Synthesis, α-Glucosidase Inhibitory Activity and Molecular Docking Study of Chalcone Derivatives Bearing a 1H-1,2,3-Triazole Unit

Abstract

Diabetes mellitus (DM) is a metabolic condition that is a major health concern around the world. The current study investigates the synthesis of a series of chalcone and 1H-1,2,3-triazole hybrid compounds and their in vitro inhibitory potential against α-glucosidase. The antidiabetic analysis revealed that compounds 4a and 4b are highly active agents with IC₅₀ of 3.90 and 4.77 µM, respectively. These results are close to quercetin (IC₅₀ = 4.24 µM) as the reference standard. Molecular docking study strongly supports the active interaction of the 4a and 4b to the enzyme through cation–π interaction and hydrogen bonding between the ligands and the active site of Saccharomyces cerevisiae α-glucosidase enzyme. This study broadened the potential of designing chalcone-triazole hybrid compounds as antidiabetic drug candidates in the pharmaceutical sector.

Introduction

Diabetes Mellitus (DM) is a metabolic illness with numerous etiologies characterized by a loss of glucose homeostasis with changes in carbohydrate, lipid, and protein metabolism as a result of deficiencies in insulin secretion and/or insulin action.¹⁾ It is one of the top ten causes of death, accounting for about 1 million fatalities worldwide.²⁾ Elevated blood glucose levels are a risk factor that can lead to a variety of problems in organs such as the heart, liver, eyes, kidneys, and nerves.³⁾ Because most of the currently available medications have a variety of side effects, effective treatment of diabetes with new drugs is seen as a critical undertaking for the medical community.⁴⁾ Therefore, from the viewpoint of medicinal chemists, it is important to design novel antidiabetic candidates through synthetic organic chemistry approach.

The growing potent literature of recent years has been paid to chalcone derivatives (Fig. 1A). Chalcones are one of the most important types of flavonoids in the plant kingdom.⁵⁾ For instance, two naturally occurring chalcones have been successfully isolated from Caesalpinia sappan and Piper hispidum, possessing biological activities.⁶⁾ From the past decades, the natural as well as the synthetic chalcones have been known to exhibit various pharmacological significance such as antibacterial,^7–9) antidiabetic,^10–12) antioxidant,^13–15) anticancer,^16–18) and anti-inflammatory^19–21) activities. Additionally, chalcone-based molecules have gained considerable attention due to their role as antitumor²²⁾ and cytotoxic²³⁾ agents.

Fig. 1. Design of Molecules Bearing Chalcone-Triazole Hybrids

Because its structure is present in a significant number of biologically active compounds, including several medications already on the market, triazole heterocycles are preferred scaffolds in medicinal chemistry²⁴⁾ (Fig. 1B). 1,2,3-Triazole skeletons have received much attention in recent years because of their ubiquitous use in chemical biology, synthetic organic chemistry, supramolecular chemistry, fluorescence imaging, drug discovery, materials, and polymer chemistry.²⁵⁾ These structures are easily accessible through click chemistry, such as azide-alkyne cycloadditions.²⁶⁾ In addition, they possess unique inherent features that provide stability to the fundamental pharmacophoric unit while also serving as a bioisostere of several chemical functionalities.²⁷⁾ Focusing on pharmacological advantages, they have been well-known for antidiabetic,^28–30) anticancer,^31–33) and antimicrobial^34–39) activities.

With the advantages of the chalcone group as well as the 1,2,3-triazole scaffold, we report here the synthesis of a series of chalcone derivative compounds integrated with 1,2,3-triazole moiety and evaluation of their activity toward the discovery of the more potent antidiabetic agents (Fig. 1C).

