ChemistryThe synthetic route for the synthesis of bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide derivatives 5a-m has been depicted in Scheme 1. Firstly, a mixture of 3-methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde 1 and 4-hydroxycoumarin 2 in acetic acid was stirred at room temperature (RT) for 5 h to afford propargilated bis-4-hydroxycoumarin derivative 3 as an appropriate motif to participate in click reaction25. On the other hand, azide derivatives 4 were prepared according to the literature25. Finally, click reaction between bis-4-hydroxycoumarin derivative 3 and azide derivatives 4 in the presence of CuSO4.5H2O/sodium ascorbate at RT led to the formation of the desired compounds 5a-m.Scheme 1Reagents and conditions for the synthesis of bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide derivatives 5a-m.In vitro anti-α-glucosidase activities of new compounds 5a-mThe in vitro anti-α-glucosidase activities of the synthesized compounds 5a-m were evaluated against yeast form of this enzyme. The obtained data demonstrated that all new compounds 5a-m were excellent α-glucosidase inhibitors in comparison to the standard inhibitor acarbose. As can be seen from Table 1, the most potent new compound 5i was 125-folds more potent than acarbose and the less potent new compound 5k was 9.2-folds more potent than acarbose. Based on the structure–activity relationship (SAR) study on compounds 5a-m, 2,4-dichloro derivative 5i showed the best inhibitory activity against α-glucosidase. The second potent compound in the latter new compounds was 4-nitro derivative 5m. Changing the position of nitro substituent from 4-position to 3-position as in the case of compound 5l, created a negligible decrease in the inhibition effect while replacement of nitro group in both position 4 and 3 with halogens bromine and chlorine as in the case of 4-bromo derivative 5k and 3-chloro derivative 5h dramatically decreased anti-α-glucosidase activity. On the other hand, introduction of bromine and chlorine atoms on 2-position of phenyl ring of N-phenylacetamide moiety, as in the cases of compounds 5g and 5j, improved inhibition effect in comparison to presence these atoms on 4 or 3-position of latter moiety. In contrast, 2-fluoro derivative 5f. considered as a moderate inhibitor in comparison to 2-chloro and 2-bromo derivatives. Among the compounds with electron donating groups, the most potent compound was 2,4-dimethyl derivative 5d. Removal of a methyl group of compound 5d at 2-position as in the case of compound 5c or 4-position as in the case of compound 5b and replacement of methyl groups with methoxy groups as in the case of compound 5e reduced the inhibitory activity by more than half.Table 1 Structures and in vitro anti-α-glucosidase activities of bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide derivatives 5a-m.Moreover, the comparison of inhibitory activity of the substituted compounds 5b-m with un-substituted compound 5a was depicted in Fig. 2. This figure showed that introduction of 2,4-dichloro, 4-nitro, 3-nitro, 2,4-dimethyl, 2-chloro, and 2-bromo substituents on the phenyl ring of N-phenylacetamide moiety improved inhibitory activity in comparison to N-phenylacetamide derivative 5a while the presence of 4-methyl, 2-methyl, 2,4-dimethoxy, 2-fluoro, 4-bromo, and 3-chloro substituents decreased anti-α-glucosidase activity.Figure 2The comparison of anti-α-glucosidase activity of un-substituted compound 5a with substituted compounds 5b-m.The comparison of IC50 values of the new compounds 5 with its template compounds A and B is showed in Fig. 324,25. As can be seen in