Synthesis and In Vitro/In Silico α‑Glucosidase Inhibitory Study of Novel Ethanones Containing Naphthalene-Linked 1,2,4-Triazole

Abstract

Type 2 diabetes mellitus is a chronic-metabolic disorder characterized by insulin resistance, resulting in persistent hyperglycemia and severe complications. α-Glucosidase inhibitors (AGIs) effectively control postprandial blood glucose level by delaying carbohydrate digestion. This study reports the synthesis of novel naphthalene-linked 1,2,4-triazole-bearing ethanones (5a–e and 7a–f) as potential AGIs. Enzymatic assay demonstrated significantly superior α-glucosidase inhibitory potency of aryl-substituted derivatives (7a−f) compared to ethyl-substituted analogs (5a−e), highlighting the advance of aromatic substituents. Compounds 7b and 7c exhibited exceptional inhibitory activity (IC₅₀ = 9.23–9.61 μM), conferring 37-fold more potency than voglibose. Molecular docking and dynamics simulations indicated predominant π-π stacking and hydrophobic interactions contributing to their stable enzyme binding. MM/GBSA binding-affinity calculation further supported their enhanced binding affinity, providing mechanistic insights into their potent activity. Collectively, these findings highlight the promise of naphthalene- and 1,2,4-triazole-bearing ethanones for the development of effective antidiabetic therapies.

Diabetes mellitus (DM) is a metabolic disorder, caused by the body unable to produce or use insulin effectively, resulting in high sugar levels in blood or hyperglycemia. Hyperglycemia triggers excessive productions of humoral mediators, cytokines, growth factors, and free fatty acids, which have been well-known as major factors involved in complications such as renal damage and cardiovascular disease. − The global incidence of DM has been dramatically increasing and is projected to reach 592 million cases by 2035, with 90% of cases being type 2, characterized by insulin resistance. ,,

Besides the conventional insulin injection, a multitude of oral diabetic agents of diverse chemical scaffolds and modes of action, such as sulfonylureas, meglitinides, biguanides, thiazolidinediones, dipeptidyl peptidase 4 inhibitors, sodium-glucose cotransporter inhibitors, and α-glucosidase inhibitors, has been currently approved for type 2 DM management (Figure ). , Among these, α-glucosidase inhibitors (AGIs) are distinct in their mechanism by targeting carbohydrate digestion and absorption and delaying the breakdown of complex carbohydrates in the small intestine, thereby attenuating postprandial hyperglycemia. AGIs have been increasingly investigated as alternative or adjunctive agents to achieve an effective and safe solution for type 2 DM management. − Albeit promising, the approved AGIs, i.e., acarbose, miglitol, or voglibose, bind reversibly to the enzymes and are quickly passed along the intestinal tract with poor retention, leading to suboptimal pharmacodynamic duration. These limitations prompted us and other laboratories to develop synthetic analogs with more sustained binding affinity and prolonged gastrointestinal residence.

1.

Representative structures of commonly used oral therapeutic agents for the management of type 2 DM.

Many progresses have been made to generate a repertoire of natural product-derived and synthetic heterocyclic compounds with varied α-glucosidase inhibitory potency. − Among the potent AGIs the triazole scaffolds have been templated for many synthetic derivatives with promising antidiabetic pharmacology, , and their derivatives have been reported to possess robust and selective intestinal α-glucosidase binding, , potentially reducing side effects and offering a new pathway for novel antidiabetic developments. For example, a panel of newly synthesized 4-(arylidene)­amino-1,2,4-triazole-5-thiol or 4-amino-1,2,4-triazole ethanones efficiently inhibited α-glucosidase at lower IC₅₀ of 1.49–6.44 μM than standard drugs. , Alternatively, naphthalene is another potential scaffold for antidiabetic drug development. Various naphthalene-derived compounds have been reported to have promising bioactivity against carbohydrate-hydrolyzing enzymes, including α-glucosidase and α-amylase, thus demonstrating promise in managing postprandial hyperglycemia. , In particular, recent studies prepared a synthetic series of naphthalene-bearing hydrazides which showed low micromolar inhibitory potency against α-glucosidase, emphasizing their therapeutic potential for type 2 diabetes treatment. , It has been shown that aryl thiotriazoles have ∼2.5-fold inhibitory activity over that of alkyl derivatives and that this improved activity stemmed from the emerging π-π, π-H, π-anion interactions with key residues within the catalytic pocket of the enzyme. , In concordance with these studies, our previous study demonstrated exceptional inhibition with IC₅₀ of 0.11 ± 0.04 μM, indicating that coupling these two moieties can provide an effective artificial scaffold for the development of antidiabetic therapies.