Results and Discussion

Chemistry

The target compounds 4a–4e were synthesized by a three-step procedure⁴⁰⁾ (Chart 1). Firstly, diazotization of p-nitroaniline 1 followed by azidation through nucleophilic aromatic substitution afforded 1-azido-4-nitrobenzene 2. Without further purification, 2 was directly submitted to the enolate-mediated triazole synthesis.⁴¹⁾ With acetylacetone in N,N-dimethylformamide in the presence of triethylamine, triazole methyl ketone 3, a common intermediate of candidate molecules, was given in good yield over two steps. With the common intermediate 3 in hand, it was submitted to the catalytic aldol condensation with the several aryl or alkenyl aldehydes to afford the target chalcone triazole hybrid compounds 4a–4e. As an aryl group conjugated with enone moiety, electron-rich aryl and electron-poor (hetero)aryl groups were successfully introduced into the products. To compare with the chalcone structures, an additional conjugated olefin was also introduced to evaluate the potential 1,4- or 1,6-addition reaction with the appropriate active sites. Thus, phenyl and electron-donative p-dimethylaminophenyl-substituted α,β,γ,δ-unsaturated ketones 4d, 4e were synthesized. All the synthesized compounds were characterized by IR, high resolution (HR)-MS, ¹H- and ¹³C-NMR.

Chart 1. Synthesis of Chalcone-Triazole Hybrids (4a–4e)

α-Glucosidase Inhibitory Activity

α-Glucosidase is a digestive enzyme that catalyzes the breakdown of polysaccharides into monosaccharides (glucose), which the colon can only convey into the blood circulation, causing a high risk of diabetes. Thus, inhibition of α-glucosidase is an essential approach in oral diabetes therapy.⁴²⁾ The triazole methyl ketone 3 and final products chalcone-triazole derivatives 4a–4e were screened for their antidiabetic potential via inhibition of α-glucosidase. The results of antidiabetic testing of the synthesized compounds are presented in Table 1. From the results, it has been revealed that all the screened compounds possess good to excellent antidiabetic activity against α-glucosidase enzyme. Parent compound triazole chalcone 3 showed good inhibitory activity with IC₅₀ of 20.06 µM. Compounds 4a (Ar = 4-methoxyphenyl) and 4b (Ar = 4-fluorophenyl) demonstrated an excellent antidiabetic activity with IC₅₀ values of 3.90 and 4.77 µM, respectively. These values are similar to Quercetin (IC₅₀ = 4.24 µM) as the reference standard. From the results, both strong electron-donating and electron-withdrawing groups are suitable for inhibition, and may be a key factor for enhancing the activity. The compound with diene skeleton gave lower activity (4d, IC₅₀ = 54.92 µM) than those of other candidates. In contrast, as demonstrated above, the presence of an electron-donating group on the benzene ring improved the activity (4e, IC₅₀ = 29.67 µM). Replacing the phenyl group in the molecule with the pyridine ring of heterocycle (4c) gave good inhibitory activity, but the activity is still lower than 4a and 4b.

Table 1. α-Glucosidase Inhibitory Activity by the Synthesized Compounds

Compound	IC50 values (µM)
3	20.06 ± 0.04
4a	3.90 ± 0.00
4b	4.77 ± 0.06
4c	23.11 ± 0.21
4d	54.92 ± 0.03
4e	29.67 ± 0.07
Quercetin	4.24 ± 0.03