this figure, anti-α-glucosidase activity range of new compounds 5 was approximately better than anti-α-glucosidase activity ranges of compounds A and B that were used for design of compounds 5.Figure 3Comparison of anti-α-glucosidase activity of new compounds 5 with templates A and B.Enzyme kinetic studyThe inhibition mechanism of the target compounds was evaluated by kinetic study. For this propose, the most potent compound 5i as a representative compound was selected. Our result is shown in Fig. 4. As can be seen in this figure, with the increase of the compound 5i concentration both of Km and Vmax were decreased and four parallel lines for four different concentrations of this compound were obtained. This finding suggested uncompetitive inhibition mode for compound 5i.Figure 4Molecular dockingMolecular docking simulations were carried on the newly synthesized compounds in the active site of α-glucosidase in order to calculate the binding energies and determine key interactions that contribute to the most stable complex conformations. The binding energies and 2D images of interaction modes were obtained by Autodock Tools (1.5.6) and BIOVIA Discovery Studio v.3.5 programs, respectively.In the first step, superimpose structure of acarbose and the most potent compound 5i was obtained (Fig. 5). As can be seen in Fig. 5, compound 5i was well placed in the active site of α-glucosidase.Figure 5Acarbose (pink) and most potent compound 5i (orange) superimposed in the active site pocket.In the second step, in addition to the most potent compound 5i, a number of new compounds that were important in terms of inhibitory effect and SAR, selected and docked in the active site of the target enzyme. Structures, IC50 values, binding energies, and interaction modes of these compounds were showed in Figs. 6 and 7.Figure 6Structures, IC50 values, and binding energies of the selected compounds 5a, 5j, 5g, and 5i.Figure 7The predicted binding modes of compounds 5a, 5j, 5g, and 5i in the active site pocket.Un-substituted compound 5a with binding energy of − 8.44 kcal/mol attached to active site of α-glucosidase (Fig. 6). IC50 value of this compound was 37.1 µM (Table 1 and Fig. 6). Introduction of bromine or chlorine atom on 2-position of N-phenylacetamide, as in the cases of compounds 5j and 5g, created a negligible increase in the inhibitory activities. This data was in agreement with the observed binding energies for compounds 5j and 5g in comparison binding energy of un-substituted compound 5a. As can be seen in Fig. 6, addition of a chlorine atom on 4-position of 2-chloro derivative 5g, as in the case of compound 5i, dramatically increased anti-α-glucosidase activity and decreased binding energy. This figure also showed that binding energy of compound 5i was lower than binding energies of compounds 5a and 5g. Therefore, the observed in vitro (IC50 value) data for the selected compounds 5a, 5j, 5g, and 5i was completely confirmed by the obtained in silico (binding energy) data.Interaction modes of the selected compounds 5a, 5j, 5g, and 5i were showed in Fig. 7. Un-substituted compound 5a established two hydrogen bonds with Asn241 and Arg312 and a non-classical hydrogen bond Pro309. This compound also formed two π-cation interactions with His239 and His279, a π-anion interaction Glu304, and several hydrophobic interactions with Arg312, His279, Pro309, and Val305. 