Synthesis and biological evaluation of 2-acylbenzofuranes as novel α-glucosidase inhibitors with hypoglycemic activity

Article abstract of DOI:10.1111/cbdd.13038

A series of benzofuran derivatives was synthesized as analogues of known natural α-glucosidase inhibitors. Their activity was evaluated in enzymatic assay and in rat model of diabetes mellitus. Newly identified inhibitors demonstrate significant potency with IC₅₀ values ranging from 6.50 to 722.2?μm, as well as hypoglycemic activity exceeding the reference drug acarbose. Docking simulations provided insight into structure-activity relationships to direct further development of these novel hypoglycemic agents.

Full text of DOI:10.1111/cbdd.13038

MR DENIS BABKOV (Orcid ID : 0000-0002-9645-3324)
Article type
: Research Article
Synthesis and biological evaluation of 2-acylbenzofuranes as
novel α-glucosidase inhibitors with hypoglycemic activity
Enzymatic study, docking, antidiabetic properties
Keywords: benzofurans, α-glucosidase, diabetes
Alexander A. Spasova*, Denis A. Babkova, Tatyana Yu. Prokhorovaa, Ekaterina A. Sturovaa, Diana
R. Muleevaa, Maxim R. Demidovb, Dmitry V. Osipovb, Vitaly A. Osyaninb, Yuri N. Klimochkinb
a Volgograd State Medical University, Pavshikh Bortsov Sq. 1, 400131 Volgograd, Russian Federation;
b Department of Organic Chemistry, Samara State Technical University, Molodogvardeiskaya St. 244,
443100 Samara, Russian Federation.
* Corresponding author: Alexander A. Spasov; +7(8442) 942-423; aspasov@mail.ru.
Abstract
A series of benzofuran derivatives was synthesized as analogues of known natural α-glucosidase
inhibitors. Their activity was evaluated in enzymatic assay and in rat model of diabetes mellitus.
Newly identified inhibitors demonstrate significant potency with IC50 values ranging from 6.50 to
722.2 μM, as well as hypoglycemic activity exceeding the reference drug acarbose. Docking
simulations provided insight to structure-activity relationships to direct further development of these
novel hypoglycemic agents.
Introduction
α-Glucosidase is an exoenzyme that hydrolyzes α-1,4 bonds at the non-reducing end of α-1,4-
glycans, cleaving glucose in its α-form. The principle of the action of oral α-glucosidase inhibitors is
based on a competitive inhibition of the enzyme and slowing the release of glucose from complex
carbohydrates, which leads to a decrease in postprandial hyperglycemia (1). Accordingly, several
α-glucosidase inhibitors are clinically approved for the treatment of diabetes and obesity. The role
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of cellular α-glucosidase in carbohydrate processing also enables a promising venue to fight other
diseases including cancer (2, 3) and viral infections (4–6).
Clinically approved drugs for the treatment of type 2 diabetes via α-glucosidase inhibition include
pseudosacharides acarbose, miglitol and voglibose (7). Their ability to improve glycemic control in
diabetic patients is well documented (8) but accompanied with gastrointestinal side effects (9).
Several cases of acarbose-induced hepatitis have been reported as well (10, 11). A wide range of
structurally different inhibitors have been reported to date (12), including transition state analogues
(13), small molecule synthetic compounds (14–17), and natural products (18–20).
Baicalein (Fig.1, 1), a 5,6,7-trihydroxyflavone isolated from marjoram leaves of O. majorana, is a
modest α-glucosidase inhibitor (IC50 32 µM) that was further modified to yield 20 times more active
compound 2 (21, 22). Anthocyanins 3 are another well-known source of α-glucosidase inhibitors
(IC50 27.6-36.2 µM) (23). Recently, hydroxyl-functionalized 2-arylbenzo[b]furans (24) and sulfur-
containing benzofurans (25) were reported as α-glucosidase inhibitors. These observations
prompted us to synthesize and explore structurally relevant benzofuran derivatives as analogues of
naturally occurring compounds.
