Newly synthesized 1,2,3-triazoles based on [1,4]-benzoxazin- 3-one: In silico evaluation of anti-inflammatory, antibacterial, antioxidant, anticancer, and antidiabetic properties, along with molecular dynamics simulation and ADME analysis

doi:10.21203/rs.3.rs-4931146/v1

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Newly synthesized 1,2,3-triazoles based on [1,4]-benzoxazin- 3-one: In silico evaluation of anti-inflammatory, antibacterial, antioxidant, anticancer, and antidiabetic properties, along with molecular dynamics simulation and ADME analysis

https://doi.org/10.21203/rs.3.rs-4931146/v1

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Based on the significant biological activity of benzoxazines and 1,2,3-triazoles, we aim to combine these active moieties to design and synthesize new compounds and evaluate their biological activity. In this context, we present the synthesis of new 1,2,3-triazoles, specifically 1,4-disubstituted, in combination with [1,4]-benzoxazin-3-one. To synthesize the target compounds, the 1,3-dipolar Huisgen cycloaddition is used as a central step. This reaction occurs between ethyl azidoacetate and the terminal alkyne of [1,4]-benzoxazin-3-one under catalytic conditions using Cu(I) (CuAAC). Followed by the condensation of hydrazine on the ester function and then a reaction with various aromatic aldehydes to form the corresponding hydrazones (4a–4j). Molecular docking revealed that the synthesis molecules exhibited potential antidiabetic, anti-inflammatory, anticancer, antibacterial, and antioxidant properties. Among them, 4a showed the highest affinity for these activities and 4b showed the highest affinity for antioxidant activity. To further evaluate its potential, 4a and 4b underwent molecular dynamics (MD) simulations over a 5 ns period. The stability and flexibility of the 4a-3W2S and 4b-3DK9 complex were evaluated using RMSF, RMSD, H-Bond, and Rg analyses, revealing notable interaction stability and flexibility. In addition, ADME analysis demonstrated favorable pharmacokinetic properties and oral absorption of the synthetic molecules, meeting the Lipinski and Veber criteria and suggesting their potential as oral drug candidates. This comprehensive assessment highlights the value of these novels [1,4]-benzoxazin-3-one derivatives and supports further research exploring their therapeutic potential.

Biological sciences/Biochemistry

Biological sciences/Computational biology and bioinformatics

Biological sciences/Drug discovery

Biological sciences/Physiology

Health sciences/Medical research

Health sciences/Molecular medicine

Physical sciences/Chemistry

3-Triazole

4]-benzoxazin-3-one

click chemistry

molecular docking

ADME

3-Dipolar cycloaddition

molecular dynamics

The discovery of new drugs is crucial for advancing healthcare and addressing the evolving challenges posed by diseases. As pathogens mutate and develop resistance to existing treatments, there is a continuous need for novel therapeutics to maintain effective healthcare standards. Additionally, emerging diseases, such as new viral outbreaks, require innovative drugs for prevention and treatment. Furthermore, the rise in chronic diseases, such as cancer, diabetes, and heart disease, also underscores the necessity for new, more effective medications that can offer better management and potential cures. Consequently, the pursuit of new drug discovery not only enhances the quality of life but also fuels scientific progress, driving economic growth through the pharmaceutical industry and related sectors^1,2. Therefore, ongoing research and investment in drug discovery are crucial for meeting future medical needs. In medicinal chemistry, [1,4]-benzoxazin-3-ones have considerable properties. These compounds are of significant interest because they exhibit a wide range of biological activities, including antimicrobial³, anticancer⁴, antifungal⁵, antithrombotic⁶, anticonvulsant⁷, antidepressant⁸, and anti-HIV-1⁹ effects. Moreover, in recent decades, 1,2,3-triazole derivatives have attracted attention not only in the field of organic chemistry but also in medicinal chemistry due to their diverse biological activities, such as antibacterial¹⁰, antioxidant¹¹, antifungal¹², anticancer¹³, anti-inflammatory¹⁴, anti-tubercular¹⁵, anti-HIV¹⁶, anti-infective¹⁷, antidepressant¹⁸ and anti-Alzheimer¹⁹ activities. The most relevant method for synthesizing 1,2,3-triazoles is the copper/Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (CuAAC), which entails the stepwise formation of carbon-nitrogen bonds between a terminal alkyne and an azide. This reaction is regio-specific, producing exclusively the 1,4-regioisomer of 1,2,3-triazoles. Considering these factors and as an extension of our research into developing new molecular entities that incorporate both [1,4]-benzoxazin-3-one and 1,2,3-triazole pharmacophores within a single molecular framework, we directed our efforts toward synthesizing these compounds to investigate and understand the combined effects of these moieties. In silico investigations play a vital role in evaluating potential therapeutics by targeting specific biological sites. Bioinformatics tools can identify the compounds with the most promising therapeutic potential, presenting them as candidates for further in vitro and in vivo studies²⁰. The development of novel drug compounds mainly relies on approaches provided by in silico techniques, which are beneficial for testing potential drug candidates, thereby reducing the time and cost of drug development²¹. Molecular docking is one of the most widely used methods to predict the binding affinities and interactions of synthetic molecules with target proteins, thereby assessing their potential biological activities²². In this study, we present the synthesis of several 1,2,3-triazoles, specifically 1,4-disubstituted derivatives based on 2H-Benzo[b][1,4]oxazin-3-one, utilizing the 1,3-dipolar cycloaddition reaction with a Cu(I) catalyst, followed by the condensation of hydrazine onto the ester function, and then a second condensation of various aromatic aldehydes leading to the corresponding hydrazones. Based on the molecular docking results, we will provide insights into the stability and flexibility of real ligand-protein complex interactions, including 4a-3W2S and 4b-3DK9, by considering physical factors such as pressure, temperature, and aqueous solvents to more closely approximate physiological conditions using molecular dynamics simulations²³. Furthermore, our analysis of ADME parameters allowed us to evaluate the pharmacokinetic properties of these molecules (4a-4j), thus determining their suitability as oral drug candidates, given their therapeutic potential²⁴.

