, a four-step procedure involving the Wittig reaction was employed to synthesize
those compounds. Specifically, 2-arylbenzo[b]furan 4b was prepared using the Wittig
reaction to convert substituted phosphonium ylide 7 and o-vanillin into stilbene 8. This was
followed by cyclization in a basic iodine solution, as shown in Scheme 2. Boron tribromide
was used to deprotect the methoxy-substituted 2-arylbenzo[b]furan 4b in a similar fashion,
and compound 4b was then converted to the desired hydroxyl-functionalized
2-arylbenzo[b]furan 5b. Bromo substituted 2-arylbenzo[b]furan 4b was coupled with ethyl
acrylate though a palladium-catalyzed Heck coupling reaction to produce (E)-ethyl acrylate
substituted 2-arylbenzo[b]furan 9a. The methoxy groups were removed from compound 9a
using the same boron tribromide treatment procedure to obtain (E)-ethyl acrylate
substituted hydroxyl-functionalized 2-arylbenzo[b]furan 5p.
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Scheme 1. Synthesis of compound 5a from palladium-catalyzed coupled phenylacetylene 2
with 2-iodo-6-methoxyphenol 3.
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Scheme 2. Synthesis of compound 5b via Wittig reaction and cyclization reactions.
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Scheme 3. Synthesis of compound 5p via palladium-catalyzed Heck coupling reaction from
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bromo substituted 2-arylbenzo[b]furan 4b.
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2.2. Inhibition of α-glucosidase
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All of the synthesized 2-arylbenzo[b]furans (Table 1) were evaluated for α-glucosidase
inhibition activity in accordance with standard procedures [24]. For this analysis, quercetin
and resveratrol were used for comparison purposes. The IC values, indicating the strength
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of α-glucosidase inhibition by 2-arylbenzo[b]furans, are summarized in Table 2.
Compounds 5a, 5e, and 5n were shown to be potent α-glucosidase inhibitors with IC50
values of 1.9-3.0 ꢀM. This is 2- to 3-times more potent than quercetin (IC = 6.6 ꢀM), a
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known α-glucosidase inhibitor. Indeed, most of the hydroxyl-functionalized
2-arylbenzo[b]furans (except 5g, 5m, and 5p) presented with potent inhibitory activity with
IC values below 10 ꢀM. Nevertheless, hydroxyl-functionalized 2-arylbenzo[b]furans 5l
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and 5m, with an (E)-ethyl acrylate substitution, only showed modest activity with IC50
values of 8.9 and 23.8 ꢀM, respectively. Compound 5p, which has only one hydroxyl group
on the 2-phenyl ring of 2-arylbenzo[b]furan, presented with potency similar to that of the
reference inhibitor resveratrol. Moreover, compounds 5b, 5c, 5f, 5h, and 5k, which have a
catechol ring on the 2-position of 2-arylbenzo[b]furan, showed similar levels of inhibitory
activity. In a comparison of compounds 5a and 5b, the bromo substituent on the catechol
ring of 2-arylbenzo[b]furan was found to confer an increase in inhibitory activity on par
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with compounds 5e and 5i. However, compounds 5f and 5k, which possess a bromo
substituent on the 4 and 5-position of 2-arylbenzo[b]furan, presented with potency similar
to that of compound 5b. Finally, the inhibitory activity of the pyrogallol ring on the
2-position of benzo[b]furan 5e is superior to that of the catechol 5k and 4-hydroxyphenyl
5p groups. The relative inhibitory strengths of compounds considered by this study were as
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follows: pyrogallol ＞ catechol ＞ 4-hydroxyphenyl. The methoxy-substituted
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2-arylbenzo[b]furans 4a, 4b, 4c, and 9a were inactive at the highest tested concentration (>
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2.3. Evaluation of antioxidant activities
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The antioxidant activity of synthesized 2-arylbenzo[b]furans was evaluated using
DPPH radical scavenging assays [25]. This widely-used method determines antioxidant
activity by measuring the hydrogen donating ability of the compound being studied. The
IC50 values are displayed in Table 3. Methoxy-substituted 2-arylbenzo[b]furans (i.e.
compounds 4a, 4b, 4c, and 9a) did not reveal any DPPH radical scavenging activity (IC50
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＞100 µM). However, most of the hydroxyl-functionalized 2-arylbenzo[b]furans (i.e.
