BDBM23406 Acarbose (3R,4R,5S,6R)-5-{[(2R,3R,4R,5S,6R)-5-{[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-{[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-en-1-yl]amino}oxan-2-yl]oxy}-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-6-(hydroxymethyl)oxane-2,3,4-triol US11292789, Acarbose
5-{5-[3,4-Dihydroxy-6-methyl-5-(4,5,6-trihydroxy-3-hydroxymethyl-cyclohex-2-enylamino)-tetrahydro-pyran-2-yloxy]-3,4-dihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy}-6-hydroxymethyl-tetrahydro-pyran-2,3,4-triol (2R,3R,4R,5R,6R)-5-((2R,3R,4R,5S,6R)-5-((2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-((1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-enylamino)-tetrahydro-2H-pyran-2-yloxy)-3,4-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-2,3,4-triol (2R,3R,4R,5S,6R)-5-((2R,3R,4R,5S,6R)-5-((2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-((1S,4S,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-enylamino)-tetrahydro-2H-pyran-2-yloxy)-3,4-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-2,3,4-triol CHEMBL1566 acarbose (2R,3R,4R,5S,6R)-5-((2R,3R,4R,5S,6R)-5-((2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-((1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-enylamino)-tetrahydro-2H-pyran-2-yloxy)-3,4-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-2,3,4-triol (3R,4R,5S,6R)-5-((2R,3R,4R,5S,6R)-5-((2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-((1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-enylamino)-tetrahydro-2H-pyran-2-yloxy)-3,4-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-2,3,4-triol Precose Glucobay BAY-G 5421 BDBM50333465
- Enzymatic Activity Assay To test whether GCTs share a common binding pocket on PPA with acarbose, we performed an experiment according to the method of Yonetani and Theorell.
- Alpha-glucosidase Assay Total volume of 100 µL reaction mixture contained 70 µL 50 mM phosphate buffer pH 6.8, 10 µL test compound (0.5 mM in methanol) followed by the addition of 10 µL enzyme solution (0.057 units) in the buffer. The contents were mixed, pre-incubated for 10 min at 37 °C and pre-read at 400 nm. The reaction was initiated by the addition of 10 µL of 0.5 mM substrate (p-nitrophenyl glucopyranoside). After 30 min of incubation at 37 °C, absorbance of p-nitrophenol released was measured. Acarbose was used as positive control.
- In Vitro α-Glucosidase Inhibitory Assay The test compounds were dissolved in DMSO to prepare the required distributing concentration. α-Glucosidase inhibitory activity was assayed using 0.1 m phosphate buffer (pH 6.8) at 37°C. The enzyme (0.1 U/ml) in phosphate-buffered saline was incubated with various concentrations of test compounds at 37°C for 15 min. Then, 1.25 mm p-nitrophenyl α-D-glucopyranoside was added to the mixture as a substrate.After further incubation at 37°C for 30 min, the absorbance was measured spectrophotometrically at 405 nm. The sample solution was replaced by DMSO as a control. Acarbose was used as a positive control. All experiments were carried out in triplicates.
- In-vitro Yeast α-Glucosidase Inhibition Assay α-Glucosidase inhibitory activity was evaluated at 37 °C, by using 0.1M phosphatebuffer (pH6.8) [28]. The enzyme(EC3.2.1.20, Saccharomyces cerevisiae purchased from sigma; catalog # G5003) (0.2 U/mL) was incubated with test compounds and phosphate buffer at 37 °C for 15 min. After 15min of incubation, 0.7 mM of substrate i.e. p-nitrophenyl-α-D-glucopyranoside (Sigma; catalog # N1377) was added and the change in absorbance was recorded at 400 nm for 30min by using spectrophotometer (SpectraMax M2, Molecular Devices, CA, USA). Test compound was replaced with DMSO in control experiments (7.5% final concentration).Acarbose was used as the standard inhibitor.
- α-Glucosidase inhibition assay α-Glucosidase inhibition assay was performed spectrophotometrically. α-Glucosidase from Saccharomyces cerevisiae (Sigma-Aldrich) was dissolved in phosphate buffer (pH 6.8, 50 mM). Test compounds were dissolved in DMSO. In 96-well microtiter plates, 20 µL of test sample, 20 µL of enzyme (20 mU/mL) and 135 µL of buffer were added and incubated for 15 min at 37 °C. After incubation, 25 µL of p-nitrophenyl-α-D-glucopyranoside (2 mM, Sigma Aldrich) was added and change in absorbance was monitored for 20 min at 400 nm. Test compound was replaced by DMSO (10% final) as control. Acarbose (Sigma-Aldrich) was used as a standard inhibitor. The assays were done in triplicate.