General

The reaction progress was monitored using Thin-Layer Chromatography (TLC) on silica gel plates, with results visualized under UV light. Melting points were determined with an Electrothermal 9100 apparatus in open capillary tubes, without correction. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity Plus spectrometer, with 1H NMR at 500 MHz and 13C NMR at 125 MHz. Chemical shifts (δ) are reported in parts per million (ppm), using tetramethylsilane (TMS) as the internal standard.

Procedure for the Preparation of Compound 2

To a solution of 4-(prop-2-yn-1-yl)-[1,4]-benzoxazin-3-one 1 (5.35 mmol) in absolute ethanol (10 mL), an azide (5.35 mmol) was added. This was followed by the addition of a mixture of CuSO₄・5H₂O (5.35 mmol) and sodium ascorbate (5.35 mmol) dissolved in water (10 mL). The reaction mixture was stirred at room temperature for 0.5 hours and monitored using thin-layer chromatography (TLC).

Procedure for the Synthesis of Compound 3

Ethyl 2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetate 2 (1.59 mmol) was dissolved in ethanol (8 mL). Hydrazine hydrate (7.9 mmol) was then added gradually to the solution while stirring continuously. The mixture was refluxed for 3 hours and then allowed to cool to 25°C. The product was isolated by filtration, dried, and crystallized from ethanol.

General Procedure for the Synthesis of Compounds 4a-4j

A boiling solution of 2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide 3 (1.82 mmol) was prepared in 5 mL of absolute ethanol. Substituted aromatic aldehyde derivatives (1.82 mmol) were then added, along with a few drops of acetic acid as a catalyst. The mixture was stirred under reflux for 30 minutes, with progress monitored by thin-layer chromatography (TLC).

N'-benzylidene-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4a ): Obtained as a white powder with an 82% yield; melting point (m.p.) 246–248°C. IR (KBr, vmax/cm − 1) shows a peak at 1677 (2C = O str.). 1H-NMR (DMSO-d6, 500 MHz) δ: 11.77 (s, 1H), 8.18 (s, 1H), 8.03 (s, 1H), 8.00-6.98 (m, 9H), 5.61 (s, 2H), 5.15 (s, 2H), 4.67 (s, 2H). 13C-NMR (125 MHz, DMSO-d6) δ: 167.83, 164.63, 145.41, 144.92, 142.57, 134.36, 130.66, 129.36, 129.00, 127.56, 126.01, 124.23, 123.20, 117.08, 116.40, 67.63, 51.03, 36.76. Elemental analysis for C₂₀H₁₈N₆O₃·1/20CH₃CH₂OH: C 61.53, N 21.53, H 4.65, O 12.29; found: C 61.48, N 21.40, H 4.70, O 12.43. (See Figures S1–S3 in the Supplementary Materials: spectra 1H, 13C NMR, DEPT-135, and IR spectra for compound 4a).

N'-(4-bromobenzylidene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4b ): A white powder with an 84% yield and a melting point of 238–240°C. The IR spectrum (KBr) displays a carbonyl stretch at 1679 cm − 1.1H-NMR (DMSO-d6, 500 MHz): δ 11.90 (s, 1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.99–6.97 (m, 8H), 5.61 (s, 2H), 5.14 (s, 2H), 4.67 (s, 2H). 13C-NMR (125 MHz, DMSO-d6): δ 167.93, 164.59, 145.46, 145.35, 143.74, 142.60, 133.74, 132.35, 129.38, 128.99, 125.98, 124.23, 123.85, 123.20, 117.11, 116.40, 67.63, 51.05, 36.76. Elemental Analysis: Expected for C₂₀H₁₈BrN₆O₃·1/20CH₃CH₂OH: C 51.19, N 17.91, H 3.65, Br 17.03, O 10.47; Observed: C 51.19, N 17.82, H 3.70, Br 16.94, O 10.35. (See Figures S4–S6 in the Supplementary Materials for the 1H, 13C NMR, DEPT-135, and IR spectra for compound 4b).

N'-(4-chlorobenzylidene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4c ): The compound is a white powder with an 86% yield and a melting point of 238–240°C. The IR spectrum (KBr) shows a carbonyl stretch at 1687 cm − 1.1H-NMR (DMSO-d6, 500 MHz): δ 11.83 (s, 1H), 8.17 (s, 1H), 8.00 (s, 1H), 7.98–6.97 (m, 8H), 5.62 (s, 2H), 5.14 (s, 2H), 4.67 (s, 2H). 13C-NMR (125 MHz, DMSO-d6): δ 167.93, 164.68, 145.41, 143.63, 142.54, 135.08, 133.37, 129.39, 129.23, 128.99, 125.99, 124.23, 123.20, 117.12, 116.44, 67.62, 51.04, 36.75. Elemental Analysis: Calculated for C₂₀H₁₈ClN₆O₃·1/25CH₃CH₂OH: C 56.54, N 19.78, H 4.03, Cl 8.34, O 11.30; Found: C 56.52, N 19.70, H 4.07, Cl 8.31, O 11.40. (See Figures S7–S9 in the Supplementary Materials for the 1H, 13C NMR, DEPT-135, and IR spectra for compound 4c).