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compounds 5a-5o, but not 5p) demonstrated radical scavenging activity in the micromolar
range, and a number of these were also potent in the low micromolar range. A comparison
of compounds with the pyrogallol ring 5e, catechol ring 5k, and 4-hydroxyphenyl ring 5p
on the 2-position of 2-arylbenzo[b]furan revealed that these compounds possess the same
order of radical scavenging activity as their α-glucosidase inhibition activity, which is
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pyrogallol ＞ catechol ＞ 4-hydroxyphenyl. In addition, compounds 5e, 5i, and 5j, which
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contain a pyrogallol ring on the 2-position of 2-arylbenzo[b]furan, demonstrated
comparable DPPH free radical scavenging activities; however, they were 2-fold less potent
than quercetin. Finally, the free radical scavenging ability of (E)-ethyl acrylate substituents
5l and 5m was slightly better than that of bromo substituents 5c, 5f, and 5k.
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2.4. Mode of α-glucosidase inhibition by hydroxyl-functionalized 2-arylbenzo[b]furans
Inhibition kinetics of hydroxyl-functionalized 2-arylbenzo[b]furans were determined
by conducting a Lineweaver-Burk plot analysis on compounds 5a and 5n, as shown in
Figure 2 [24]. In this figure, the concentrations of 1/(4-NPGP) are displayed on the X-axis,
and 1/V values obtained from the Lineweaver-Burk plot are shown along the Y-axis. The
plots did not intersect either the X- or Y-axis, suggesting that both 5a and 5n are
mixed-type mode inhibitors with respect to 4-NPGP for α-glucosidase. We also examined
Dixon plots of how compounds 5a and 5n affect α-glucosidase. As shown in Figure 3, these
plots further confirm that compounds 5a and 5n are mixed-type α-glucosidase inhibitors.
The Ki values of 5a and 5n were 4.21 ± 0.03 ꢀM and 3.19 ± 0.1 ꢀM, respectively, while the
Ki′ values of these compounds were 13.66 ± 1.38 ꢀM and 11.81 ± 1.08 ꢀM, respectively.
Ki is the equilibrium constant for the inhibitor binding to α-glucosidase, and Ki′ is the
equilibrium constant for the inhibitor binding to the α-glucosidase-4-NPGP complex.
Previous studies have reported that in a reversible mixed-competitive inhibition reaction,
the Ki values are usually smaller than the Ki′ values, and the results from this current study
are in strong agreement with those findings. This indicates that the inhibitor-enzyme
binding affinity exceeds the binding affinity of the inhibitor-enzyme-substrate complex and
compounds 5a and 5n are mixed-competitive inhibitors of α-glucosidase. The binding sites
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and mechanism underlying inhibition have yet to be determined; however, the result of
mixed-competitive inhibition against α-glucosidase suggests that compounds 5a and 5n
may bind to either α-glucosidase or the α-glucosidase-4-NPGP complex.
2.5. Molecular modeling
The docking experiments were performed base on the maltose binding model of S.
cerevisiae α-glucosidase, as shown in Figure 4. [26] There are three catalytic acidic
residues in the active site of α-glucosidase and they are Asp 215, Glu 277, and Asp 352.
Both binding models of compounds 5a and 5n showed that Asp 215 is involved in the
interactions between the compound and enzyme. For compound 5a, there are two residues
(Arg 213 and Asp 215) formed two hydrogen bonds with the compound. One catalytic
acidic residue, Asp 215, is involved in the binding of compound 5a. In a competitive
inhibitor maltose binding model, Arg 213 also formed hydrogen bond with maltose.
Compound 5a and maltose may show similar binding properties with α-glucosidase. For
compound 5n, three residues (Asp 69, Asp 215, and Arg 442) formed three hydrogen bonds
with the compound. In addition to the catalytic acidic residue (Asp 215), Asp 69 and Arg
442 were shown to interact with maltose [26]. Compound 5n may occupy the glucose
binding site of α-glucosidase through hydrogen bonding with these three residues. The
moiety -CH=CHCO Et of 5n is extend into the pocket formed by Gln 22, Trp 58, Phe 301,
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3. Conclusions
An ideal anti-diabetic drug should possess both hypoglycemic and antioxidant
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properties and be free from adverse side effects. This study prepared a series of
hydroxyl-functionalized 2-arylbenzo[b]furan compounds from the core structure of
tournefolic acid A using one-pot palladium-catalyzed coupling methods. The synthesized
2-arylbenzo[b]furans were evaluated for α-glucosidase inhibition and antioxidant activity.