Molecular Docking Study

Molecular docking studies were carried out to predict the binding interactions of the synthesized compounds in the active site of Saccharomyces cerevisiae α-glucosidase enzyme. The binding site of the quercetin and our compounds were predicted according to the previous studies, which they are using the similar compounds as we used in this study suggesting that the binding site should be similar each other.^43,44) In this study, the docking study takes place in the catalytic site of the α-glucosidase, which is located in the Asp214, Glu276, and Asp349, the catalytic triad of the respective enzyme. Furthermore, these catalytic triads are surrounded by several amino acid residues, such as Lys155, Phe157, Phe158, Phe177, Thr215, Leu218, Ala278, and Arg312, which are responsible for providing the non-covalent interactions of either enzyme substrate and inhibitor that stabilized the binding interactions in its active site.⁴⁴⁾ As displayed in Table 2, the compound 4b was predicted to have the lowest ΔG_binding value among all at −9.8 kcal/mol, followed by 4a, quercetin, 4c, 4d, and 4e, with the ΔG_binding value of −9.1, −9.0, −8.7, −8.7, and −8.5 kcal/mol, respectively. From the post-docking analysis, as depicted in Fig. 2, the binding pose of 4a was observed to form a strong π–π interaction between the aromatic site of 4a ligand with the negatively charged –COO⁻ group in the side chain of Asp349, while also formed a hydrogen bonding with the Asp68 and Arg312. As for the ligand 4b, although there were no non-covalent interactions made between the ligand and the catalytic triad, the binding pose seems to be stable due to the formed hydrogen bond (Arg312) and cation–π interactions (Lys155, Phe157, Leu218, Ala278, Arg312, Asp408).

Table 2. The Docking Result of Synthesized Compounds in the Active Site of α-Glucosidase

No.	Compounds	ΔGbinding (kcal/mol)	Binding residues
1	4a	−9.1	H-Bond: Asp68, Arg312
			π–π interaction: Tyr71, His111, Lys155, Phe157, Phe177, Asp349
2	4b	−9.8	H-Bond: Arg312
			Cation–π interaction: Lys155, Phe157, Leu218, Ala278, Arg312, Asp408
3	4c	−8.7	H-Bond: Lys155, Glu276, Phe311Cation–π interaction: Lys155, Phe157, Phe158, Thr215, Leu218, Ala278, Arg312
4	4d	−8.7	H-Bond: Lys155, Phe311, Arg439
			Cation–π interaction: Lys155, Phe157, Phe158, Phe177, Leu218, Ala278, Arg312
5	4e	−8.5	H-Bond: Glu276, Glu278, Thr307, Arg312, Asp349
			Cation–π interaction: Phe157, Phe158, Phe177, Phe239, His279, Glu304, Ser308, Pro309
S1	Quercetin	−9.0	H-Bond: Phe157, Asp214, Asp349
			Cation–π interaction: Phe157, Arg312, Arg439

Fig. 2. (a) The Predicted 3D Structure of Saccharomyces cerevisiae α-Glucosidase Enzyme with Ligand 4a (Yellow) and 4b (Blue), and the Binding Interactions of (b) 4a and (c) 4b in the Catalytic Site of α-Glucosidase Enzyme, Respectively

Interestingly, the nitro group of all of the synthesized ligands, apart from 4e, form a strong interaction with the side chain of Lys155 residue through cation–π interaction (4a, 4b) and hydrogen bonding interaction (4c, 4d). This phenomenon is caused by the fact that Lys residue has a protonated aliphatic amine group (–NH₃⁺) in its side chain, which is susceptible to the electrostatic interaction from the negatively charged functional group, such as nitro group. Furthermore, strong interaction with Arg312 was also observed in all docked ligands, either through hydrogen bonding (4a, 4b, 4e) or cation–π interactions (4b, 4c, 4d, quercetin). As a result, the binding poses of the ligands in their nitrobenzene and triazole rings are similarly positioned, whereas the difference lies in the remaining group. For instance, ligand 4a forms additional hydrogen bonding with Asp68, while ligand 4c interacts with Glu276 and Phe311 through hydrogen bonding as well. Additionally, all the binding interactions involve the α, β-unsaturated ketone group from the chalcone structure, which is existed in all of the synthesized ligands. Therefore, these two residues might play an important role in the binding stability of the synthesized compounds in their function as the α-glucosidase inhibitor.