N'-(4-nitrobenzylidene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4d ): The compound is a white powder with an 82% yield and a melting point of 266–268°C. The IR spectrum (KBr) indicates a carbonyl stretch at 1685 cm − 1. The 1H-NMR (DMSO-d6, 500 MHz) shows δ 12.05 (s, 1H), 8.24 (s, 1H), 8.22 (s, 1H), 8.09–6.97 (m, 8H), 5.67 (s, 2H), 5.15 (s, 2H), and 4.67 (s, 2H). The 13C-NMR (125 MHz, DMSO-d6) provides δ 168.30, 164.65, 145.44, 142.60, 140.65, 133.98, 129.02, 129.00, 128.54, 125.97, 124.52, 123.24, 123.20, 117.07, 116.38, 67.63, 51.09, and 36.76. Elemental analysis calculated for C₂₀H₁₇N₇O₅·1/20C₃H₇NO: C 55.17, N 22.52, H 3.94, O 18.37; found: C 55.12, N 22.49, H 3.98, O 18.40. (See Figures S10–S12 in the Supplementary Materials for the 1H, 13C NMR, DEPT-135, and IR spectra for compound 4d).

N'-(4-methoxybenzylidene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4e): The compound is a white powder with an 86% yield and a melting point of 222–224°C. The IR spectrum (KBr) shows a carbonyl stretch at 1679 cm − 1. The 1H-NMR (DMSO-d6, 500 MHz) displays δ 11.62 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 7.99–6.97 (m, 8H), 5.58 (s, 2H), 5.14 (s, 2H), and 4.67 (s, 2H). The 13C-NMR (125 MHz, DMSO-d6) gives δ 167.58, 164.64, 145.40, 144.83, 142.49, 134.24, 130.20, 129.38, 129.16, 126.01, 124.25, 123.20, 117.07, 116.40, 114.84, 67.63, 51.00, 36.76, and 55.84. Elemental analysis calculated for C₂₁H₂₀N₆O₄·1/20CH₃CH₂OH: C 59.99, N 19.99, H 4.79, O 15.22; found: C 59.95, N 19.88, H 4.84, O 15.33. (See Figures S13–S15 in the Supplementary Materials for the 1H, 13C NMR, DEPT-135, and IR spectra for compound 4e).

N'-(3-ethoxy-2-hydroxybenzylidene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4f ): The compound is a white powder with a yield of 84% and a melting point of 242–244°C. The IR spectrum (KBr) reveals a carbonyl stretch at 1660 cm − 1. 1H-NMR (DMSO-d6, 500 MHz) shows δ 11.69 (s, 1H), 9.14 (s, 1H), 8.40 (s, 1H), 8.33 (s, 1H), 8.04–6.75 (m, 7H), 5.58 (s, 2H), 5.14 (s, 2H), 4.67 (s, 2H), 4.02 (q, 2H, J = 7 Hz) and 1.26 (t, 3H, J = 7 Hz). 13C NMR (125 MHz, DMSO-d6) gives δ 167.54, 164.66, 147.62, 146.70, 145.40, 142.52, 126.04, 124.24, 123.17, 120.95, 119.83, 119.67, 118.21, 117.10, 116.40, 67.64, 64.68, 51.00, 36.76 and 15.14. Elemental analysis calculated for C₂₂H₂₂N₆O₅·1/25H₂O: C 58.66, N 18.66, H 4.92, O 17.76; found: C 58.56, N 18.62, H 4.93, O 17.88. (See Figures S16–S18 in the supplemental material for 1H, 13C NMR, DEPT-135 and IR spectra for compound 4f).

2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)-N'-((E)-3 phenylallylidene)acetohydrazide ( 4g ): The compound is a white powder with an 80% yield and a melting point of 266–268°C. The IR spectrum (KBr) displays a carbonyl stretch at 1677 cm − 1. The 1H-NMR (DMSO-d6, 500 MHz) shows δ 11.66 (s, 1H), 8.02 (s, 1H), 7.99–6.90 (m, 12H), 5.49 (s, 2H), 5.14 (s, 2H), and 4.67 (s, 2H). The 13C-NMR (125 MHz, DMSO-d6) provides δ 167.44, 164.66, 147.56, 145.40, 142.72, 142.56, 140.10, 136.28, 129.38, 129.01, 127.70, 126.04, 125.29, 124.23, 123.21, 117.07, 116.40, 67.63, 50.85, and 36.76. Elemental analysis calculated for C₂₂H₂₀N₆O₃·1/25CH₃CH₂OH: C 63.45, N 20.18, H 4.84, O 11.53; found: C 63.34, N 20.15, H 4.85, O 11.66. (See Figures S19–S22 in the Supplementary Materials for the 1H, 13C NMR, DEPT-135, and IR spectra for compound 4g).