A DPPH radical scavenging assay revealed that most of these compounds possess
antioxidant properties. Some of the hydroxyl-functionalized 2-arylbenzo[b]furans also
showed remarkable inhibitory activity against α-glucosidase with potency exceeding that of
quercetin. Further investigation of binding kinetics indicated that the mechanism of
α-glucosidase inhibition by compounds 5a and 5n was mixed-competitive. This suggests
that the hydroxyl-functionalized 2-arylbenzo[b]furans may bind to either the α-glucosidase
or α-glucosidase-4-NPGP complex. Furthermore the docking study has predicted that
compounds 5a and 5n bind to the active site of the Saccharomyces cerevisiae α-glucosidase
through both hydrophobic and hydrogen interactions. Compound 5a and maltose may have
binding properties that are similar to α-glucosidase, and compound 5n may occupy the
glucose binding site of α-glucosidase through hydrogen bonding with the three amino acid
residues of Asp 69, Asp 215, and Arg 442. Taken together, our results suggest that
hydroxyl-functionalized 2-arylbenzo[b]furans 5a and 5n are promising candidates for the
further development of diabetes treatments.
4. Experimental section
4.4. Chemistry synthesis
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All reactions were conducted in dried glassware under an oven at 120 C overnight
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and cooled in a desiccator. All reagents were used as received from commercial suppliers
unless otherwise stated. Dichloromethane (DCM) and N,N’-dimethylformamide (DMF)
were dried over calcium hydride for 48 h prior to distillation. Tetrahydrofuran (THF) was
distilled from sodium/benzophenone ketyl under nitrogen. The proton NMR spectra were
obtained on Bruker Avance 400 (400 MHz), Varian Unity Inova 500 (500 MHz) and
Varian VNMRS600 (600 MHz) spectrometers. All NMR chemical shifts were reported as δ
values in parts per million (ppm), and coupling constants (J) were given in hertz (Hz). The
splitting pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; br,
broad; m, unresolved multiplet due to the field strength of the instrument; dd, doublet of
doublet; dt, doublet of triplet; and ddd, doublet of doublet of doublet. Melting points were
measured on a Yanaco MP-S3 micro melting point apparatus and are uncorrected. Fourier
transform infrared spectra were collected with an Avatar 320 spectrometer. Mass spectra
were carried out on ThermoQuest Finnigan and Microsaic 4000MiD mass spectrometers.
Purification was performed using preparative separations in flash column chromatography
(Merck silica gel 60, particle size of 230-400 mesh). Analytical TLC was carried out on
precoated plates (Merck silica gel 60, F254). The compounds analyzed on the TLC plates
were visualized using a UV light, I vapor, or basic aqueous potassium permanganate
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(KMnO ) with heating.
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4.4.1. General procedure for synthesis of 2-(3,4-dimethoxyphenyl) -7-methoxybenzofuran
(4a) via palladium-catalyzed coupled reaction
A solution of 4-ethynyl-1,2-dimethoxybenzene 2 (105 mg, 0.65 mmol),
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2-iodo-6-methoxyphenol 3 (135 mg, 0.54 mmol), bis(triphenylphosphine) palladium(II)
chloride (19 mg, 0.027 mmol), copper(I) iodide (5 mg, 0.027 mmol) and triethylamine
(0.15 mL) in N,N’-dimethylformamide (5 mL) under nitrogen atmosphere was heated at 70
°C for 24 h until complete by TLC. The reaction mixture was quenched with water and
extracted with ethyl acetate. The organic layers were combined, dried over with MgSO and
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concentrated. The residue was purified by column chromatography to give the
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2-arylbenzo[b]furan 4a (79 mg, 52 %) as a white solid. H NMR (500 MHz, CDCl ): 7.45
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(dd, J = 8.0, 2.0 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.13 (s, 1H), 7.10 (t, J = 8.0 Hz, 1H),
6.90 (d, J = 8.0 Hz, 1H), 6.88 (s, 1H), 6.77 (dd, J = 8.0, 2.0 Hz, 1H), 4.02 (s, 3H), 3.97 (s,
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3H), 3.90 (s, 3H). C NMR (125 MHz, CDCl ): 156.1, 149.5, 149.1, 145.1, 143.8, 131.1,
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4.4.2. General procedure for the synthesis of (E)-2-(2-bromo-4,5-dimethoxystyryl)
-6-methoxyphenol (8) via Wittig reaction
To a solution of phosphorus ylide 7 (297 mg, 0.52 mmol) in THF (20 mL) was cooled