2-Bromo derivative 5j formed three classical hydrogen bonds with His279, Glu304, and Thr307 and a non-classical hydrogen bond Thr307. Compound 5j created two π-cation interactions with His239 and a π-anion interaction Asp408. This compound established hydrophobic interactions with Pro309 and Arg312. Compound 5g with 2-chloro substituent interacted with Thr307 via a hydrogen bond and Thr301, Arg312, and His239 via three non-classical hydrogen bonds. This compound formed a π-anion interaction with Glu304 and a π-cation interaction with His279. Compound 5g also established several hydrophobic interaction with Ser308, His279, Pro309, and Phe311. Finally, the most potent compound 5i formed the following interactions with the active site of α-glucosidase: six hydrogen bonds with Phe157, His239, Thr307, Gly306, Gln322, and Thr301, two non-classical hydrogen bonds with Arg312 and His279, a π-anion interaction with Glu304, and several hydrophobic interactions with Arg312, Pro309, Val316, Thr301, Ala326, and His279.Molecular dynamicsThe interaction between a substrate and the active site of an enzyme, similar to other molecular events, exhibits a dynamic nature. Thus, simulating and analyzing enzyme–substrate complex behavior in a natural-like environment, incorporating water and ions, can yield insights into the stability and flexibility of receptor-ligand complexes. In this study, molecular dynamics (MD) simulation was utilized to analyze the docking files of both acarbose, acting as the standard inhibitor, and compound 5i, identified as the most potent inhibitor against α-glucosidase in vitro. The aim was to assess the stability and flexibility of the enzyme-ligand complex within an explicitly hydrated environment throughout the simulation time. Two types of simulations were conducted. Initially, a 10 ns simulation was run for all complexes. It was found that both acarbose and 5i were stable at the α-glucosidase active site during this phase. Therefore, the simulation was extended by another 10 ns to explore deeper into the behavior of these compounds within the active site. They remained stable throughout the extended simulation. Following this, the simulation trajectories of these compounds underwent additional analysis using various tools.To assess the stability of the complexes, root-mean-square deviation (RMSD) and radius of gyration (Rg) calculations were conducted for all structures in the trajectory. Graphs depicting changes over time were generated using these calculations. Furthermore, the root mean square fluctuation (RMSF) of the backbone atoms in α-glucosidase and the heavy atoms in the ligands were computed to evaluate the residual flexibility of the enzyme and the flexibility of the ligand atoms, respectively. According to Fig. 8, the RMSD calculations suggest that α-glucosidase maintains a consistently low RMSD value, staying below 3 Å throughout the entire simulation time. This pattern implies a stable protein structure. Specifically, the average RMSD values for α-glucosidase in complex with acarbose and/or 5i were 1.73 Å and 2.23 Å, respectively. Additionally, the RMSD values of acarbose and/or 5i in complex with α-glucosidase consistently remained below 2 Å, with average RMSD values of 1.91 Å for acarbose and 1.45 Å for 5i. These results indicate stable structures for both α-glucosidase and the ligands. Certain ligands and conditions have the potential to reduce protein compactness and stability. In this study, the radius of gyration (Rg) of α-glucosidase in complex with acarbose and 5i was computed throughout the simulation, serving as an indicator of protein compactness (Fig. 8). The average Rg of α-glucosidase when bound to acarbose and 5i was found to be 25.3 and 24.9 Å, respectively. Throughout the simulation, Rg values fluctuated within the narrow range of 23.8 to 25.5 Å, suggesting the presence of stable