Conclusion

A series of chalcone-1H-1,2,3-triazole hybrid compounds were successfully synthesized and tested for their in vitro antidiabetic potential against α-glucosidase enzyme. Compounds having electron-donative or—withdrawing group show higher activity towards the enzyme than that of compound without substituent at para-position. Compounds 4a and 4b were the most active agents in this series, demonstrated an excellent activity with low IC₅₀ and close to the reference standard quercetin. Furthermore, molecular docking study revealed that 4a and 4b were observed to make an intensive cation–π interaction as well as hydrogen bond stabilization. The current investigation clearly identified compounds 4a and 4b as interesting leads for future development of active antidiabetic inhibitors.

Experimental

Caution!

Organic azides are potentially hazardous and explosive. Although we have never had such serious occurrences in our research, all manipulation should be done carefully behind a safety shield in a hood to avoid an explosion. Sodium azide must be transferred with a plastic spatula. Extra attention should be paid for azido compounds with (C + O)/N < 3, according to Smith’s ratio.

General

¹H- and ¹³C-NMR spectra were recorded using a Bruker Avance Neo 500 MHz spectrometer at Integrated Laboratory and Research Center (ILRC), Universitas Indonesia. Chemical shifts are reported in ppm with the deuterated dimethylsulfoxide (DMSO-d₆) solvent resonance as the internal standard (¹H: δ 2.51, ¹³C: δ 39.53 ppm). The abbreviations used are as follows: s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). Melting points were measured using an Electrothermal IA9100. IR spectra were measured using a Shimadzu IRPrestige-21 FTIR spectrometer. High Resolution Mass Spectra (HR-MS) were recorded using Xevo G2-XS QTof (Waters, U.S.A.) with detection mode by electrospray ionization-time-of-flight (ESI-TOF) at Forensic Laboratory Centre of Indonesia National Police. The reaction progress was monitored by silica gel TLC (Merck TLC Silica Gel 60 F₂₅₄), visualized under UV lamp. Column chromatography was performed using Merck silica gel 60. All reagents were purchased from Sigma-Aldrich (U.S.A.) and Merck (Germany). Anhydrous solvents such as methanol and ethanol were purchased from Merck and PT. SMARTLAB Indonesia. Distilled water was used for reaction solvent, quenching reactions, and separation sequences.

Synthesis of 1-(5-Methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)ethan-1-one (3)³⁷⁾

Triazole methyl ketone (3) was prepared according to the previous method with modification.⁴⁰⁾ In a 200 mL round bottom flask, 4-nitroaniline (20 mmol, 2.76 g) was suspended in 100 mL hydrochloric acid (18% (v/v)) at room temperature, and then ethanol (10 mL) was added. The solution was cooled to 0 °C, and sodium nitrite (1.5 equivalent (equiv.), 30 mmol, 2.07 g) was added in small portions. After stirring at 0 °C for 30 min, sodium azide (1.5 equiv., 30 mmol, 1.95 g) was added portionwise and the mixture was warmed up to room temperature and stirred for additional 4 h. After completion, the reaction mixture was extracted with ethyl acetate to wash with saturated sodium bicarbonate aqueous solution and brine. After drying over anhydrous MgSO₄, the solvent was removed under reduced pressure, and the desired azide (1-azido-4-nitrobenzene) was obtained as a yellow solid with a mass greater than the quantitative yield. This material was used for the next step without further purification.

In a 30 mL round bottom flask containing acetylacetone (40 mmol, 4.0 g) and triethylamine (40 mmol, 5.58 mL) in dimethylformamide (2.5 M, 8 mL), was added 1-azido-4-nitrobenzene (obtained from the last step) portionwise at room temperature. The mixture was stirred at this temperature for 24 h. After completion, it was extracted with ethyl acetate to wash with water and brine. The combined organic layer was dried over sodium sulfate and evaporated to give the crude product. The crude material was then purified by silica gel column chromatography (hexane/ethyl acetate = 20/1 to 2/1) to afford 1-(5-methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)ethan-1-one (4.43 g; 90% yield over two steps) as a yellow solid.