- α-Glucosidase Inhibition Assay For α-glucosidase inhibition assay, α-glucosidase enzyme (Cat No. 5003-1KU Type I; isolated from Saccharomyces cereviciae) was used. Acarbose was the reference standard and Pierre's methodology was employed [Pierre et al., J. Clin. Chem., 24:208-211]. First absorbance was taken at 400 nm after the 10 min incubation of a mixture of phosphate buffer (70 mL, pH 6.8), test compound (10 mL, 0.5 mM) and enzyme (10 mL, 0.0234 units, Sigma Inc.). Later, 10 mL of p-nitrophenyl-α-D-glucopyranoside (0.5 mM, code No. N1377 from Sigma Inc) was added. After 30 min incubation at 37 °C, the absorbance was retaken at 400 nm.
- In vitro Assay of alpha-Glucosidasee Inhibitory Activity The test compounds were dissolved in DMSO to prepare the required distributing concentration. α-Glucosidase inhibitory activity was assayed by using 50 mM phosphate buffer (pH 6.8) at 37 °C. The enzymatic reaction mixture composed of 20 µL α-glucosidase (0.2 U), 10 µL of various concentrations of test compounds and 140 µL of phosphate buffer was incubated at 37 °C for 10 min. Then 0.5 mM p-nitrophenyl α-D-glucopyranoside (30 µL) was added to the mixture as a substrate. After further incubation at 37 °C for 30 min. The absorbance was measured spectrophotometrically at 405 nm. The sample solution was replaced by DMSO as a control. Acarbose was used as a positive control. All experiments were carried out in triplicates.
- α-Glucosidase Assay The α-glucosidase inhibition activity was performed with slight modifications according to method. Total volume of 100 µL reaction mixture contained, 70 µL 50 mM phosphate buffer, pH 6.8, 10 µL (0.5 mM) test compound, followed by the addition of 10 µL (0.0234 units, Sigma Inc.) yeast α-glucosidase enzyme. Thecontents were mixed, preincubated for 10 min at 37 °C and preread at 400 nm. The reaction was initiated by the addition of 10 µL of 0.5 mM substrate (p-nitrophenyl glucopyranoside, Sigma Inc.). After 30 min of incubation at 37 °C, absorbance of the yellow color produced due to the formation of p-nitrophenol was measured at 400 nm using Synergy HT (BioTek, USA) 96-well microplate reader. Acarbose was used as positive control.
- In Vitro α-Glucosidase Inhibitory Assay The α-glucosidase inhibition assay had been carried out using baker's yeast α-glucosidase (EC 3.2.1.20) and p-nitrophenyl α-d-glucopyranoside. The test compounds were dissolved in DMSO to prepare the required distributing concentration. α-Glucosidase inhibitory activity was assayed by using 0.1 M phosphate buffer (pH 6.8) at 37 °C. The enzyme (0.1 U/mL) in phosphate buffer saline was incubated with various concentrations of test compounds at 37 °C for 15 min. Then 1.25 mM p-nitrophenyl α-d-glucopyranoside was added to the mixture as a substrate. After further incubation at 37 °C for 30 min. The absorbance was measured spectrophotometrically at 405 nm. The sample solution was replaced by DMSO as a control. Acarbose was used as a positive control. All experiments were carried out in triplicates.
- α-Glucosidase Inhibition Assay Briefly, the sample solution (25 μL, 1.0 mM solution in DMSO) was mixed with enzyme solution (25 μL, 0.5 U/mL prepared in 0.1 M phosphate buffer, pH 6.8). The mixture was incubated at 37 ± 1 °C for 10 min to allow inhibition of enzyme by test samples. After the incubation, the substrate p-nitrophenyl α-D-glucopyranoside (25 μL, 0.5 mM, 0.1 Mphosphate buffer, pH 6.8) was added to the mixture and allowed to incubate at 37 ± 1 °C for 30 min. The reaction was then terminated by the addition of aqueous solution of sodium carbonate (100 μL, 0.2 M). Finally, absorbance was measured with a microplate reader at 405 nm. The uninhibited enzyme solution was taken as blank and an appropriate DMSO control was used. Acarbose was used as the positive control. The concentration of DMSO in the final solution was maintained below 5.0% so that the enzyme activity was not destroyed.