protein structures.Figure 8Superimposed RMSD of Cα atoms of α-glucosidase in complex with 5i (green) and acarbose (blue) (A). Superimposed RMSD of 5i (green) and acarbose (blue) in complex with α-glucosidase (B). Time dependence of the radius of gyration (Rg) graph of α-glucosidase in complex with 5i (green) and acarbose (blue) (C).In a protein, all atoms and residues experience some level of fluctuation. The fluctuation patterns of α-glucosidase residues in complex with acarbose and 5i are depicted in Fig. 9. According to this figure, the RMSF of α-glucosidase residues in the complex with acarbose and 5i are notably similar and almost identical. α-Glucosidase, a large protein with 579 residues and multiple domains with distinct structures and functions, displays varying degrees of fluctuation in different parts, as shown in Fig. 9. The enzyme’s active site is located in a cleft between two domains, namely the A domain and B domain. Residues from these domains, involved in non-bonded interactions with ligands, exhibit minimal fluctuations. Conversely, residues situated in loop regions, such as the B domain loop and the active site lid, display higher levels of fluctuation, as expected. In Fig. 9, the fluctuation of heavy atoms in acarbose and 5i is depicted too. Notably, the RMSF of all heavy atoms in these ligands remains below 1.5 Å. This limited fluctuation suggests a stable complex with α-Glucosidase, indicating that intermolecular interactions effectively restrict their movements.Figure 9RMSF graph of the Cα atoms of α-glucosidase in complex with 5i (green) and acarbose (blue) (A). Close-up representation of α-glucosidase active site (B). RMSF graph of the heavy atoms of 5i (C) and acarbose (D) in complex with α-glucosidase. Structure of these compounds are illustrated.Pharmacokinetic and toxicity predictionPharmacokinetic (ADME) and toxicity properties of acarbose and the most potent compound 5i were predicted by an online server and the obtained data were listed in Table 2. As can be seen in this table, acarbose and compound 5i did not follow of Lipinski ‘Rule of five’. These compounds had poor permeability to Caco-2 and acceptable permeability to blood brain barrier (BBB) and skin. Acarbose did not have human intestinal absorption (HIA) while Compound 5i had high HIA. In term of in silico toxicity evaluation, our new compound 5i demonstrated good properties in comparison to acarbose. As can be seen Table 2, acarbose had carcinogen effect on mouse and was mutagen while compound 5i did not have carcinogenicity and was non-mutagen.Table 2 ADME and toxicity prediction of acarbose and the most potent compound 5i.ConclusionIn this work, we have synthesized a class of thirteen compounds 5a-m based on bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide scaffold and they assessed for α-glucosidase inhibitory potential. All the synthesized compounds were recognized as excellent inhibitors of α-glucosidase in comparison to standard inhibitor acarbose. Among these series, compound 5i showed considerable activity against α-glucosidase and found many folds more potent than acarbose (125-fold). Additionally, to find out the binding interactions and binding energies molecular docking and molecular dynamics studies were conducted against α-glucosidase. The obtained results confirmed observed in vitro data and shown that selected compounds with favorable binding energies interacted with α-glucosidase. In silico pharmacokinetics and toxicity studies predicted that in term of pharmacokinetics, the most potent compound 5i was approximately similar to acarbose and in term of toxicity, compound 