Methods and Materials
Chemistry
IR spectra were recorded on a Shimadzu IRAffinity-1 spectrometer in KBr pellets. 1Н and 13C NMR
spectra (including DEPT experiments) were acquired on a JEOL JNM-ECX 400 spectrometer (400
and 100 MHz, respectively) in DMSO-d6 or in CDCl3 with TMS as internal standard. Elemental
analysis was performed on a EuroVector EA-3000 automated СНNS-analyzer. Melting points were
determined by capillary method on an SRS OptiMelt MPA100 apparatus and are uncorrected. The
reaction progress was monitored by TLC on aluminum foil-backed silica gel plates (Merck, Kiesgel
60 F254) with visualization under UV light and in iodine vapor.
The synthesis of compounds 6a-c, 8, 11
Dihydrobenzofurans were prepared according to previously described method (26).
trans-(4-Chlorophenyl)(3-phenyl-2,3-dihydrobenzofuran-2-yl)methanone (6a).
To a mixture of 2-(acetoxy(phenyl)methyl)phenyl acetate 4a (1.180 g, 4.15 mmol) and 1-(2-(4-
chlorophenyl)-2-oxoethyl)pyridin-1-ium bromide 5a (1.297 g, 4.15 mmol) in acetonitrile (20 ml) DBU
(1.26 g, 8.3 mmol) was added. The solution obtained was refluxed under argon atmosphere for 10
h and then was evaporated in vacuo. The residue was purified by column chromatography using
CHCl3 as eluent, further recrystallization from ethanol gave 6a as colorless crystals (1.069 g, 76%).
mp: 109–110 ºC (EtOH). FT-IR (KBr; cm-1): 3024, 1690 (C=O), 1593, 1481, 1462, 1400, 1238,
1165, 1092, 1011, 968, 887, 841, 818, 756, 702. 1H-NMR (400 MHz, CDCl3): δ 5.01 (d, J = 6.6 Hz,
1H), 5.73 (d, J = 6.6 Hz, 1H), 6.90 (td, J1 = 0.9 Hz, J2 = 7.6, 1H), 6.96 (d, J = 8.0 Hz, 1H), 7.01 (d, J
= 7.4 Hz, 1H), 7.19–7.36 (m, 6H), 7.43 (d, J = 8.9 Hz, 2H), 7.90 (d, J = 8.7 Hz, 2H). 13C-NMR (100
MHz, CDCl3): δ 50.7 (CH), 90.7 (CH), 110.1 (CH), 121.9 (CH), 125.5 (CH), 127.6 (CH), 128.2
(2CH), 129.0 (CH), 129.12 (2CH), 129.14 (2CH), 129.2 (C), 130.9 (2CH), 132.9 (C), 140.4 (C),
142.1 (C), 158.9 (C), 193.7 (C=O). Elemental analysis for C21H15ClO2: Calculated: C, 75.34; H,
4.52; Found: C, 75.25; H, 4.48.
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(1,2-Dihydronaphtho[2,1-b]furan-2-yl)(3-hydroxyadamantan-1-yl)methanone (6b). A mixture of
2-((dimethylamino)methyl)naphthalen-1-ol 4b (604 mg, 3 mmol) and 1-(2-(3-hydroxyadamantan-1-
yl)-2-oxoethyl)pyridin-1-ium bromide 5b (1.057 g, 3 mmol) was refluxed in 10 ml of a mixture of
acetonitrile-DMF (3:1) for 10 h and then was evaporated in vacuo. The residue was purified by
column chromatography using CHCl3, further recrystallization from CCl4 gave 6b as colorless
crystals (721 mg, 69%); mp: 137–139 ºC (СCl4). FT-IR (KBr cm-1): 3600–3200 (OH), 3059 (CH Ar),
2920, 2855 (CH Ad), 1705 (C=O), 1632, 1601, 1578, 1520, 1466, 1373, 1335, 1312, 1261, 1246,
1184, 1161, 1138, 1115, 1076, 1015, 976, 957, 907, 810, 745. 1H-NMR (400 MHz, CDCl3): δ 1.55–
1.98 (m, 13H, Ad, OH), 2.33–2.35 (m, 2H, Ad-2), 3.58 (dd, J1 = 7.8 Hz, J2 = 15.6 Hz, 1H), 3.66 (dd,
J2 = 10.6 Hz, J2 = 15.6 Hz, 1H), 5.64 (dd, J1 = 7.8 Hz, J2 = 10.6 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H),
7.32 (ddd, J1 = 1.2 Hz, J2 = 6.9 Hz, J3 = 8.2 Hz, 1H), 7.47 (ddd, J1 = 1.2 Hz, J2 = 6.9 Hz, J3 = 8.2
Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H). 13C-NMR (100
MHz, CDCl3): δ 30.3 (2CH), 32.2 (CH2), 35.1 (CH2), 36.8 (2CH2), 44.4 (2CH2), 45.4 (CH2), 49.5
(CAd-1), 68.5 (CAd-3), 82.4 (CH), 111.9 (CH), 117.0 (C), 122.7 (CH), 123.3 (CH), 127.0 (CH), 128.8
(CH), 129.5 (CH), 129.6 (C), 130.5 (C), 156.7 (C), 187.3 (C=O). Elemental analysis for C23H24O3:
Calculated: C, 79.28; H, 6.94. Found: C, 79.34; H, 6.93.