Yellow solid; Rf value 0.70 (hexane/ethyl acetate = 3/1); m.p. 126–128 °C (Lit.132–133 °C)⁴⁵⁾; IR (KBr, disc) ν_max 3344, 3090, 1680, 1598, 1557, 1521, 1429, 1345, 1069, 979, 855, 753, 652 cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.49 (d, 2H, J = 9.1 Hz), 7.99 (d, 2H, J = 9.1 Hz), 2.66 (s, 3H), 2.60 (s, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 193.8, 148.4, 143.6, 140.3, 138.7, 127.0, 125.6, 28.2, 10.3; HR-MS (ESI-TOF) Calcd for C₁₁H₁₁N₄O₃ [M + H]⁺ 247.0831. Found 247.0827.

Synthesis of Chalcones Bearing a 1,2,3-Triazole Unit (General Procedure)

Synthesis of the chalcone-triazole derivatives was performed according to previous method.⁴⁶⁾ In a 10 mL round bottom flask, the mixture containing 1-(5-methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)ethan-1-one (1.0 mmol, 246 mg) and aromatic aldehyde (1.0 mmol) in ethanol (0.5 M with respect to methyl ketone, 2 mL) was stirred and cooled with an ice bath. A solution of sodium hydroxide (0.6 g in 3 mL ethanol) was added dropwise to the mixture with continuous stirring for 10 min. The reaction mixture was then warmed to room temperature and stirred for 72 h. It was diluted with ice-cold water (4 mL) and then was neutralized with HCl solution (12% (v/v)). The formed solid was filtered, washed with excess ice-cold water, and then dried to give pure (E)-chalcones 4a–e bearing a 1,2,3-triazole scaffold.

(E)-3-(4-Methoxyphenyl)-1-(5-methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)prop-2-en-1-one (4a)

The reaction of 3 (1.0 mmol, 246 mg) with 4-methoxybenzaldehyde (1.0 mmol, 121 µL), following the general procedure, gave the titled compound 4a in 97% yield (354 mg). Brown solid; Rf value 0.57 (hexane/ethyl acetate = 2/1); m.p. 200–202 °C; IR (KBr, disc) ν_max 3094, 2939, 2841, 1660, 1594, 1512, 1426, 1344, 1285, 1254, 1175, 1031, 996, 856, 834 cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.50 (d, 2H, J = 9.1 Hz), 8.02 (d, 2H, J = 9.1 Hz), 7.90 (d, 1H, J = 15.9 Hz), 7.84 (d, 1H, J = 16.0 Hz), 7.81 (d, 2H, J = 8.8 Hz), 7.05 (d, 2H, J = 8.8 Hz), 3.84 (s, 3H), 2.68 (s, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 183.7, 162.1, 148.4, 143.91, 143.87, 140.4, 139.7, 131.2, 127.4, 127.0, 125.6, 120.6, 115.1, 55.9, 10.5; HR-MS (ESI-TOF) Calcd for C₁₉H₁₇N₄O₄ [M + H]⁺ 365.1250. Found 365.1241.

(E)-3-(4-Fluorophenyl)-1-(5-methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)prop-2-en-1-one (4b)⁴¹⁾

The reaction of 3 (1.0 mmol, 246 mg) with 4-fluorobenzaldehyde (1.0 mmol, 107 µL), following the general procedure, gave the titled compound 4b in 98% yield (345 mg). Yellow solid; Rf value 0.66 (hexane/ethyl acetate = 2/1); m.p. 192–195 °C (Lit. 196–199 °C)⁴⁵⁾; IR (KBr, disc) ν_max 3117, 3095, 1667, 1590, 1524, 1508, 1427, 1344, 1231, 1157, 1033, 998, 857, 835, 752 cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.51 (d, 2H, J = 9.0 Hz), 8.03 (d, 2H, J = 9.0 Hz), 7.98 (d, 1H, J = 15.9 Hz), 7.94 (dd, 2H, J = 8.8 Hz), 7.88 (d, 1H, J = 16.0 Hz), 7.32 (dd, 2H, J = 8.8 Hz), 2.69 (s, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 183.7, 164.0 (d, J = 249.8 Hz), 148.4, 143.8, 142.7, 140.3, 140.0, 131.6 (d, 2C, J = 8.6 Hz), 127.0 (2C), 125.6 (2C), 122.98, 122.97, 116.6 (d, 2C, J = 21.8 Hz), 10.5; HR-MS (ESI-TOF) Calcd for C₁₈H₁₄FN₄O₃ [M + H]⁺ 353.1050. Found 353.1061.