5i was better than acarbose. Future studies on bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide scaffold could involve additional optimization of the analogs and in vivo assessments to understand its efficacy of this scaffold as a new class of anti-diabetic agents.ExperimentalGeneral synthesis for bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide derivatives 5a-mA mixture of 3,3′-((3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) 3 (1 mmol), in situ prepared azide derivatives 4 (1 mmol), and sodium ascorbate (1 mmol), and CuSO4.5H2O (7 mol %) in H2O/t-BuOH (10 ml, 1:1) was stirred at RT for 24–48 h25. After that, reaction mixture was poured into crushed ice and precipitated products 5a-m were filtered off, washed with cold water, and purified by recrystallization in ethyl acetate.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-phenylacetamide (5a)Brown solid; Yield: 84%; MP = 203–205 °C; IR (KBr, vmax) 3378 (NH), 3020 (CH Aromatic), 2885 (CH Aliphatic), 1661 (C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 10.38 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.71 (d, J = 7.8 Hz, 2H, HAr), 7.48 (d, J = 8.3 Hz, 2H, HAr), 7.44–7.35 (m, 2H, HAr), 7.27–7.19 (m, 2H, HAr), 7.19–7.06 (m, 4H, HAr), 6.98 (t, J = 7.0 Hz, 1H, HAr), 6.84 (d, J = 8.2 Hz, 1H, HAr), 6.57 (s, 1H, HAr), 6.53 (d, J = 8.4 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.23 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm; 13C NMR (75 MHz, DMSO-d6) δ 168.18, 165.01, 164.67, 152.94, 148.95, 145.74, 138.89, 135.96, 131.34, 129.38, 126.74, 124.58, 124.21, 123.36, 120.42, 119.65, 119.30, 115.93, 113.59, 111.90, 104.08, 62.17, 52.66, 46.19, 36.24 ppm; Anal. Calcd: C37H28N4O9; C, 66.07; H, 4.20; N, 8.33; Found; C, 66.32; H, 4.38; N, 8.56.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(o-tolyl)acetamide (5b)Brown solid; Yield:78%; MP = 207–209 °C; IR (KBr, vmax) 3357 (NH), 3070 (CH Aromatic), 2980 (CH Aliphatic), 1677 (C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 9.69 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.71 (d, J = 7.8 Hz, 2H, HAr), 7.39 (t, J = 7.7 Hz, 2H, HAr), 7.33 (d, J = 7.9 Hz, 1H), 7.20–7.08 (m, 5H, HAr), 7.08–6.94 (m, 2H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.58 (s, 1H, HAr), 6.53 (d, J = 8.4 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.28 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3), 2.12 (s, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.18, 165.02, 164.86, 152.94, 148.94, 145.74, 135.94, 132.04, 131.34, 130.90, 126.52, 126.00, 125.18, 124.57, 123.36, 120.41, 119.29, 115.92, 113.58, 111.86, 104.08, 62.14, 52.37, 46.18, 36.24, 18.28 ppm; Anal. Calcd: C38H30N4O9; C, 66.47; H, 4.40; N, 8.16; Found; C, 66.62; H, 4.59; N, 8.37.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(p-tolyl)acetamide (5c)Cream solid; Yield: 77%; MP = 190–192 °C; IR (KBr, vmax) 3252 (NH), 3025 (CH Aromatic), 2970 (CH Aliphatic), 1660 (C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 10.29 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.71 (d, J = 7.7 Hz, 2H, HAr), 7.46–7.30 (m, 4H, HAr), 7.19–7.07 (m, 4H, HAr), 7.02 (d, J = 8.1 Hz, 2H, HAr), 6.84 (d, J = 8.3 Hz, 1H, HAr), 6.57 (s, 1H, HAr), 6.52 (d, J = 8.4 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.21 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3), 2.15 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6) δ 168.21, 165.03, 164.41, 152.94, 148.95, 145.75, 136.38, 135.96, 133.19, 131.34, 129.75, 124.58, 123.36, 120.43, 119.66, 119.30, 115.93, 113.59, 111.90, 104.09, 62.18, 52.65, 46.19, 36.24, 20.91 ppm. Anal. Calcd: C38H30N4O9; C, 66.47; H, 4.40; N, 8.16; Found; C, 66.59; H, 4.63; N, 8.31.