(1,2-Dihydronaphtho[2,1-b]furan-2-yl)(4-hydroxyphenyl)methanone (6c). A mixture of 2-
((dimethylamino)methyl)naphthalen-1-ol 4b (604 mg, 3 mmol) and 1-(2-(4-hydroxyphenyl)-2-
oxoethyl)pyridin-1-ium bromide 5c (882 mg, 3 mmol) was heated in DMF (5 ml) at 90 °С for 15 h.
Solution obtained was cooled, poured into water, the formed solid was filtered off, washed with
water and dried. The product was purified by column chromatography using CHCl3/EtOAc (3:1),
further recrystallization from isopropyl alcohol gave 6c as colorless crystals (435 mg, 50%); mp:
147–149 ºС (i-PrOH). FT-IR (KBr cm-1): 3206, 1666 (С=О), 1632, 1601, 1578, 1516, 1466, 1369,
1285, 1242, 1173, 968, 810. 1H-NMR (400 MHz, DMSO-d6): δ 3.54 (dd, J1 = 6.9 Hz, J2 = 15.9 Hz,
1Н), 3.85 (dd, J1 = 11.2 Hz, J2 = 15.9 Hz, 1Н), 6.38 (dd, J1 = 6.9 Hz, J2 = 11.2 Hz, 1Н), 6.88–6.92
(m, 2Н), 7.20 (d, J = 8.8 Hz, 1Н), 7.28–7.32 (m, 1Н), 7.43–7.47 (m, 1Н), 7.61 (d, J = 8.2 Hz, 1Н),
7.75 (d, J = 8.8 Hz, 1Н), 7.85 (d, J = 8.2 Hz, 1Н), 7.93–7.96 (m, 2Н), 10.56 (s, 1Н, ОН). 13C-NMR
(100 MHz, DMSO-d6): δ 32.1 (СН2), 82.7 (СН), 112.4 (СН), 116.1 (2СН), 117.9 (С), 123.4 (СН),
123.7 (СН), 125.9 (С), 127.4 (СН), 129.1 (СН), 129.4 (С), 129.6 (СН), 130.7 (С), 132.1 (2СН),
157.1 (С), 163.4 (С), 194.0 (С=О). Elemental analysis for C19H14O3: Calculated: C, 78.61; H, 4.86.
Found: C, 78.75; H, 4.84.
2-Nitrobenzofuran 8 was prepared from the diacetate of salicylic alcohol 4c and potassium
trinitromethanide 7 (27).
(3-Hydroxybenzofuran-2-yl)(phenyl)methanone (11). Phenacyl bromide 10 (1.09 g, 5.5 mmol)
and K2CO3 (2.09 g, 15 mmol) were added to a solution of methyl salicylate 9 (0.76 g, 5.0 mmol) in
dry acetone (6 ml). The mixture obtained was heated under reflux for 6 h, cooled, filtered and
washed with acetone. Then the solid was suspended in water (30 ml) and acidified with 3M HCl.
The precipitated yellow crude product was filtered and crystallized from EtOH. The mother liquor
was evaporated in vacuo and purified by column chromatography using CHCl3/EtOAc (4:1) as
eluent for additional crop of the product 11. Total yield 1.07 g (90%). Spectral characteristics and
melting point are identical to literature data (28).
