Synthesis and Biological Evaluation of New Substituted 3-[ 4-( Phenylsulfonamido ) benzoyl ]-2 H-1-benzopyran-2one Derivatives as α-Glucosidase Inhibitors

A series of new substituted 3-[4-(phenylsulfonamido)benzoyl]-2H-1-benzopyran-2-one derivatives bearing groups methoxy, tertbutyl, and atoms of halogens at the para-position of the A-ring were synthesized and in vitro biological activities were evaluated as nonsugar α-glucosidase inhibitors. Most of the test compounds demonstrated significant α-glucosidase inhibitory activity relative to that of Acarbose (IC 50 = 29.26 μM).The para-substitution with a methoxy group or halogens could notably increase the potency. Compounds 17, 18, and 23, with IC 50 values of 0.025 μM, 0.014μM, and 0.018 μM, respectively, may be of significance for the further development of new nonsugar α-glucosidase inhibitors.


Introduction
-Glucosidase (EC 3.2.1.20),located in the brush-border surface membrane of intestinal cells, is a key enzyme catalyzing the final step in the digestive process of carbohydrates into glucose [1].This enzyme has drawn a special interest in the pharmaceutical research community because the inhibition of its catalytic activity can retard the liberation of glucose from dietary complex carbohydrates and delay glucose absorption, resulting in reduced postprandial plasma blood glucose level and suppression of postprandial hyperglycemia [2].Thus, -glucosidase inhibitors exhibit high promise as therapeutic agents for the treatment of metabolic disorders, such as type II non-insulin-dependent diabetes mellitus, obesity, and hyperglycemia [3].-Glucosidase inhibitors are also known to be promising as antiviral and antitumor agents that interfere with the biosynthesis of N-linked oligosaccharide chains [4].
Classically, efforts for the development of a new set of -glucosidase inhibitors have focused mainly on sugar mimics, such as disaccharides, iminosugars, carbasugars, and thiosugars.However, numerous disadvantages exist for this strategy, including poor activity, low natural abundance, or complicated stereochemistry, which makes them difficult to approach synthetically.A more modern approach is to investigate nonsugar -glucosidase inhibitors [5][6][7][8][9].
We had previously reported that 3-[4-(phenylsulfonamido)benzoyl]-2H-1-benzopyran-2-one derivatives [10], designed by incorporating the phenylsulfonamide chalcone substructure into the benzopyran backbone shown in Figure 1 [11], have the potential to act as a new class of nonsugar -glucosidase inhibitors.The preliminary study on the structure-activity relationship had focused on the C-ring, which showed that the modification of this substructure with halogen or bulky groups can reduce the inhibitory activity, whereas introducing diethylamino group at C7 and methoxy and hydroxy groups at C6 and C7, respectively, can increase the potency significantly.In addition, we found that compounds bearing the methyl group displayed higher activities than those bearing a hydrogen atom, such as compounds 7u and 7j (Figure 1).This observation In the present study, a series of new substituted 3-[4-(phenylsulfonamido)benzoyl]-2H-1-benzopyran-2-one derivatives (Scheme 1), bearing halogen atoms, methoxy, or tertbutyl groups at the para-position of the A-ring while keeping the favorable substituents on the C-ring unchanged, were synthesized and assayed on yeast -glucosidase to explore the effect of these substituents on the -glucosidase inhibitory activity and obtain a comprehensive understanding of the structure-activity relationships of this class of compounds.
All target compounds were evaluated spectrophotometrically at 490 nm on yeast -glucosidase to evaluate their potential as -glucosidase inhibitors.Acarbose was included as a reference compound.The in vitro -glucosidase inhibitory activity of the test compounds was assayed as described previously [10].The test compounds were initially assayed for their ability to inhibit -glucosidase at a concentration of 1 g⋅mL −1 .Compounds that displayed more than 50% inhibition were selected for concentration dependent activity evaluation and calculation of IC 50 values, as shown in Table 1.