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2,4-dimethylphenyl)acetamide (5d)Cream solid; Yield: 81%; MP = 195–197 °C; IR (KBr, vmax). 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 9.62 (s, 1H, NHAmid), 8.10 (s, 1H, HTriazol), 7.72 (d, J = 7.8 Hz, 2H, HAr), 7.46–7.35 (m, 2H, HAr), 7.21–7.08 (m, 5H, HAr), 6.92 (s, 1H, HAr), 6.90–6.81 (m, 2H, HAr), 6.58 (s, 1H, HAr), 6.53 (d, J = 8.4 Hz, 1H, HAr), 6.11 (s, 1H, HBis), 5.26 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3), 2.14 (s, 3H, CH3), 2.07 (s, 3H, CH2) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.24, 165.06, 164.80, 152.95, 148.96, 145.76, 135.96, 135.14, 133.37, 132.01, 131.39, 127.02, 126.70, 125.25, 124.59, 123.38, 120.43, 119.31, 115.94, 113.62, 111.90, 62.18, 52.37, 46.19, 36.25 ppm; Anal. Calcd: C39H32N4O9; C, 66.85; H, 4.60; N, 8.00; Found C, 67.02; H, 4.78; N, 8.21.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2,4-dimethoxyphenyl)acetamide (5e)Cream solid; Yield: 76%; MP = 199–201 °C; IR (KBr, vmax) 3400 (NH), 3020(CH Aromatic), 2975(CH Aliphatic) 1701(C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 9.50 (s, 1H, NHAmid), 8.08 (s, 1H, HTriazol), 7.71 (d, J = 7.8 Hz, 2H, HAr), 7.61 (d, J = 8.8 Hz, 1H, HAr), 7.46–7.33 (m, 2H, HAr), 7.19–7.06 (m, 4H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.57 (s, 1H, HAr), 6.57–6.48 (m, 2H, HAr), 6.38 (dd, J = 8.8, 2.7 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.27 (s, 2H, CH2), 4.95 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 3.41 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.19, 165.00, 164.57, 157.46, 152.93, 151.76, 148.93, 145.76, 135.95, 131.34, 126.68, 124.58, 123.81, 123.36, 120.42, 120.03, 119.29, 115.93, 113.57, 111.87, 104.53, 104.08, 99.31, 62.17, 56.20, 55.83, 55.75, 52.51, 46.19, 36.22 ppm; Anal. Calcd: C39H32N4O11; C, 63.93; H, 4.40; N, 7.65; Found; C, 64.05; H, 4.58; N, 7.82.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-fluorophenyl)acetamide (5f)Cream solid; Yield: 65%; MP = 217–219 °C; IR (KBr, vmax) 3291 (NH), 3065 (CH Aromatic), 2980(CH Aliphatic), 1675 (C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 10.22 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.88 7.76 (m, 1H, HAr), 7.72 (d, J = 7.7 Hz, 2H, HAr), 7.45–7.35 (m, 2H, HAr), 7.25–7.11 (m, 5H, HAr), 7.11–7.02 (m, 2H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.58 (s, 1H, HAr), 6.54 (d, J = 8.7 Hz, 1H, HAr), 6.11 (s, 1H, HBis), 5.33 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.23, 165.32, 165.05, 155.53, 152.94, 152.26, 148.96, 145.76, 135.97, 131.36, 126.80, 126.01, 124.99, 124.58, 124.21, 123.37, 120.42, 119.31, 116.20, 115.94, 113.63, 111.90, 104.09, 62.19, 52.45, 46.19, 36.25 ppm; Anal. Calcd: C37H27FN4O9; C, 64.35; H, 3.94; N, 8.11; Found; C, 64.51; H, 4.08; N, 8.34.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-chlorophenyl)acetamide (5g)Cream solid; Yield: 81%; MP = 194–196 °C; IR (KBr, vmax) 3264 (NH), 3025(CH Aromatic), 2960 (CH Aliphatic),1662(C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 9.98 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.71 (d, J = 7.8 Hz, 2H, HAr), 7.64 (d, J = 8.1 Hz, 1H, HAr), 7.52–7.33 (m, 3H, HAr), 7.29–7.20 (m, 1H, HAr), 7.21–7.04 (m, 5H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.58 (s, 1H, HAr), 6.53 (d, J = 8.5 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.34 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.20, 165.38, 165.04, 152.94, 148.95, 145.75, 135.94, 134.62, 131.36, 130.08, 128.02, 127.16, 126.72, 126.35, 124.58, 123.38, 120.40, 119.30, 115.94, 113.61, 111.90, 104.10, 62.18, 52.39, 46.19, 36.23 ppm; Anal. Calcd: C37H27ClN4O9; C, 62.85; H, 3.85; N, 7.92; Found; C, 63.04; H, 4.07; N, 8.10.