(E)-1-(5-Methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)-3-(pyridin-3-yl)prop-2-en-1-one (4c)

The reaction of 3 (1.0 mmol, 246 mg) with 3-pyridinecarboxaldehyde (1.0 mmol, 94 µL), following the general procedure, gave the titled compound 4c in 84% yield (280 mg). Pale yellow solid; Rf value 0.33 (hexane/ethyl acetate = 1/1); m.p. 218–220 °C; IR (KBr, disc) ν_max 3089, 3057, 1662, 1602, 1528, 1502, 1425, 1343, 1289, 1024, 854 cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.99 (d, 1H, J = 1.9 Hz), 8.65 (dd, 1H, J = 4.7, 1.3 Hz), 8.51 (d, 2H, J = 8.9 Hz), 8.33 (dt, 1H, J = 7.9, 1.7 Hz), 8.14 (d, 1H, J = 16.1 Hz), 8.03 (d, 2H, J = 8.9 Hz), 7.92 (d, 1H, J = 16.1 Hz), 7.52 (dd, 1H, J = 7.9, 4.7 Hz), 2.69 (s, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 183.6, 151.8, 151.0, 148.5, 143.7, 140.6, 140.3, 135.4, 130.7, 127.1 (2C), 125.6, 124.9, 124.6 (3C), 10.5; HR-MS (ESI-TOF) Calcd for C₁₇H₁₄N₅O₃ [M + H]⁺ 336.1097. Found 336.1100.

(2E,4E)-1-(5-Methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)-5-phenylpenta-2,4-dien-1-one (4d)

The reaction of 3 (1.0 mmol, 246 mg) with cinnamaldehyde (1.0 mmol, 126 µL), following the general procedure, gave the titled compound 4d in 80% yield (288 mg). Red-brown solid; Rf value 0.82 (hexane/ethyl acetate = 2/1); m.p. 220–222 °C; IR (KBr, disc) ν_max 3055, 1651, 1576, 1526, 1449, 1344, 1032, 851 cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.50 (d, 2H, J = 8.8 Hz), 8.02 (d, 2H, J = 9.0 Hz), 7.67 (dd, 1H, J = 15.3, 13.4 Hz), 7.58 (d, 1H, J = 16.0 Hz), 7.42 (dd, 1H, J = 16.0, 7.7 Hz), 7.40–7.33 (m, 5H), 7.27 (d, 1H, J = 15.3 Hz), 2.67 (s, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 183.9, 148.4, 144.6, 143.8, 142.9, 140.4, 139.8, 136.8, 129.8, 129.4, 127.9, 127.7, 127.1, 126.6, 125.6, 10.7; HR-MS (ESI-TOF) Calcd for C₁₉H₁₇N₄O₄ [M + H]⁺ 361.1301. Found 361.1343.