Building on the additive effect of 1,2,4-triazole-3-thiol and naphthalene moieties in α-glucosidase inhibitory activity, we set out to synthesize an array of naphthalene-linked 1,2,4-triazole-bearing ethanones via the 1,2,4-triazole-3-thione intermediate (compound 3) through condensation between acyl hydrazides (compound 2) and ethyl/phenyl isothiocyanate (Scheme ). As such, we successfully generated a series of 2-{[5-(naphthalen-1-ylmethyl)-4-ethyl-4H-1,2,4-triazol-3-yl]­thio}-1-aryl ethanones (5a–e) and 2-{[5-(naphthalen-1-ylmethyl)-4-phenyl-4H-1,2,4-triazol-3-yl]­thio}-1-aryl/coumarin-3-yl ethanones (7a–f). The chemical structures were elucidated using FTIR, NMR, and HRMS (see Supporting Information for full experimental details).

1. Illustrative Scheme for Synthesizing Compounds 5a–e and 7a–f .

^a The key intermediates 3 and 6 were synthesized via esterification of 2-(naphthalen-1-yl)­acetic acid (1) with ethanol in the presence of sulfuric acid, followed by hydrazinolysis to yield 2-(naphthalen-1-yl)­acetohydrazide (2). Subsequent cyclization of 2 with ethyl or phenyl isothiocyanate afforded 3 and 6, respectively. Final coupling of 3 and 6 with 2-bromo-1-arylethanone derivatives (4a–e) or 3-(bromoacetyl)­coumarin (4f) provided the target compounds 5a–e and 7a–f.

Next, we profiled the inhibitory activity of synthesized derivatives against Saccharomyces cerevisiae-derived α-glucosidase. In general, the synthesized compounds outperform voglibose (IC₅₀ ≅347.04 μM), whose activities were arbitrarily divided into upper (IC₅₀ ≅9.23–65.69 μM) (7b, 7c, 7d, 7f, 5d, 7a) and lower (IC₅₀ ≅177.82–345.67 μM) (5b, 5c, 5a, 5e) tiers (Table ). The potency of our compounds stratifies well with their respective structures where compounds with an electron withdrawing group (EWG) on the triazole ring (i.e., R₁ = C₆H₅) (upper tier) were more active than those with an electron donating group (EDG) (i.e., R₁ = C₂H₅) (lower tier), with the exception of 5d (Figure ). Further structural and activity stratification was observed among the compounds within each tier with the least potent compounds, i.e., 7a (IC₅₀ ≅ 65.69 μM) and 5e (IC₅₀ ≅ 345.67 μM), carrying an unsubstituted phenyl (−C₆H₅) and 3-nitrophenyl (3-NO₂C₆H₅) groups, respectively. Substitution of the para hydrogen with halogen (e.g., chloro, bromo) or nitro (−NO₂) effectively boosts α-glucosidase inhibition. Similarly, replacement of phenyl with a coumarinyl moiety improves inhibition activity. Indeed, the activity of 7b (IC₅₀ ≅ 9.23 μM), 7c (≅9.61 μM), 7d (≅21.16 μM), and 7f (≅25.41 μM) was enhanced ∼14.7–37.6 fold compared to voglibose. Of note, compounds in the lower tier benefit limitedly from such substitutions, alleviating the activity of 5b (≅177.82 μM), and 5c (≅223.77 μM) to a much lesser extent of ∼ 1.6–2.0 fold compared to voglibose. The identity and position of substituted groups on the aromatic moiety greatly influence the overall activity. For example, in the upper tier, halogenated phenyl ketone, i.e., −C₆H₅ Cl (7b) and −C₆H₅ Br (7c), exhibit greater potency compared to nonhalogenated phenyl ketone, i.e., −C₆H₅ NO ₂ (7d). Conversely, in the lower tier, −C₆H₅ Cl (5b) and −C₆H₅ Br (5c) are less favored than −C₆H₅ NO ₂ (5e). Moreover, para substitution seems to be more favored over that of meta as evident by a large activity boost of 5d (4-NO₂C₆H₅) relative to that of 5e (3-NO₂C₆H₅), bringing its potency closer to that of 7d (4-NO₂C₆H₅).