N'-(4-hydroxybenzylidene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4h ): The compound is a white powder with an 80% yield and a melting point of 178–180°C. The IR spectrum (KBr) exhibits a carbonyl stretch at 1679 cm − 1. The 1H-NMR (DMSO-d6, 500 MHz) shows δ 11.55 (s, 1H), 9.89 (s, 1H), 8.06 (s, 1H), 8.00 (s, 1H), 7.99–6.76 (m, 8H), 5.56 (s, 2H), 5.14 (s, 2H), and 4.67 (s, 2H). The 13C-NMR (125 MHz, DMSO-d6) provides δ 167.41, 164.66, 159.95, 145.38, 142.52, 129.54, 129.30, 128.96, 125.99, 125.38, 124.22, 123.25, 123.20, 117.10, 116.21, 67.63, 50.98, and 36.76. Elemental analysis calculated for C₂₀H₁₈N₆O₄·1/30CH₃CH₂OH: C 59.11, N 20.68, H 4.46, O 15.75; found: C 59.08, N 20.60, H 4.50, O 15.82. (See Figures S23–S26 in the Supplementary Materials for the 1H, 13C NMR, DEPT-135, and IR spectra for compound 4h).

N'-((1-methyl-1H-pyrrol-2-yl)methylene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4i ): The compound is a white powder with a yield of 86% and a melting point of 228–230°C. The IR spectrum (KBr) shows a carbonyl stretch at 1662 cm − 1. 1H-NMR (DMSO-d6, 500 MHz) provides δ 11.41 (s, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 7.97–6.04 (m, 7H), 5.51 (s, 2H), 5.14 (s, 2H), 4.67 (s, 2H) and 3.79 (s, 2H). 13C NMR (125 MHz, DMSO-d6) shows δ 167.15, 164.61, 145.41, 142.56, 138.26, 129.13, 128.97, 127.07, 125.93, 124.23, 123.19, 122.97, 117.07, 116.39, 108.61, 67.63, 51.13, 36.74 and 37.21. Elemental analysis calculated for C₁₉H₁₉N₇O₃ 1/22H₂O: C 58.01, N 24.92, H 4.87, O 12.20; found: C 57.89, N 24.87, H 4.88, O 12.36. (See Figures S27–S29 in the supplemental material for 1H, 13C, DEPT-135 and IR NMR spectra for compound 4i).

N'-(furan-2-ylmethylene)-2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)acetohydrazide ( 4j ): The compound is a white powder with a yield of 82% and a melting point of 256–258°C. The IR spectrum (KBr) shows a carbonyl stretch at 1662 cm − 1. The 1H-NMR spectrum (DMSO-d6, 500 MHz) displays δ 11.70 (s, 1H), 8.06 (s, 1H), 8.02 (s, 1H), 7.97–6.60 (m, 7H), 5.51 (s, 2H), 5.15 (s, 2H), and 4.67 (s, 2H). In the 13C-NMR spectrum (125 MHz, DMSO-d6), the δ values are 167.56, 164.64, 149.39, 145.81, 145.41, 142.54, 135.07, 129.01, 126.02, 124.18, 123.17, 117.09, 116.35, 114.51, 112.75, 67.62, 50.83 and 36.75. Elemental analysis calculated for C₁₈H₁₆N₆O₄ 1/20H₂O: C 56.84, N 22.10, H 4.24, O 16.83; found: C 56.81, N 21.96, H 4.29, O 16.93. (See Figures S30–S32 in the supplemental material for 1H, 13C, DEPT-135 and IR NMR spectra for compound 4j).

In silico evaluation of the biological activities of the synthetic molecules (4a-4j).

Ligand Preparation

The synthetic molecules (4a-4j) (Sch. 2) were modeled in 3D using Avogadro 1.2. The standard inhibitors, namely acarbose (CID: 41774), zileuton (CID: 60490), vincristine (CID: 5978), methotrexate (CID: 126941) and xanthene (CID: 7107), were obtained in 3D SDF format from the PubChem database (available online: https://pubchem.ncbi.nlm.nih.gov/). To assess the molecular docking of these ligands to target proteins, ligands were first converted to pdb format using PyMoL Molecular Graphics System (version 2.5.3) and then processed to pdb format via Autodock Tools (ADT; version 1.5.7, The Scripps Research Institute)⁵⁸. Visualization of molecular interactions between ligands and receptors and comparison of binding affinities with standard inhibitors was performed using Discovery Studio Visualizer (Biovia, 2021), which helped generate the graphical representations⁵⁹.

Preparation of Protein

The crystal structures of human proteins α-amylase (PDB ID: 1B2Y), α-glucosidase (PDB ID: 5NN8), Dihydrofolate reductase (PDB: 1DRF), epidermal growth factor receptor (PDB: 3W2S), glutathione reductase (PDB: 3DK9) and lipoxygenase (PDB: 1N8Q) were retrieved from the Protein Data Bank (PDB) (available online: www.rcsb.org)⁵⁸. All crystal structures were individually organized by removing water molecules and carefully adding polar hydrogen and Kollman charges via AutoDockTools (ADT; program version 1.5.7.)⁶⁰. For the molecular docking process, a grid with a point spacing of 0.375 Å and dimensions of 40 × 40 × 40 was created and centered on the x, y and z coordinates, to encompass both the peripheral regions and the active sites of proteins. Finally, the prepared macromolecules were stored in PDB format to facilitate the subsequent molecular analysis process.

Molecular Dynamic Simulations

Molecular dynamics (MD) simulations were performed on a high-potential activity complex from the docking study (4a-3W2S) and (4b-3DK9) using GROMACS 2024.2^50,51. The simulations, which lasted for 5 ns, employed the CHARMM36 force field [61], with ligand parameters obtained from the CGenff servers (available at www.cgenff.com)^62,63. The complex was solvated with the SPC/E water model⁶², and sodium and chloride ions were added for charge neutralization. The system underwent energy minimization with a cutoff of 100 kJ/mol/nm, followed by equilibration in NVT and NPT ensembles for 1 ns, where the temperature was kept at 310 K and the pressure at 1 bar^53,54. Post-simulation analyses, including root mean square fluctuation (RMSF), root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond analysis (Hb), were conducted using the XMGrace tools^45,55.