α-Glucosidase inhibition assay
In a 96-well plate the 0.01 mg/mL enzyme solution (EC 3.2.1.20, expressed in S. cerevisiae,
Sigma; catalog #G5003) was incubated with test compounds in 0.1 M phosphate buffer (pH 6.8) at
37 °C for 5 min. Then, 25 μL of 5 mM substrate solution, i.e. p-nitrophenyl-α-D-glucopyranoside
(Sigma; catalog #N1377), was added and the change in absorbance was recorded for 15 min at
400 nm with Infinite M200 PRO microplate reader (Tecan, Austria)(29). Test compounds were
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replaced with 0.1 M phosphate buffer (pH 6.8) in negative control experiments. Acarbose was used
as the positive control.
Oral maltose tolerance test
All animal studies were conducted with a previous approval from the VolgSMU Animal Care and
Use Committee. Male Wistar rats aged 9–12 weeks were injected i.p. with 45 mg/kg streptozotocin
(STZ) dissolved in 0.1 M citrate buffer (pH 4.5) 1 week before the experiment. Rats were fasted
overnight and then administered orally with 2 g/kg maltose. Immediately after, 5 mg/kg acarbose,
2.2 mg/kg 6c, or 1.8 mg/kg 11 suspended in 1% sodium carboxymethyl cellulose solution were
orally administered to the respective groups consisting of five rats. Control group received the
same volume of vehicle. Blood sampling from the tail vein was performed at 0, 60 and 120 min
(30). The blood glucose levels were measured at each time interval using Biosen C_line glucose
analyzer (EKF Diagnostics, Germany).
Docking simulations
Ligands were prepared with Marvin Scetch 16.2.22 (ChemAxon) (31). Protein (PDB ID: 1LWJ) and
ligand structures followed standard preparation procedure using AutoDocTools 1.5.6. We used
AutoDock Vina 1.1.2 (32) to perform all docking runs. Cubic grid box centered on cognate ligand
was adjusted to include the entire concave region around the ligand and the solvent accessible
entrance of the pocket. Only top-score binding pose were used in subsequent analysis. First,
saxagliptin was docked in the native protein conformation to assess the ability of docking and
scoring functions to reproduce crystallographic binding pose. Second, auxiliary ligand was docked
in the same binding site. Protein-ligand interactions were analyzed with Discovery Studio 4.5
Visualizer (Accelrys) (33).
Statistical analysis
The GraphPad Prism 6 (GraphPad Software Inc.) was employed for data analysis and graph
preparation. The experimental data of biological activity was expressed as mean values standard
error of mean (mean SEM). The IC50 value is defined as the concentration of an inhibitor which
caused 50% reduction of the enzyme activity under specific assay conditions and was calculated
with a nonlinear regression analysis. Each point in the constructed graphs represents the mean of
three experiments. Mann-Whitney U-test or two-way ANOVA were used for comparisons of sample
values versus control. P < 0.05 was considered statistically significant.
Results
Chemistry
The synthesis of condensed furans and dihydrofurans is presented in Scheme 1. Compounds 6a-c
and 8 were synthesized via formation of ortho-quinone methide intermediates from corresponding
2-(acetyloxy)benzyl acetates 4a and 4c (for 6a and 8) or naphtholic Mannich base 4b (for 6b and
6c) and further cascade heterocyclization (34). Dihydroarenofurans 6a-c were formed through
Michael-type addition of in situ generated pyridinium ylides from salts 5a-c to ortho-quinone
methides followed by intramolecular nucleophilic substitution. Compound 6a was obtained
exclusively as the trans-diastereomer; such diastereoselectivity was explained in previous
publication (26). 2-Nitrobenzofuran 8 was synthesized according to earlier developed method with
the use of potassium nitroformate 7 in the presence of triethylamine in aqueous methanol (27).
Benzofuran 11 was prepared from methyl salicylate 9 and phenacyl bromide 10 in dry acetone in
the presence of K2CO3 in 90% yield. The structures of all synthesized compounds were confirmed
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with elemental analysis and spectroscopic methods including 1H, 13C and DEPT NMR
spectroscopy and IR spectroscopy.
Inhibitory effect of 2-acylbenzofuran derivatives against α-glucosidase activity
Synthesized compounds were first evaluated in vitro for α-glucosidase inhibition as previously
described (35). Results are represented in Table 1. A bulky substituent might be required for
activity. Indeed, as the structure-activity relationships show, a 2-benzoyl substituent provides
optimal steric properties in comparison to adamantane counterpart 6b and 2,3-disubstituted 6a.