1. Inhibition Activity of Compounds 5a–e and 7a–f against α-Glucosidase.

Compounds	R1	R2	IC50 Value (μM)
Voglibose	-	-	347.04 ± 0.11
5a	-C2H5	-H	312.12 ± 0.08
5b	-C2H5	4-ClC6H5	177.82 ± 0.17
5c	-C2H5	4-BrC6H5	223.77 ± 0.13
5d	-C2H5	4-NO2C6H5	38.30 ± 0.16
5e	-C2H5	3-NO2C6H5	345.67 ± 0.09
7a	-C6H5	-H	65.69 ± 0.09
7b	-C6H5	4-ClC6H5	9.23 ± 0.04
7c	-C6H5	4-BrC6H5	9.61 ± 0.06
7d	-C6H5	4-NO2C6H5	21.16 ± 0.06
7f	-C6H5	Coumarin-3-yl	25.41 ± 0.06

2.

Structure–activity relationship (SAR) of synthesized compounds.

Molecular docking studies in the model S. cerevisiae-derived α-glucosidase (AlphaFold ID: P38158) indicated that the most potent compounds (7b and 7c) adopt a favorable orientation in the binding site (Figure A,B). This orientation promotes π-π stacking interaction with Phe157 and hydrogen bonding with Arg312, effectively positioning their halogenated phenyl moiety within an active site-surrounding subpocket formed by Asp214, Glu276, and Asp349. Such an arrangement likely blocks access to this region, thus enhancing their inhibitor activity. In contrast, the least potent in upper tier 7a and the most potent in lower tier 5d showed reversed orientations that disrupted key interactions. As a result, the subpocket remained accessible, which may possibly explain their reduced inhibitory activity against α-glucosidase (Figure C,D).

3.

Molecular docking studies of selected compounds within the active site of S. cerevisiae-derived α-glucosidase. Docking poses of compounds 7b (A), 7c (B), 7a (C), and 5d (D) within the α-glucosidase binding pocket. Compounds 7b and 7c form π-π interactions with Phe157 and additional hydrogen bonding with Arg312, positioning the halogenated phenyl moiety within the subpocket formed by Asp214, Glu276, and Asp349. In contrast, compounds 7a and 5d adopt a reversed orientation that disrupts these key interactions, potentially reducing binding stability.

We next performed molecular dynamics (MD) simulations to further evaluate the structural stability and dynamics behavior of 7b and 7c in a complex with α-glucosidase with voglibose serving as a reference inhibitor. The root-mean-square deviation (RMSD) analysis over a 50 ns simulation indicated that ligand-bound complexes exhibited lower fluctuations and more stable conformations compared to those of the Apo form (Figure A). Notably, the complex of compound 7c maintained the lowest RMSD values (≈0.15–0.20 nm), suggesting effective stabilization of the enzyme structure. Conversely, compound 7b showed slightly higher RMSD fluctuations, indicating a comparatively less stable complex and possibly different binding interactions or conformational dynamics.

4.

Ligand-protein interaction analyses of α-glucosidase complexes with compounds 7b, 7c, and voglibose. (A) RMSD plots of α-glucosidase in complexes with 7b, 7c, voglibose, and the Apo form during 50 ns molecular dynamics simulations. Interaction profiles of voglibose (B), 7b (C), and 7c (D). Voglibose appears to adopt a substrate-mimicking mechanism by primarily forming hydrogen bonds with key residues, whereas compounds 7b and 7c exhibit extensive π-π stacking interactions with Phe157, Phe177, His239, His279, and His300, along with π–cation and hydrogen bonding. These interactions suggest a distinct binding mode for 7b and 7c, contributing to their enhanced binding stability compared to voglibose.