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to 0 C under nitrogen and lithium tert-butoxide (83 mg, 1.04 mmol) was added
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portionwise. The mixture was stirred at 0 C for 30 min. A solution of
6-bromo-2-hydroxy-3-methoxybenzaldehyde (79 mg, 0.52 mmol) in THF (5 mL) was
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added dropwise at 0 C and the reaction mixture was warmed up to room temperature and
stirred for 24 h. Sat’d aqueous NH Cl solution was added to the reaction mixture and
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extracted with EtOAc (15 mL × 3). The combined organic layers were washed with brine,
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dried with MgSO , filtered and concentrated. The residue was purified by column
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chromatography to yield 8 (97 mg, 51%) as a white solid. H NMR (500 MHz, CDCl ):
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7.00 (s, 1H), 6.75 (d, J = 12.0, 1H), 6.72-6.65 (m, 2H), 6.60 (t, J = 8.0 Hz, 1H), 5.77 (s, 1H),
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3.86 (s, 3H), 3.84 (s, 3H), 3.43 (s, 3H). C NMR (125 MHz, CDCl ): 148.8, 147.6, 146.6,
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143.5, 129.6, 129.5, 124.8, 123.0, 122.1, 119.1, 115.0, 114.4, 113.2, 109.5, 56.0, 56.0, 55.6.
+
ESMS m/z: 388.7 (M + 23) .
4.4.3.
General procedure for the synthesis of 2-(2-bromo-4,5-dimethoxyphenyl)
-7-methoxybenzofuran (4b)
To a solution of (E)-2-(2-bromo-4,5- dimethoxystyryl)-6-methoxyphenol 8 (250 mg,
0.68 mmol) in 15 mL of THF was mixed with potassium carbonate (568 mg, 4.1 mmol) and
iodine (1.04 g, 4.1 mmol). The mixture was stirred at room temperature for 3 h until
complete by TLC. Sat’d NaHSO aqueous solution was added to the solution, and the
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mixture was extracted with ethyl acetate. The organic layers were combined and dried over
MgSO . The residue was purified by flash column chromatography to afford the title
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compound (206 mg, 83%) as a yellowish solid. Mp = 106-109 C. IR νmax: 3439, 2953,
-1 1
1511, 1254, 1180 cm . H NMR (500 MHz, CDCl ): 7.44 (s, 1H), 7.38 (s, 1H), 7.20 (d, J =
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8.0 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 7.12 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.02 (s, 3H),
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3.95 (s, 3H), 3.90 (s, 3H). C NMR (125 MHz, CDCl ): 153.4, 149.4, 148.3, 145.2, 143.4,
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130.7, 123.6, 123.4, 116.6, 113.6, 112.2, 111.6, 106.7, 106.3, 56.2, 56.1, 56.0. ESMS m/z:
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5-Bromo-7-methoxy-2-(3,4,5-trimethoxyphenyl)benzofuran (4c). Yield: 81%.
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Amorphous powder. IR νmax: 3441, 2957, 1531, 1251, 1132 cm . H NMR (500 MHz,
CDCl ): 7.28 (d, J = 1.5 Hz, 1H), 7.05 (s, 1H), 6.89 (d, J = 1.5 Hz, 1H), 6.86 (s, 1H), 4.01
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(s, 3H), 3.94 (s, 6H), 3.88 (s, 3H). C NMR (125 MHz, CDCl ): 157.0, 153.6, 145.5, 143.0,
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139.1, 125.4, 116.0, 115.8, 110.1, 102.5, 100.7, 61.0, 56.3, 56.2, 29.7. ESMS m/z: 393.2 (M
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+ 1) , 395.2 (M + 1) , 415.2 (M + 23) , 417.2 (M + 23) .
4.4.4.
General procedure for the synthesis of 4-bromo-5-(7-hydroxybenzofuran
-2-yl)benzene-1,2-diol (5b)
To a solution of 4b (200 mg, 0.55 mmol) in dry dichloromethane (15 mL) at -60 °C
under N was added BBr (0.48 ml, 4.96 mmol) dropwise. The reaction mixture was then
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allowed to warm up to -40 C and stirred for another 2 h until complete by TLC. The
reaction was carefully mixed with addition of sat’d aqueous NaHCO (20 mL) at 0 °C and
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stirred for 30 min. This mixture was extracted with ethyl acetate twice (15 mL × 2) and the
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organic portion was combined, washed further with brine, and dried with MgSO . The
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residue was filtered, concentrated and purified by column chromatography to yield 5b as an
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off-white solid (122 mg, 69%). Mp = 205-210 C. IR νmax: 3253, 1596, 1489, 1174 cm .