Discussion
Except for compound 10 (IC 50 = 7.568 M) in the halogen substituted series, most of the tested compounds showed stronger inhibitory activity than that of compound 7a, previously studied in [10] (IC 50 = 3.283 M).Compound 18 (IC 50 = 0.014 M) presented as the strongest inhibitor which was nearly 235 times higher than compound 7a.The activities of compounds 13 (IC 50 = 0.075 M), 17 (IC 50 = 0.025 M), and 21 (IC 50 = 0.036 M) were increased about 3, 8, and 6 times, respectively, compared with the activity of compound 7j, previously described in [10] (IC 50 = 0.199 M).This observation suggested that the halogen atoms at the para-position of the A-ring could greatly enhance the activity.However, this fact was contrary to the disadvantageous effect observed for the halogen groups presented on the C-ring [10].
The activity of compound 22 (IC 50 = 0.073 M) in the methoxy substituted series (R 3 = OCH 3 ) was 45-fold higher than that of compound 7a.Compound 23 displayed an IC 50 value of 0.018 M, which was 320-fold more potent than compound 7 h and was ranked as the strongest inhibitor in this series.Compounds 24 (IC 50 = 0.037 M) and 25 (IC 50 = 0.069 M) also exhibited a much better inhibitory activity compared with compounds 7i and 7j.Interestingly, most compounds in this series showed higher activities than those in the methyl substituted series.The activities of compounds 22, 23, and 24 were higher than those of compounds 7l (IC 50 = 3.577 M), 7s (IC 50 = 1.125 M), and 7t (IC 50 = 0.347 M), respectively.All these observations indicated that the methoxy group substituted at the para-position of the A-ring was favorable for increasing the -glucosidase inhibitory activity of 3-[4-(phenylsulfonamido)benzoyl]-2H-1-benzopyran-2-one derivatives.
The activity of compound 26 with tert-butyl group at the para-position of the A-ring disappeared, suggesting that this bulky group is unfavorable for binding with -glucosidase, which is in agreement with what we had found on the C-ring [10].

Experimental
4.1.Chemistry.Melting points were recorded using YRT-3 melting point apparatus.The 1 H-NMR spectra were recorded in DMSO- 6 on a Bruker ARX-300 spectrometer, and chemical shifts () were expressed in ppm downfield from the TMS and were used as the internal standard.Coupling constant (J) values were in Hz.The IR spectra were determined as KBr pellets on the Bruker IFS-55 spectrometer and were expressed in cm −1 .The progress of the reactions was monitored by TLC using several solvent systems with different polarities.The TOF-HRMS spectra were recorded on a Bruker Micro-TOFQ.All solvents and reagents were of analytical grade.

General Procedure for the Synthesis of Compounds 10-26.
Compounds 5-9 (1.4 mmol) reacted with substituted salicylaldehyde (1.6 mmol) separately; five drops of piperidine, one drop of glacial acetic acid, and ethanol (10 mL) were added and stirred under reflux in an argon atmosphere for 0.5 h.The mixture was cooled to room temperature, filtered   to collect the solid (if there was no precipitate, 25 mL water was added), and then washed with a small amount of ethanol.
In most cases, the products were sufficiently pure.Products with impurities were purified via column chromatography, petroleum ether, and ethyl acetate systems with different polarities as eluent.

Assay of the In Vitro 𝛼-Glucosidase Inhibitory Activity.
A 100 L reaction system containing 0.02 U of -glucosidase, 67 nM sodium phosphate buffer (pH 6.8), and a test compound were preincubated at 37 ∘ C for 10 min.A negative control in the absence of a test compound and a blank control in the absence of either an enzyme or the test compound were run simultaneously.The reaction was initiated by the addition of 0.1 M maltose, and the reaction mixture was incubated at room temperature for 10 min.Then, the 200 L glucosedetecting agent was added, and the absorbance (A) at 490 nm was recorded on a SPECTRAmax Plus 384 reader (MD, USA).The test compounds were initially assayed for their inhibition of -glucosidase at a concentration of 10 g/mL.Calculate inhibition ratio according to OD value; inhibition ratio = [1 − (OD sample − OD blank )/(OD negative − OD blank )] × 100%.Each monoconcentration had two replicates when preliminary screened.If an inhibition ratio of more than 70% was observed, diluted 10 times and re-screened.If an inhibition ratio of re-screening was more than 70%, measured IC 50 value.The active compounds were consequently tested at six gradient dilute concentrations, with each concentration having two replicates.On the basis of inhibition ratio, the IC 50 values were calculated using 4-Parameter Logistic Model of Xlfit software.