ADME Studies

Understanding the pharmacokinetic properties of substances encompassing absorption, distribution, metabolism, and excretion (ADME)—is essential for grasping how a compound behaves within the body⁶⁴. These phases outline the journey of a substance from its initial absorption to its eventual elimination. Computational tools are increasingly vital for predicting these ADME characteristics, evaluating a molecule's ability to cross cellular barriers, interact with key transporters and enzymes involved in absorption and excretion, and assessing its metabolic stability. In our evaluation process, we use the Swiss ADME platform (available at www.swissadme.ch)⁵⁸. This tool allows us to thoroughly analyze the physicochemical properties of synthetic molecules (4a-4j), assess their potential as therapeutic agents, and gain insights into their pharmacokinetic profiles, providing a detailed overview of their ADME characteristics.

Chemical Synthesis of 2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)-N'-(benzylidene) acetohydrazide derivatives 4a–4j

The synthesis of 1,4-disubstituted 1,2,3-triazoles linked to [1,4]-benzoxazin-3-one was carried out through a multi-step process. Initially, dipolarophile 1 was prepared by alkylation of [1,4]-benzoxazin-3-one was reacted with propargyl bromide in DMF using potassium carbonate at room temperature, following the established protocol in the literature²⁵. The resulting dipolarophile was then reacted with ethyl azidoacetate in the presence of Cu(I), yielding ethyl 2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl) acetate 2. This reaction has several advantages, including mild reaction conditions, exhibiting high regioselectivity in a CuAAC process, and being environmentally friendly. The acid hydrazide 3 was synthesized by reacting the ethyl ester 2 with hydrazine hydrate in ethanol under reflux. The final derivatives, 2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)-N'-(benzylidene)acetohydrazide derivatives 4, were synthesized through the condensation of acid hydrazide 3 with various aromatic aldehydes (Sch. 1).

In silico biological activities of the synthesized molecules (4a-4j)

In the field of medicinal chemistry, in silico analysis plays a crucial role in predicting the biological activities of newly synthesized molecules. Here, we focus on a series of synthesized compounds, labeled 4a-4j (Sch. 2), whose potential antibacterial, anticancer, anti-inflammatory and antidiabetic activities are predicted by their affinity for the protein involved in each biological activity and in comparison, with known standard inhibitors. This comparison allowed us to identify the compounds with the best activity and safety profiles for further preclinical studies.

Antidiabetic activity

α-amylase is an essential enzyme involved in carbohydrate metabolism, found in humans as well as animals, bacteria, plants, and fungi. It is crucial for digestion, facilitating the hydrolysis of 1,4-glucan bonds in starch, maltodextrins and maltooligosaccharides. The enzyme initiates its activity in the mouth with salivary α-amylase, which breaks down starch into oligomers. This process continues in the intestine where pancreatic α-amylase further decomposes these oligomers into smaller oligosaccharides²⁶. These are then converted into glucose by glucosidases in the intestine, thus allowing the absorption of glucose into the bloodstream, where it plays an essential role in regulating blood sugar levels. Many studies investigate α-amylase as a therapeutic target for diabetes treatment. By modulating the activity of this enzyme, it is possible to influence the rate of starch degradation and thus manage postprandial blood glucose levels²⁷. Therefore, α-amylase is not limited to its digestive function but also represents a potential target in improving diabetes management, underscoring its importance in medical and therapeutic applications²⁸.

In this study, the molecular docking results for ligands binding to α-amylase (PDB: 1B2Y) showed that the 4a molecule had a superior binding affinity (-9.3 kcal/mol) compared to acarbose (-8.2 kcal/mol), which is a commonly used α-amylase inhibitor. This was followed by 4b, 4e, and 4c, which exhibited binding affinities of -9.0, -8.5, and − 8.3 kcal/mol, respectively (Table 1). The 4a molecule stood out for its good affinity towards α-amylase, interacting efficiently with several amino acid residues of the enzyme. Specifically, it demonstrated hydrogen bond interactions with Arg 421 and Arg 398 which were located in the active site (Fig. 1).

α-glucosidase is a vital enzyme in carbohydrate metabolism, found in a range of organisms including humans, animals, plants, and microorganisms. It is essential for digestion, as it catalyzes the hydrolysis of α-glucose residues from the non-reducing ends of carbohydrates like starch and disaccharides. This process yields simple sugars that are absorbed into the bloodstream, thereby affecting blood sugar levels. α-glucosidase primarily acts in the small intestine, working at the brush border to transform complex carbohydrates into glucose and other monosaccharides²⁹. This enzymatic process is crucial for supplying the body with usable energy and regulating normal blood sugar levels. Given its key role in carbohydrate digestion and absorption, α-glucosidase has emerged as an important target for diabetes treatment. Inhibiting this enzyme can effectively reduce the rate of glucose absorption, leading to more gradual increase in blood sugar levels following meals³⁰. This mechanism has been utilized in the creation of α-glucosidase inhibitors; a group of oral antidiabetic drugs designed to manage postprandial blood glucose levels in individuals with diabetes. Thus, α-glucosidase not only facilitates digestion but also serves as a potential therapeutic target in diabetes management, underscoring its importance in both physiological and therapeutic contexts³¹.