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H NMR (500 MHz, CD OD): 7.43 (s, 1H), 7.30 (s, 1H), 7.09 (s, 1H), 7.06 (dd, J = 7.8, 1.2
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Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.72 (dd, J = 7.8, 1.2 Hz, 1H). C NMR (125 MHz,
CD OD): 154.8, 148.0, 146.3, 144.1, 143.4, 132.2, 124.6, 123.6, 121.5, 117.2, 113.2, 111.5,
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4.4.4.1. 4-(7-Hydroxybenzofuran-2-yl)benzene-1,2-diol (5a). Yield: 72%. Mp = 149-152
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C. IR νmax: 3253, 1596, 1489, 1206, 1069 cm . H NMR (600 MHz, CD OD): 7.33 (d, J
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= 2.0 Hz, 1H), 7.26 (dd, J = 8.5, 2.0 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 6.98 (dd, J = 15.0,
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8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 6.88 (dd, J = 7.5, 2.0 Hz, 1H). C NMR
(150 MHz, CD OD): 157.6, 147.3, 146.6, 144.6, 143.3, 132.8, 124.5, 124.0, 118.2, 116.6,
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113.1, 112.8, 111.0, 100.5. ESMS m/z: 241.4 (M - 1) .
4.4.4.2. 4-(Benzofuran-2-yl)-5-bromobenzene-1,2-diol (5c). Yield: 64%. Mp = 111-113
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C. IR νmax: 3333, 1614, 1506, 1256 cm . H NMR (500 MHz, CDCl ): 7.59 (d, J = 7.5
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Hz, 1H), 7.48 (s, 1H), 7.47 (dd, J = 7.5, 0.5 Hz, 1H), 7.40 (d, J = 0.5 Hz, 1H), 7.28 (dt, J =
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7.5, 1.0 Hz, 1H), 7.22 (dt, J = 7.5, 1.0 Hz, 1H), 7.21 (s, 1H), 5.59 (s, 1H), 5.38 (s, 1H). C
NMR (125 MHz, CDCl ): 154.0, 152.9, 144.5, 142.8, 128.9, 124.5, 122.9, 121.2, 120.8,
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116.1, 111.4, 110.9, 105.9. ESMS m/z: 305.2 (M - 1) .
4.4.4.3. 5-(Benzofuran-2-yl)-4-bromobenzene-1,2,3-triol (5d). Yield: 62%. Mp = 159-162
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C. IR νmax: 3419, 1613, 1507, 1452, 1187 cm . H NMR (600 MHz, CD OD): 7.58 (d, J
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= 7.2 Hz, 1H), 7.45 (dd, J = 7.8, 0.6 Hz, 1H), 7.31 (d, J = 0.6 Hz, 1H), 7.26 (dt, J = 7.2, 1.2
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Hz, 1H), 7.20 (dt, J = 7.2, 1.2 Hz, 1H), 7.01 (s, 1H). C NMR (150 MHz, CD OD): 155.8,
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155.4, 146.1, 145.2, 136.2, 130.4, 125.2, 123.6, 123.0, 122.0, 111.6, 109.3, 100.9. ESMS
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m/z: 321.0 (M -1) .