- Baker's Yeast α-Glucosidase Inhibition Assay The enzyme inhibition was evaluated according to the method previously reported by Rahim et al. [25] with slight modification. Various concentration of test compounds (10 µL) were dissolved in DMSO (ranging from 200 to 6.25 µg/mL) and premixed with 95 µL of 50 mM phosphate buffer (pH 6.8). Then, 25 µL of enzyme(0.0625 U/mL) in phosphate buffer saline was added into each well and the plate was incubated at 37 °C for 10 min. Afterward, 25 µl of PNPG in phosphate buffer saline (5 mM) were added and pre-read of the plate was taken by using a microplate reader (Spectrostar Nano BMG Labtech, Germany). The reaction mixture was then incubated at 37 °C for 30 min and change in absorbance at 405 nm was monitored up to 30 min. For negative control, the test samples were replaced with 10 µL of DMSO and acarbose was used as positive control
- Baker's Yeast alpha-Glucosidase Inhibition Assay The enzyme inhibition was evaluated according to the method previously reported by Taha et al. with slight modification. Various concentration of test compounds (10 uL) were dissolved in DMSO (ranging from 200 to 6.25 ug/mL) and premixed with 95 uL of 50 mM phosphate buffer (pH 6.8). Then, 25 uL of enzyme (0.0625 U/mL) in phosphate buffer saline was added into each well and the plate was incubated at 37 °C for 10 min. Afterward, 25 uL of PNPG in phosphate buffer saline (5 mM) were added and pre-read of the plate was taken by using a microplate reader (Spectrostar Nano BMG Labtech, Germany). The reaction mixture was then incubated at 37 °C for 30 min and change in absorbance at 405 nm was monitored up to 30 min. For negative control, the test samples were replaced with 10 uL of DMSO and acarbose was used as positive control. All experiments were triplicated and the results were expressed as the mean +/- S.E.M of three determinations.
- Baker's Yeast α-Glucosidase Inhibition Assay The enzyme inhibition was evaluated according to the method previously reported by Zawawi et al. [Zawawi et al., Bioorg. Chem., 23:3119] with slight modification. Various concentration of test compounds (10 μL) were dissolved in DMSO (ranging from 200 to 6.25 μg/mL) and premixed with 95 μL of 50 mM phosphate buffer (pH 6.8). Then, 25 μL of enzyme (0.0625 U/mL) in phosphate buffer saline was added into each well and the plate was incubated at 37 °C for 10 min. Afterward, 25 μl of PNPG in phosphate buffer saline (5 mM) were added and pre-read of the plate was taken by using a microplate reader (Spectrostar Nano BMG Labtech, Germany). The reaction mixture was then incubated at 37 °C for 30 min and change in absorbance at 405 nm was monitored up to 30 min. For negative control, the test samples were replaced with 10 μL of DMSO and acarbose was used as positive control. All experiments were triplicated and the results were expressed as the mean ± S.E.M of three determinations.
- Property Evaluation 3 Using the samples prepared according to Example 1 and Comparative Examples 2 and 3, inhibitory activity against α-glucosidase was evaluated to analyze activity based on structural analogs.50 μl of phosphate buffer (100 mM, pH=6.8), 10 μl of α-glucosidase (1 U/ml) and 20 μl of each of samples at various concentrations (0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2 and 0.25 mg/ml) of Example 1 and Comparative Example 2 were fed into 96-well plates and pre-incubated at 37° C. for 15 minutes. Next, 20 μl of P-NPG (5 mM) was added as a substrate and further incubated at 37° C. for 20 minutes. This reaction was stopped by adding 50 μl of Na2CO3 (0.1 M). The absorbance of released p-nitrophenol was measured at 405 nm using a multi-mode plate reader. Acarbose was set as a positive control. Each experiment was performed three times. An inhibition rate (%) was evaluated using the following equation (inhibition (%)=[1−(sample/control)]×100). IC50 values were calculated by regression analysis.