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-chlorophenyl)acetamide (5h)Cream solid; Yield: 68%; MP = 215–217 °C; IR (KBr, vmax) 3235 (NH), 3030 (CH Aromatic), 2970 (CH Aliphatic), 1684 (C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 9.98 (s, 1H, NHAmid), 8.12 (s, 1H, HTriazol), 7.71 (d, J = 7.8 Hz, 2H, HAr), 7.64 (d, J = 8.0 Hz, 1H, HAr), 7.57–7.29 (m, 4H, HAr), 7.23 (t, J = 7.7 Hz, 1H, HAr), 7.20–7.08 (m, 4H, HAr), 6.84 (d, J = 8.5 Hz, 1H, HAr), 6.58 (s, 1H, HAr), 6.53 (d, J = 8.3 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.34 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.21, 165.37, 165.04, 152.94, 148.95, 145.75, 135.97, 134.62, 131.34, 130.08, 128.02, 127.16, 126.71, 126.35, 124.58, 123.36, 120.42, 119.31, 115.93, 113.62, 111.91, 104.10, 62.20, 52.40, 46.19, 36.23 ppm; Anal. Calcd: C37H27ClN4O9; C, 62.85; H, 3.85; N, 7.92; Found; C, 63.02; H, 3.98; N, 8.11.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2,4-dichlorophenyl)acetamide (5i)Brown solid; Yield: 79%; MP = 211–213 °C; IR (KBr, vmax) 3378 (NH), 3065 (CH Aromatic), 2990 (CH Aliphatic), 1703 (C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 10.06 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.75–7.64 (m, 2H, HAr), 7.64–7.57 (m, 1H, HAr), 7.45–7.29 (m, 4H, HAr), 7.19–7.06 (m, 4H, HAr), 6.83 (d, J = 8.4 Hz, 1H, HAr), 6.58 (s, 1H, HAr), 6.53 (d, J = 7.5 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.35 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm 13C NMR (75 MHz, DMSO-d6) δ 168.18, 165.55, 165.00, 152.93, 148.94, 145.72, 135.98, 133.85, 131.33, 130.20, 129.52, 128.16, 127.53, 127.28, 124.57, 123.34, 120.42, 119.29, 115.92, 113.61, 111.90, 104.08, 62.18, 52.38, 46.19, 36.23.ppm; Anal. Calcd: C37H26Cl2N4O9; C, 59.93; H, 3.53; N, 7.56; Found; C, 60.6; H, 3.69; N, 7.74.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-bromophenyl)acetamide (5j)Cream solid; Yield: 88%; MP = 205–207 °C; IR (KBr, vmax) 3253 (NH), 3030 (CH Aromatic), 2975(CH Aliphatic), 1661(C=O) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 9.91 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.71 (d, J = 9.2 Hz, 2H, HAr), 7.62–7.51 (m, 2H, HAr), 7.44–7.35 (m, 2H, HAr), 7.32–7.24 (m, 1H, HAr), 7.20–7.08 (m, 4H, HAr), 7.07–7.03 (m, 1H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.57 (s, 1H, HAr), 6.53 (d, J = 8.6 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.32 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.20, 165.29, 165.02, 152.94, 148.94, 145.75, 135.96, 133.27, 131.34, 128.60, 127.90, 127.34, 126.72, 124.58, 123.36, 120.42, 119.30, 118.03, 115.93, 113.61, 111.91, 104.09, 62.15, 52.39, 46.19, 36.23 ppm; Anal. Calcd: C37H27BrN4O9; C, 59.13; H, 3.62; N, 7.45; Found; C, 59.35; H, 3.79; N, 7.61.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-bromophenyl)acetamide (5k)Brown solid; Yield: 69%; MP = 217–219 °C; IR (KBr, vmax) 3371 (NH), 3040 (CH Aromatic), 2985 (CH Aliphatic), 1669 (C=O) Cm−1 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 10.52 (s, 1H, NHAmid), 8.11 (s, 1H, HTriazol), 7.71 (d, J = 7.8 Hz, 2H, HAr), 7.54–7.35 (m, 7H, HAr), 7.21–7.06 (m, 4H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.57 (s, 1H, HAr), 6.53 (d, J = 8.2 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.24 (s, 2H, CH2), 4.96 (s, 2H, CH2), 3.41 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.24, 165.07, 164.92, 152.94, 148.96, 145.75, 138.26, 135.96, 115.94, 113.62, 111.91, 104.10, 62.18, 52.68, 46.20, 36.24 ppm; Anal. Calcd: C37H27BrN4O9; C, 59.13; H, 3.62; N, 7.45; Found; C, 59.32; H, 3.83; N, 7.67.