4.4.4.4. 5-(5-Bromo-7-hydroxybenzofuran-2-yl)benzene-1,2,3-triol (5e). Yield: 59%. Mp
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= 224-227 C. IR νmax: 3445, 1646, 1445, 1314, 1197 cm . H NMR (500 MHz, CD OD):
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7.14 (d, J = 2.0 Hz, 1H), 6.90 (s, 2H), 6.80 (d, J = 2.0 Hz, 1H), 6.78 (s, 1H). C NMR (125
MHz, CD OD): 159.1, 147.3, 144.2, 143.6, 135.9, 134.2, 122.4, 116.6, 115.2, 114.1, 105.6,
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4.4.4.5. 4-(4-Bromo-7-hydroxybenzofuran-2-yl)benzene-1,2-diol (5f). Yield: 67%. Mp =
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82-86 C. IR νmax: 3220, 2924, 1486, 1186 cm . H NMR (400 MHz, CD OD): 7.33 (d, J
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= 2.5 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H),
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6.83 (s, 1H), 6.61 (d, J = 8.5 Hz, 1H). C NMR (125 MHZ, CD OD): 158.5, 148.0, 146.8,
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144.3, 143.2, 133.4, 127.1, 123.2, 118.5, 116.7, 113.2, 112.3, 103.1, 100.2. ESMS m/z:
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4.4.4.6. 4-Bromo-5-(4-bromo-7-hydroxybenzofuran-2-yl)benzene-1,2,3-triol (5g). Yield:
o
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55%. Mp = 171-174 C. IR νmax: 3378, 1609, 1486, 1294, 1190 cm . H NMR (600 MHz,
CD OD): 7.24 (s, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.07 (s, 1H), 6.66 (d, J = 7.8 Hz, 1H), 4.60
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(br). C NMR (150 MHz, CD OD): 156.2, 146.2, 145.2, 143.9, 143.2, 136.6, 132.7, 127.1,
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122.3, 112.7, 109.5, 106.2, 103.4, 101.0. ESMS m/z: 415.0 (M - 1) .
4.4.4.7.
4-Bromo-5-(4-bromo-7-hydroxybenzofuran-2-yl)benzene-1,2-diol (5h). Yield:
o
-1 1
61%. Mp = 210-214 C. IR νmax: 3260, 1592, 1484, 1277, 1199 cm . H NMR (500 MHz,
CD OD): 7.43 (s, 1H), 7.26 (s, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.66 (d, J = 8.4
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Hz, 1H), 5.03 (br). C NMR (125 MHz, CD OD): 155.6, 148.5, 146.4, 143.9, 143.3, 132.7,
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127.2, 122.9, 121.6, 117.2, 112.8, 110.8, 106.0, 103.4. ESMS m/z: 399.0 (M - 1) .
4.4.4.8. 5-(7-Hydroxybenzofuran-2-yl)benzene-1,2,3-triol (5i). Yield: 67%. Mp = 200-203
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C. IR νmax: 3332, 1747, 1595, 1447, 1310, 1192 cm . H NMR (500 MHz, CD OD): 7.24
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(s, 1H), 7.15 (d, J = 7.8 Hz, 1H), 7.07 (s, 1H), 6.66 (d, J = 7.8 Hz, 1H), 4.60 (br). C NMR
(125 MHz, CD OD): 156.2, 146.2, 145.2, 143.9, 143.2, 136.6, 132.7, 127.1, 122.3, 112.7,
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4.4.4.9. 5-(Benzofuran-2-yl)benzene-1,2,3-triol (5j). Yield: 68%. Mp = 187-188 C. IR
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νmax: 3386, 1612, 1522, 1453, 1188 cm . H NMR (600 MHz, CD OD): 7.51 (dd, J = 7.2,
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1.2 Hz, 1H), 7.43 (dd, J = 7.2, 1.2 Hz, 1H), 7.20 (dt, J = 7.2, 1.2 Hz, 1H), 7.16 (dt, J = 7.2,
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1.2 Hz, 1H), 6.89 (s, 2H), 6.85 (d, J = 1.2 Hz, 1H). C NMR (125 MHz, CD OD): 158.0,
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155.9, 147.3, 135.6, 131.0, 124.6, 123.8, 122.8, 121.5, 111.6, 105.3, 100.2. ESMS m/z:
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4.4.4.10. 4-(5-Bromo-7-hydroxybenzofuran-2-yl)benzene-1,2-diol (5k). Yield: 51%. Mp =
o
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221-224 C. IR νmax: 3231, 2925, 1615, 1446, 1249, 1203 cm . H NMR (400 MHz,
CD OD): 7.30 (d, J = 2.0 Hz, 1H), 7.25 (dd, J = 8.5, 2.0 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H),
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6.84 (d, J = 8.5 Hz, 1H), 6.82 (s, 1H), 6.80 (d, J = 2.0 Hz, 1H). C NMR (125 MHz,
CD OD): 159.0, 147.8, 146.7, 144.2, 143.6, 134.2, 123.4, 118.4, 116.7, 116.6, 115.2, 114.1,
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4.4.4.11. (E)-Ethyl 3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-4-yl) acrylate (5l).