Subsequent ligand-protein interaction analyses provided a mechanistic rationale for these observed differences in the structural stability. Voglibose primarily formed hydrogen bonding with critical residues, including Asp68, Arg312, Asp408, Arg439, and the catalytic residues Asp214 and Asp349, suggesting a possible substrate-mimicking mechanism (Figure B). In contrast, 7b and 7c showed markedly different interaction profiles (Figure C,D). Both inhibitors predominantly formed π-π stacking interactions with aromatic residues, including Phe157, Phe177, His239, His279, and His300, along with supplementary π cation and hydrogen bonding. These diverse interactions suggest a distinct binding mechanism in comparison to that of voglibose. The combination of extensive hydrophobic contacts and larger molecular size enabled 7b and 7c to snugly fit into the active site, contributing to their enhanced binding stability and inhibitory potency. Consistent with these interaction profiles, binding-affinity calculations using the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method revealed significantly stronger interactions for 7b (ΔG ≈ – 55.05 ± 2.76 kcal/mol) and 7c (ΔG ≈ – 58.23 ± 2.68 kcal/mol) compared to voglibose (ΔG ≈ – 20.16 ± 3.49 kcal/mol) (Table ).

2. Binding Free Energy Calculation for Voglibose, 7b, and 7c in Complexes with α-Glucosidase.

Ligand	ΔG binding (kcal/mol)
Voglibose	–20.16 ± 3.49
7b	–55.05 ± 2.76
7c	–58.23 ± 2.68

In summary, we have successfully synthesized a novel series of ethanones containing triazole-naphthalene hybrids, evaluated their α-glucosidase inhibitory activities, and elucidated their structure–activity relationships (SARs) through molecular docking and molecular dynamics simulations. Compounds 7b and 7c harboring S-halogenated phenyl and N-phenyl moieties emerged as the most potent inhibitors, exhibiting favorable binding orientations stabilized by predominant π-π stacking and hydrophobic contacts, effectively blocking access to the enzyme’s activity site. MD simulations confirmed their stable binding, while MM/GBSA calculations underscored their superior binding free energies compared to voglibose. Collectively, these findings highlight the significance of specific structural features, such as naphthalene and triazole cores with S-halogenated phenyl and N-phenyl substituents, in improving inhibitory potency against α-glucosidase, thereby offering promising scaffolds for the development of more effective antidiabetic drugs.

While encouraging, our study has several limitations that need to be addressed in further investigations. First, the number of synthesized derivatives is limited, constraining the ability to establish comprehensive and generalizable SAR. Future studies should systematically synthesize and evaluate a larger set of structural analogs to provide deeper insights and establish a more precise SAR profile. Second, our enzymatic assays and computational analyses used yeast-derived α-glucosidase, which may be structurally different from its human counterpart, thereby potentially compromising direct extrapolation to clinical efficacy. Further investigation should, therefore, validate the inhibitory potency using human-derived α-glucosidase or appropriate mammalian models. Third, our biological evaluations only relied on in vitro assays, which may not be reflective of these derivatives’ therapeutic efficacy and pharmacological behavior. Subsequent in vivo studies, along with detailed pharmacokinetic and toxicity profiling, are critical for clinical translation. Ultimately, cointegration of computational, medicinal chemistry, and pharmacological approaches will facilitate the development of these novel scaffolds into clinically viable antidiabetic agents.

Safety. No unexpected or unusually high safety hazards were encountered.

Glossary

Abbreviation

DM

diabetes mellitus

AGIs

α-glucosidase inhibitors

SGLT2

Sodium-Glucose Cotransporter-2

DPP-4

Dipeptidyl Peptidase-4

TZDs

Thiazolidinediones

FTIR

Fourier Transform Infrared

NMR

Nuclear Magnetic Resonance

HRMS

High-Resolution Mass Spectrometry

EWG

Electron withdrawing group

EDG

Electron donating group

IC₅₀

half-maximal inhibitory concentration

MM/GBSA

Molecular Mechanics/Generalized Born Surface Area

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.5c00387.

• Experimental details, synthesis and characterization, computational methods, and additional spectroscopic data including FTIR, ¹H NMR, ¹³C NMR, and HRMS (PDF)

∇.

US DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

○.

Center for Advanced Bioenergy and Bioproducts; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.