Introduction of a 2-(4-hydroxybenzoyl) group resulted in sharp potency increase rendering
compound 6c as the most potent with IC50 value of 6.50 µM.
Docking studies
In order to rationalize the activity data and gain structural insight into the binding of the novel
inhibitors to α-glucosidase, they were docked into the active site of the enzyme and compared to
the acarbose binding conformation. Due to the unavailability of three-dimensional X-ray structures
of α-glucosidase enzymes commonly used in biological assays, such as the native or complexed
protein of S. cerevisiae, we employed a recently constructed model of an α-glucosidase
homologue based on a 4-α-glucanotransferase of T. maritima (36), the latter showing a high
sequential identity with the α-glucosidase of S. cerevisiae (37).
Fig. 2 shows the experimental binding mode of acarbose and the predicted top-scored
conformation of 6c in the active site of α-glucosidase. As revealed by interaction analysis,
acarbose forms an extensive hydrogen bond network, while inhibitor 6c seem to be stabilized
through the interactions with aromatic side chains of Trp131, Phe150 and Trp218. It is of interest
that, the simple yet well-tuned stacking interactions provide affinity that is sufficient for inhibiting the
enzyme. Along with that simple scaffold of identified 2-acylbenzofuranes provide multiple
opportunities for modification to further enhance the potency. Considering the hydrophilic nature of
the binding site introduction of hydrogen bond donors is the most apparent way to improve the
affinity. However, there may be an enthalpic penalty for displacement of buried water molecules
(38).
Hypoglycemic activity of 2-acylbenzofurane derivatives
In order to confirm the potency of the newly identified α-glucosidase inhibitors in animals, the oral
maltose tolerance test was carried out using the male Wistar rat streptozotocin diabetes model
(39). All groups demonstrated marked hyperglycemia prior to the experiment. In the control group,
the blood glucose level increased from 15.44 2.07 to 21.66 4.30 mM 60 minutes after the maltose
challenge (Fig. 3). Administration of the compounds 6c and 11 was found to effectively prevent
postprandial hyperglycemia. Moreover, they proved to be significantly superior to acarbose at the
specified time points (p < 0.05).
Discussion
We have developed an efficient and versatile synthetic route to the novel 2-acylbenzofuran
derivatives as analogues of naturally occurring flavonoids. An alternative method was reported
earlier (40), however our attempts to reproduce the described results were unsuccessful. Among
the target derivatives four compounds were identified as inhibitors of α-glucosidase. Compound 6c
inhibits α-glucosidase in vitro with IC50 of 6.50 µM and exceeds hypoglycemic activity of acarbose
in streptozotocin-induced diabetic rats. Recently, structurally related 2-arylbenzo[b]furans (41) and
oxadiazole-substituted benzofurans (42) have been reported as potent inhibitors of α-glucosidase.
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However, animal studies were not reported to support these findings. Considering the in vivo
hypoglycemic activity the most active compound 6c could be further pursued for the improved
potency and ADMET properties. Docking experiments have elucidated the binding of 6c to the
enzyme and could facilitate future optimization efforts.
Acknowledgments
The biological part of this work received financial support from the Russian Science Foundation
(project 14-25-00139); the synthetic part was supported by the Russian Foundation for Basic
Research (project 15-43-02304 r_povolzhye_a).
Conflict of Interest
The authors declare no conflicts of interest.
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Figure legends and Tables
Figure 1: Flavonoid α-glucosidase inhibitors.
Scheme 1: Synthesis of the target compounds.
Figure 2: Proposed binding mode of compound 6c (B) in comparison with acarbose (A).
Figure 3: Effect of compounds 6b and 11 on plasma glucose levels during oral maltose tolerance
test in diabetic rats (n = 5). Statistical significance versus diabetic control (*p < 0.05) and diabetic
animals treated with acarbose (#p < 0.05).
Table 1: Activity of the target compounds against α-glucosidase in vitro
Comp.
IC50 (µM)
451.4
722.2
6.50
6a
6b
6c
8
n.a.
11
167.0
543.6
Acarbose
* Statistical significance (p < 0.001) versus negative control (Mann-Whitney U-test); n.a. – not
analyzed.
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This article is protected by copyright. All rights reserved.