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-nitrophenyl)acetamide (5l)Brown solid; Yield: 81%; MP = 216–218 °C; IR (KBr, vmax) 3343 (NH), 3040 (CH Aromatic), 2980 (CH Aliphatic), 1674 (C=O), 1560–1355 (NO2) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.50 (s, 1H, OH), 10.89 (s, 1H, NHAmid), 8.13 (s, 1H, HTriazol), 7.92–7.78 (m, 2H, HAr), 7.71 (d, J = 7.7 Hz, 2H, HAr), 7.61–7.45 (m, 2H, HAr), 7.45–7.33 (m, 2H, HAr), 7.21–7.06 (m, 4H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.57 (s, 1H, HAr), 6.53 (d, J = 8.9 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.30 (s, 2H, CH2), 4.97 (s, 2H, CH2), 3.42 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.21, 165.60, 165.04, 152.94, 148.96, 148.43, 145.73, 139.96, 135.97, 131.34, 130.90, 125.69, 124.57, 123.36, 120.42, 119.30, 118.78, 115.93, 113.84, 113.61, 111.91, 104.09, 62.16, 52.69, 46.20, 36.23 ppm; Anal. Calcd: C37H27N5O11; C, 61.92; H, 3.79; N, 9.76; Found; C, 62.11; H, 3.96; N, 9.98.2-(4-((4-(Bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)-2-methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-nitrophenyl)acetamide (5m)Brown solid; Yield: 81%; MP = 224–226 °C; IR (KBr, vmax) 3331 (NH), 3055 (CH Aromatic), 2975 (CH Aliphatic), 1669 (C=O), 1545–1345 (NO2) Cm−1; 1H NMR (300 MHz, DMSO-d6) δ 17.51 (s, 1H, OH), 10.99 (s, 1H, NHAmid), 8.22–8.07 (m, 3H, HTriazol, HAr), 7.78–7.68 (m, 4H, HAr), 7.45–7.35 (m, 2H, HAr), 7.24–7.04 (m, 4H, HAr), 6.84 (d, J = 8.4 Hz, 1H, HAr), 6.58 (s, 1H, HAr), 6.53 (d, J = 8.3 Hz, 1H, HAr), 6.10 (s, 1H, HBis), 5.33 (s, 2H, CH2), 4.98 (s, 2H, CH2), 3.42 (s, 3H, OCH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ 168.26, 167.90, 165.85, 165.09, 152.94, 148.98, 145.73, 144.99, 143.02, 135.97, 131.36, 126.79, 125.58, 124.58, 123.38, 120.42, 119.49, 119.31, 115.94, 113.63, 111.91, 104.10, 62.16, 52.79, 46.20, 36.26 ppm; Anal. Calcd: C37H27N5O11; C, 61.92; H, 3.79; N, 9.76; Found; C, 62.04; H, 4.02; N, 10.09.In vitro α-glucosidase inhibition and kineticsThe in vitro α-glucosidase inhibitory activity of bis-4-hydroxycoumarin-phenoxy-1,2,3-triazole-N-phenylacetamide derivatives 5a-m was determined according to the previously reported methods25,26. For this purpose, enzyme (Saccharomyces cerevisiae α-glucosidase) solution (20 μL), target compounds (dissolved in DMSO with 10% final concentration) in the various concentrations (20 μL), potassium phosphate buffer (PPB/135 μL) were incubated for 10 min at 37 °C in a 96-well plat25. Then, substrate (p-nitrophenyl-α-glucopyranoside/4 mM/25 μL) was added to the latter plate and the obtained mixture was incubated at 37 °C for 20 min. Finally, absorbance was measured (405 nm, Gen5, Power wave xs2, BioTek, USA) and IC50 values of the compounds 5a-m were calculated (logit method). Kinetics was carried out to determine the inhibition mechanism of the most potent compound 5i. The enzyme solution (0.2 U/mL) with different concentrations of compound 5i (0, 1.5, 3, and 6 μM) was incubated for 15 min at 37°C25. After that, the enzymatic reaction was initiated by adding various concentrations of substrate (1–4 mM) and change in absorbance was measured for 20 min at 405 nm (Spectra Max M2, Molecular Devices, CA, USA)25.Molecular docking and molecular dynamicsThe molecular docking and molecular dynamics studies in the active site of α-glucosidase were carried out using by previously described methods26,27.ADME and toxicity predictionIn silico druglikeness study and ADME/T prediction were obtained from the preADMET online server (https://preadmet.bmdrc.org/)28.