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Yield: 53%. Mp = 156-160 C. IR νmax: 3408, 1678, 1613, 1506, 1269, 1177 cm . H
NMR (500 MHz, CD OD): 7.84 (d, J = 16.0 Hz, 1H), 7.36 (d, J = 2.0, 1H), 7.32 (dd, J =
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= 8.5 Hz, 1H), 6.37 (d, J = 16.0 Hz, 1H), 4.94 (br), 4.21 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0
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Hz, 3H). C NMR (125 MHz, CD OD): 169.6, 159.2, 147.9, 146.6, 145.7, 144.3, 132.4,
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4.4.4.12. (E)-Ethyl 3-(2-(3,4-dihydroxyphenyl)-7-hydroxybenzofuran-5-yl)acrylate (5m).
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Yield: 57%. Mp = 183-186 C. IR νmax: 3342, 1686, 1629, 1282 cm . H NMR (600 MHz,
CD OD): 7.67 (d, J = 15.5 Hz, 1H), 7.32 (d, J = 2.0 Hz, 1H), 7.26 (dd, J = 8.5, 2.0 Hz, 1H),
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7.25 (br), 6.96 (d, J = 2.0 Hz, 1H), 6.89 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.38 (d, J = 15.5
1
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Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.31 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CD OD):
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4.4.4.13. (E)-Ethyl 3-(7-hydroxy-2-(3,4,5-trihydroxyphenyl)benzofuran-5-yl)acrylate (5n).
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Yield: 64%. Mp = 234-239 C. IR νmax: 3412, 1685, 1629, 1451, 1287 cm . H NMR (600
MHz, CD OD): 7.67 (d, J = 16.2 Hz, 1H), 7.25 (d, J = 1.2 Hz, 1H), 6.94 (d, J = 1.2 Hz,1H),
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6.91 (s, 2H), 6.85 (s, 1H), 6.39 (d, J = 16.2 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 1.32 (t, J =
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4.4.4.14. (E)-Ethyl 3-(4,5-dihydroxy-2-(7-hydroxybenzofuran-2-yl)phenyl)acrylate (5o).
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-1 1
Yield: 62%. Mp = 171-176 C. IR νmax: 3256, 2925, 1685, 1368, 1297 cm . H NMR
(400 MHz, CD OD): 8.09 (d, J = 16.0, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.08 (t, J = 8.0 Hz,
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16.0 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H). C NMR (125 MHz,
CD OD): 169.2, 155.3, 149.1, 147.8, 145.0, 144.7, 143.6, 132.3, 126.1, 125.3, 124.8, 117.8,
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116.5, 114.4, 113.0, 111.7, 107.5, 61.6, 14.6. ESMS m/z: 339.1 (M - 1) .
4.4.4.15. 5-Bromo-2-(4-hydroxyphenyl)benzofuran-7-ol (5p). Yield: 52%. Mp = 255-258
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C. IR νmax: 3358, 1584, 1469, 1209 cm . H NMR (600 MHz, CD OD): 7.73 (dd, J = 6.6,
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6.80 (d, J = 1.8 Hz, 1H). C NMR (150 MHz, CD OD): 159.7, 158.9, 144.3, 146.6, 134.2,
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127.7, 122.9, 116.7, 116.6, 115.2, 114.1, 99.9. ESMS m/z: 305.1 (M - 1) .
4.4.5. General procedure for the synthesis of (E)-Ethyl 3-(4,5-dimethoxy-2-(7-
methoxybenzofuran-2-yl)phenyl)acrylate (9a).
To a dry pressure tube was added 4a (180 mg, 0.50 mmol), Pd(OAc) (5 mg, 0.02
2
mmol), triphenylphosphine (16 mg, 0.06 mmol) and Et N (0.14 mL, 0.99 mmol) in
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Ethyl acrylate was degassed before being added to the reaction mixture. The tube was then
sealed and the mixture was heated to 110 °C with stirring for 24 h. The reaction was cooled
to ambient temperature and the solvent was removed under high vacuum. The residue was
purified by column chromatography to yield 9a (136 mg, 71%) as a white solid. IR νmax:
-1 1
3448, 2929, 1716, 1519, 1274, 1205 cm . H NMR (500 MHz, CDCl ): 8.12 (d, J = 15.6
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Hz, 1H), 7.32 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.10 (s, 1H), 6.82
(d, J = 8.0 Hz, 1H), 6.71 (s, 1H), 6.34 (d, J = 15.5 Hz, 1H), 4.24 (q, J = 7.0 Hz, 2H), 4.03 (s,
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3H), 3.97 (s, 3H), 3.94 (s, 3H), 1.31 (t, J = 7.0 Hz, 3H). C NMR (125 MHz, CDCl ):
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167.0, 153.4, 150.5, 149.5, 145.3, 144.1, 142.9, 130.8, 126.0, 124.4, 123.7, 118.5, 113.5,
+
111.1, 109.3, 107.5, 106.9, 60.4, 56.2, 56.1, 56.0, 14.3. ESMS m/z: 787.2 (2M + 23) .