In this study, the results obtained from molecular docking of ligands to α-glucosidase (PDB: 5NN8) revealed that all (4a-4j) molecules present a higher affinity for α-glucosidase than acarbose (-7.2 kcal/mol) (Table 1), the 4a molecule stood out for its good affinity towards α-glucosidase, interacting efficiently with several amino acid residues of the enzyme. Specifically, it demonstrated hydrogen bonding interactions with Arg 585 (Fig. 1). Molecular docking results indicate that molecules 4a-4j exhibit significantly high affinity for α-amylase and α-glucosidase enzymes compared to known inhibitors such as acarbose. Notably, molecule 4a exhibits remarkable affinity for these targets. They suggest that these molecules could provide therapeutic benefits for the treatment of diabetes. To confirm their therapeutic potential, in vivo and in vitro studies are needed to evaluate their efficacy as drug candidates and their safety in complex biological systems for the development of more effective antidiabetic therapies.

Anti-cancer activity

The epidermal growth factor receptor (EGFR) is a transmembrane protein that belongs to the receptor tyrosine kinase (RTK) family within the erythroblastic leukemia viral oncogene homolog B (ErbB) group. This group includes EGFR (ErbB-1), HER2 (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4)³². ErbB-1 plays a vital role in various normal cellular functions, including growth, differentiation, and survival. However, in cancer, abnormal expression or activity of EGFR is frequently observed and is linked to the advancement of numerous tumor types, such as non-small cell lung cancer, colorectal cancer, head and neck cancer, and pancreatic cancer. Inhibiting EGFR affects not only the EGFR/RAS/RAF signaling pathway, essential for tumor cell survival and proliferation but also disrupts the EGFR/PI3K/AKT/mTOR pathway³³. The latter pathway is crucial for regulating various cellular processes, including growth, survival, proliferation, and migration. Targeting EGFR is a central therapeutic approach in cancer treatment. Discovering new molecules with high affinity for this protein is a significant and active research focus³⁴.

In this study, results obtained from molecular docking of ligands to EGFR (PDB: 3W2S) revealed that all 4a-4j molecules present a higher affinity for epidermal growth factor receptor protein than Vincristine (-8.1 kcal/mol) (Table 1), the 4a molecule stood out for its good affinity towards EGFR, interacting efficiently with several amino acid residues of the enzyme. Specifically, it demonstrated hydrogen bonding interactions with Arg 841 which was located in the active site (Fig. 1). According to the results, 4a-4j molecules exhibit a high affinity for epidermal growth factor receptor (EGFR), especially 4a. These observations suggest that 4a-4j molecules may offer therapeutic benefits for cancer treatment by effectively targeting EGFR. This could be explored using in vivo and in vitro studies to evaluate and confirm their anticancer effects in various cancer types.

Antioxidant activity

Glutathione reductase (GR) is a vital enzyme that aids cells in combating oxidative stress, a factor linked to the development of various diseases. Targeting glutathione (GSH), a key antioxidant, could provide therapeutic advantages due to its role in mitigating excessive oxidative stress associated with many conditions. A promising strategy for developing targeted therapies for cancer and antimicrobial treatments involves inhibiting glutathione reductase³⁵. This approach capitalizes on the dependence of bacteria and cancer cells on the glutathione system for their antioxidant protection. When used strategically, it can work in synergy with other treatment methods, leading to a more precise and effective strategy for addressing cancer and bacterial infections³⁶.

In this study, the results obtained by the molecular docking of the ligands to human enzyme glutathione reductase (PDB: 3DK9) revealed that all the molecules (4a, 4b, 4c, 4d, 4e, 4f, 4h and 4j) present an affinity higher for glutathione reductase than Xanthene (-6.2 kcal/mol) (Table 1), the 4b molecule stood out for its good affinity towards glutathione reductase, interacting efficiently with several amino acid residues of the enzyme. More specifically, it demonstrated hydrogen bond interactions with Gln 182 and Asn 294, unlike xanthene which showed no hydrogen bonds (Fig. 2). All molecules (4a, 4b, 4c, 4d, 4e, 4f, 4h, and 4j) showed higher affinity for glutathione reductase compared to xanthene. Among them, molecule 4b stands out for its efficient interaction with key residues of the enzyme, suggesting its potential as a targeted treatment against oxidative stress. This molecule could be particularly promising when used in synergy with other therapies. In vitro and in vivo studies are needed to confirm its efficacy and therapeutic potential.

Antibacterial activity

Dihydrofolate reductase (DHFR) is an essential enzyme found in various species, playing a vital role in folate metabolism, which is crucial for synthesizing nucleotides required for DNA replication. Its primary function is within the thymidylate production cycle, where it catalyzes the conversion of dihydrofolate (DHF) to tetrahydrofolate (THF)³⁷ This reaction is vital because THF serves as a cofactor in synthesizing purine and pyrimidine bases, fundamental components of DNA. DHFR inhibitors specifically target this metabolic pathway. By blocking the conversion of DHF to THF, these inhibitors prevent the regeneration of folate metabolites necessary for DNA formation. This interruption of the nucleotide base synthesis cycle results in a deficiency of essential components for DNA replication, which can lead to a bactericidal or bacteriostatic effect, depending on the concentration of the inhibitor and the sensitivity of the target bacterial species³⁸. In addition to their significance in bacteriology, DHFR inhibitors play a vital role in chemotherapy. They are employed to suppress the growth of cancer cells by utilizing the same mechanism³⁹. This highlights the importance of DHFR not only as an antibacterial target but also as a therapeutic target in cancer treatment⁴⁰. Research continues to develop new, more selective, and less toxic DHFR inhibitors to optimize their effectiveness and minimize side effects in various clinical settings.