(2E,4E)-5-(4-(Dimethylamino)phenyl)-1-(5-methyl-1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)penta-2,4-dien-1-one (4e)

The reaction of 3 (1.0 mmol, 246 mg) with 4-(dimethylamino)cinnamaldehyde (1.0 mmol, 175 mg), following the general procedure, gave the titled compound 4e in 94% yield (381 mg). Red-brown solid; Rf value 0.40 (hexane/ethyl acetate = 3/1); m.p. 248–250 °C; IR (KBr, disc) ν_max 3092, 2908, 1650, 1556, 1522, 1426, 1367, 1343, 1153, 1031, 855 cm⁻¹; ¹H-NMR (500 MHz, DMSO-d₆) δ: 8.49 (d, 2H, J = 9.0 Hz), 8.01 (d, 2H, J = 9.0 Hz), 7.64 (dd, 1H, J = 15.9, 10.2 Hz), 7.49 (d, 2H, J = 8.9 Hz), 7.43 (d, 1H, J = 15.0 Hz), 7.13 (dd, 1H, J = 15.6, 10.5 Hz), 7.08 (d, 1H, J = 14.8 Hz), 6.73 (d, 2H, 8.9 Hz), 2.98 (s, 6H), 2.66 (s, 3H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 183.6, 151.6, 148.4, 145.8, 144.4, 144.0, 139.3, 129.6, 127.0, 125.6, 124.1, 123.4, 122.7, 115.4, 112.4, 64.8, 10.4; HR-MS (ESI-TOF) Calcd for C₂₂H₂₂N₅O₃ [M + H]⁺ 404.1723. Found 404.1724.

α-Glucosidase Inhibition Test

The method of glucosidase inhibitory activity evaluation was carried out according to previous work with modification.⁴⁷⁾ A solution of p-nitrophenyl-α-D-glucopyranoside (5 mM, 250 µL) and phosphate buffer pH 7 (0.1 M, 495 µL) were added to a reaction tube containing standard or various concentration of sample solution (5 µL) in DMSO. After homogeny solution obtained, it was pre-incubated for 5 min at 37 °C, and reaction was started with addition of α-glucosidase solution (0.062 U, 250 µL). Incubation was then continued for 15 min. Reaction was stopped by addition of sodium carbonate solution (0.2 M, 1.0 mL). Enzyme activity was measured based on the formation p-nitrophenol, absorption reading at λ = 400 nm. Quercetin was used as comparison standard. Percentage of inhibitory activity was measured using an equation:

  

• Where: C = Absorbance of blank (DMSO); S = Absorbance of sample (absorbance difference between with and without enzyme).

Molecular Docking Study

Docking studies were performed to predict the molecular interactions and binding poses of the synthesized compounds towards the binding site of α-glucosidase enzyme. Since the crystallographic structure of Saccharomyces cerevisiae lysosomal α-glucosidase (UniProtID: P53341) is not available yet in the protein data bank, the protein structure was predicted and generated using AlphaFold.⁴⁸⁾ As with the ligands, the molecular structure of quercetin (CID: 5280343) and 4b (CID: 155806145) were obtained from PubChem website,⁴⁹⁾ while 4a, 4c, and 4d structures were prepared and drawn using MarvinSketch software.⁵⁰⁾ Prior to the docking simulation, all ligands were optimized and minimized using DataWarrior v5.5.0 software by MMFF94x with the RMS gradient was set to 0.001 kcal/mol·A². The docking procedure was carried out using AutoDock Vina tool in PyRx v0.8.0 software^51,52) with the exhaustiveness value of 8 and generating 10 poses at maximum for each docked ligands. The binding site of α-glucosidase was set in the catalytic site of the enzyme (Asp214, Glu276 and Asp349). Finally, the binding interaction analysis and the 3D visualization of the docking results were carried out using BIOVIA Discovery Studio Visualizer 2021 and ChimeraX 1.4⁵³⁾ software, respectively.

Acknowledgments

This work was fully supported by the Directorate of Research and Development, Universitas Indonesia through Hibah Publikasi Internasional Terindeks (PUTI) Q2 2022 with contract No. NKB-645/UN2.RST/HKP.05.00/2022. The authors thank Ms. Pratiwi Puji Lestari, M.Si. for recording ¹H- and ¹³C-NMR spectra. We also send our gratitude to Mr. Azhar Darlan for his help in carrying out the HR-MS measurements.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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