4.5. Inhibition assay for α-glucosidase activity
All synthetic compounds were evaluated for α-glucosidase inhibition activity. We
purchased α-glucosidase (isolated from Saccharomyces cerevisiae) and 4-nitrophenyl
α- -glucopyranoside (4-NPGP) from Sigma Chemical Co. (St. Louis, MO, USA).
D
Inhibitory activity was measured according to Chu, Wu and Hsieh (2014). Briefly, the
quantity of 4-nitrophenol released from 4-NPGP was measured using a UV-Vis
spectrophotometer at 405 nm. The reaction mixture, comprising 20 ꢀL of the test
compound at various concentrations (0 to 100 ꢀM), was premixed with 120 ꢀL of 100 mM
o
phosphate buffer solution (pH 7.0). Following incubation at 30 C for 10 min, 40 ꢀL of
12.5 mM 4-NPGP was added, and the absorbance at 405 nm was measured using a
VersaMax microplate reader (Molecular Devices Corporation, Sunnyvale, CA, USA).
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Resveratrol and quercetin were used as positive controls in this α-glucosidase inhibition
assay. IC values were defined as the concentration of compound required to inhibit 50%
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of α-glucosidase activity under assay conditions.
4.6. DPPH radical scavenging assays
The free radical scavenging activity of each hydroxyl-functionalized
2-arylbenzo[b]furan was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free
radicals. The reactions were performed in 96-well microplates with each well containing
150 ꢀL of the final reaction mixture. The test compound was dissolved in MeOH at varying
concentrations (0 to 100 ꢀM) and mixed with 0.1 mM DPPH at 37 °C for 5 min. The
absorbance was read using a microplate spectrophotometer at 517 nm. Antioxidant activity
was determined according to the IC value of DPPH.
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4.7. Kinetics involved in the inhibition of α-glucosidase
Lineweaver-Burk plot analysis was performed to determine the inhibition mode of
hydroxyl-functionalized 2-arylbenzo[b]furans 5a and 5n, and kinetics were measured using
increasing concentrations of 4-NPGP as a substrate in the absence or presence of various
concentrations of hydroxyl-functionalized 2-arylbenzo[b]furans 5a and 5n. Dixon plot
analysis was used to determine the competitive inhibition constant (Ki) and uncompetitive
inhibition constant (Ki′). Ki expresses the equilibrium constant for the binding of
hydroxyl-functionalized 2-arylbenzo[b]furans 5a and 5n to α-glucosidase, and Ki′ is the
equilibrium constant of hydroxyl-functionalized 2-arylbenzo[b]furans 5a and 5n binding to
α-glucosidase-4-NPGP complex. This study of kinetics was conducted using various
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concentrations of hydroxyl-functionalized 2-arylbenzo[b]furans, 5a and 5n, and 4-NPGP.
The initial velocity was expressed as the absorbance rate/min at 405 nm.
4.8. Docking experiments
The 3D-structural model of the Saccharomyces cerevisiae α-glucosidase (protein
sequence entry: NP_011803, PDB code: 3A4A) was used in docking experiments. The
models of compounds 5a and 5n were docked into the active site of the α-glucosidase based
on the binding mode of maltose in S. cerevisiae α-glucosidase. On the basis of the
structures of α-glucosidase, compounds 5a and 5n were manually docked into the active
site with the program Coot to generate an initial binding pose of compounds 5a and 5n in S.
cerevisiae α-glucosidase, respectively. These models of S. cerevisiae α-glucosidase-5a
complex and S. cerevisiae α-glucosidase-5n complex were optimized by energy
minimization with the program Discovery Studio, and the resulting models with most low
potential energy were selected. The structure figures were generated with the program
PyMOL (Schrödinger, New York, NY).
Acknowledgements
This study was supported by the National Science Council (Grant NSC
102-2113-M-077-002-MY2).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at
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