In this study, the results obtained by molecular docking of ligands to human enzyme dihydrofolate reductase (PDB: 1DRF) revealed that all molecules (4a-4j) exhibit higher affinity for glutathione reductase than Methotrexate (-5.5 kcal/mol) (Table 1), the 4a molecule stood out for its good affinity towards dihydrofolate reductase, interacting efficiently with several amino acid residues of the enzyme. Specifically, it demonstrated interactions by hydrogen bonds with Asp 95 and Arg 91 (Fig. 2). Docking results revealed that molecule 4a is distinguished by a particularly high affinity for dihydrofolate reductase (DHFR) compared to other molecules (4a-4j) and methotrexate. This affinity suggests that molecule 4a could act as an effective DHFR inhibitor, with significant potential for antibacterial treatment. Further in vitro and in vivo studies are needed to confirm its therapeutic efficacy as a drug.

Anti-inflammatory

Lipoxygenases (LOX) are enzymes crucial for the metabolism of polyunsaturated fatty acids and are found in various species, including humans, other mammals, and certain plants. The names of LOX enzymes are based on the specific carbon atom they oxygenate; examples include 5-LOX, 12-LOX, and 15-LOX in animals, and 9-LOX and 13-LOX in plants⁴¹. The inhibition of lipoxygenase enzymes, in particular 5-lipoxygenase (5-LOX), has a direct and significant impact on the production of leukotrienes, which are lipid mediators playing a key role in inflammation⁴². This enzyme facilitates the conversion of arachidonic acid, a polyunsaturated fatty acid released from cell membranes, into 5-HPETE, which can be directly transformed into leukotriene B4 (LTB4). Additionally, 5-HPETE can be converted into leukotriene A4 (LTA4), which is then processed by various enzymes into cysteinyl leukotrienes (LTC4, LTD4, LTE4)⁴³. Notably, these leukotrienes are essential in various physiological processes and are particularly associated with conditions like allergic disorders and systemic inflammatory diseases, including rheumatoid arthritis and cancer⁴⁴. The importance of finding molecules with high affinity for the enzyme lipoxygenase is crucial in the development of therapeutic strategies to treat inflammation-related diseases⁴⁵.

In this study, the results obtained by molecular docking of ligands to the lipoxygenase enzyme (PDB: 1N8Q) revealed that all molecules (4a-4j) exhibit a higher affinity for glutathione reductase than Zileuton (-6.4 kcal/mol) (Table 1), the 4a molecule stands out for its good affinity towards lipoxygenase (-11.0 kcal/mol), interacting effectively with several amino acid residues of the enzyme. It demonstrated hydrogen bond interactions with Val 256, Leu 273, Thr 274, Tyr 275, and Lys 278 (Fig. 2). The docking results showed that molecule 4a could act as a potential lipoxygenase inhibitor, highlighting its potential for the development of treatments against inflammatory diseases. Further in vitro and in vivo studies are essential to confirm these results and to advance the development of drugs based on this molecule.

Table 1

Molecular binding affinities (Kcal/mol) between synthetic molecules (4a-4j) and human proteins.
	Antidiabetic		Anti-inflammatory	Anti-cancer	Antibacterial	Antioxidant
	α-amylase (1B2Y)	α-glucosidase (5NN8)	LOX (1N8Q)	EGFR (3W2S)	DHFR (1DRF)	GR (3DK9)
Compounds	Affinity (Kcal/mol)		Affinity (Kcal/mol)	Affinity (Kcal/mol)	Affinity (Kcal/mol)	Affinity (Kcal/mol)
Inhibitor standard	-8.2	-7.2	-6.4	-8.1	-5.5	-6.2
4a	-9.3	-9.2	-11.0	-9.7	-7.6	-7.4
4b	-9.0	-8.6	-10.3	-9.2	-7.1	-9.5
4c	-8.3	-8.2	-10.4	-8.7	-6.9	-7.0
4d	-7.9	-8.0	-10.6	-9.6	-6.6	-6.9
4e	-8.5	-7.9	-9.9	-9.2	-6.1	-6.7
4f	-8.1	-8.0	-8.5	-8.7	-6.6	-6.6
4g	-7.7	-8.0	-9.1	-8.3	-5.9	-6.2
4h	-8.1	-7.7	-9.9	-8.9	-6.4	-6.7
4i	-7.9	-7.9	-9.3	-8.6	-6.0	-5.8
4j	-8.2	-7.6	-9.5	-8.3	-6.3	-6.5

LOX; Lipoxygenases, EGFR; The epidermal growth factor receptor, DHFR; Dihydrofolate reductase, GR; Glutathione reductase.

Molecular dynamic simulation

The complex (4a-3W2S) was subjected to dynamic simulation after molecular docking using the CROMACS software. To understand the dynamic behavior of molecular systems under similar physiological conditions⁴⁶. The binding affinity between ligand-proteins and the stability of the complex (4a-3W2S) was evaluated using RMSD plots, which measure the deviation of the atoms of the protein and the ligand during the simulation (5 ns) about their positions⁴⁷. However, the RMSD plot shows the stability of the protein during the simulation, and the complex (4a-3W2S) showed significant fluctuations of 4.5 Å revealing a potentially low affinity or an ability to adopt different conformations⁴⁸ (Fig. 3. A). The Root Mean Square Fluctuation (RMSF) reflects the root mean square fluctuations of the atomic positions in the trajectory. However, significant fluctuations in atoms during MD were recorded in the RMSF plot⁴⁹ (Fig. 3. B), revealing that there are different regions of flexibility and stability at the structural level⁵⁰. Additionally, intermolecular hydrogen bonding networks are essential for understanding conformational changes and complex stability during MD⁵¹. Changes were observed in the complex (4a-3W2S) with an interaction number fluctuating between 1 and 6, forming an average hydrogen bond number of 3 in (5 ns), demonstrating moderately stable interactions⁵² (Fig. 3. C). The radius of gyration (Rg) was used to calculate the compactness of the protein-ligand complex structure throughout the simulation. The Rg plot shows fluctuations with an average value of 2.02 nm, indicating that the complex maintains a stable overall structure with variations. These fluctuations also demonstrate the flexibility of the complex^41,43 (Fig. 3. D).

The RMSD plot of the 4b-3DK9 complex showed stability at the beginning of the simulation with insignificant fluctuations around 1–2.5 Å. However, between 1 ns and 5 ns, significant fluctuations of up to 4.8 Å appear, suggesting conformational flexibility of the complex⁵³ (Fig. 4. A). RMSF reveals notable fluctuations up to 2.5 Å, indicating that these regions of the ligand are more flexible. In contrast, other parts of the ligand showed small fluctuations of approximately 1 Å, suggesting more stable regions⁵⁴ (Fig. 4. B). The number of hydrogen bonds in the 4b-3DK9 complex fluctuates between 0 and 4 over 5 ns. Towards the end of the simulation, the number of hydrogen bonds decreases, stabilizing mainly around the 0–1 bond, indicating instability or low binding affinity during the simulation⁵⁵ (Fig. 4. C). The radius of gyration (Rg) of the complex indicated that the overall structure remained relatively stable despite fluctuations reaching 2.04 nm (Fig. 4. D).

Results of ADME Analysis

In silico ADME studies are crucial for optimizing pharmaceutical development, providing a cost-effective method to predict a drug's behavior within the body. By utilizing computer models for early pharmacokinetic profiling, these studies accelerate the selection of drug candidates and refine development processes. They help minimize the risk of adverse effects, decrease the likelihood of drug development failures, and improve the chances of clinical success, making them essential in contemporary drug discovery and development. These studies also ensure adherence to Lipinski's rule of five and other drug similarity criteria, such as Veber's rule. Lipinski's guidelines suggest that compounds suitable for oral administration should not violate more than one of the following conditions: (1) more than 10 hydrogen bond acceptors (oxygen or nitrogen atoms), (2) an octanol-water partition coefficient (log P or MLogP) exceeding 5, (3) more than 5 hydrogen bond donors (oxygen or nitrogen atoms with one or more hydrogen atoms), (4) a molecular weight greater than 500 daltons and (5) Polar surface Area > 140 Å² ⁴⁵. In our study, it was noted that all the compounds examined adhered to Lipinski's criteria, with each having no more than one violation. Furthermore, the compounds were assessed according to Veber's rule, which takes into account the number of rotatable bonds and polar surface area. Importantly, none of the molecules breached Veber's criteria, highlighting their strong potential as viable candidates for oral drug development. In Table 2, it is noted that none of the molecules cross the blood-brain barrier (BBB), as illustrated in (Fig. 5). Moreover, only compounds 4a, 4b, 4c, and 4g were identified as non-substrates for P-glycoprotein (PGP), as shown in (Sch. 1). Additionally, these compounds exhibited favorable intestinal absorption, enhancing their suitability for oral administration. The cytochrome P450 enzyme system, predominantly found in the liver, is crucial for drug detoxification. Our analysis revealed that none of the compounds acted as inhibitors or substrates for the CYP450 enzymes, specifically CYP1A2 and CYP2D6 (Table 2). This suggests a lower risk of interference with drug metabolism, thereby improving the safety profile of these compounds⁵⁶. As a result, all the identified compounds were assigned a bioavailability score of 0.55. (Fig. 6) displays the bioavailability radar charts for these compounds. In these plots, the pink area represents the space for oral bioavailability. To be considered drug-like, a compound's profile must fall entirely within this area. Notably, in this study, compound 4f falls within the desired range for oral bioavailability⁵⁷, indicating its potential as a drug candidate.

Table 2

Assessment of the pharmacokinetic characteristics (ADME) of the synthetic compounds (4a-4j) (in silico).
	Physicochemical Properties					Lipophilicity		Druglikeness		Pharmacokinetics
Compounds	MW g/mol	HBA	HBD	TPSA Å²	Rotatable Bonds	M logP	W logP	Lipinski’s Violat-ion	Verber’s Violat-ion	GI Absorp-tion	BBB Permea-tion	CYP1A2 Inhibitor	CYP2D6 Inhibitor
4a	388.42	5	1	84.64	7	1.69	1.81	0	0	High	No	No	No
4b	469.29	6	1	101.71	7	1.70	1.58	0	0	High	No	No	No
4c	424.84	6	1	101.71	7	1.59	1.47	0	0	High	No	No	No
4d	435.39	8	1	147.53	8	1.11	0.73	1	1	Low	No	No	No
4e	420.42	7	1	110.94	8	0.82	0.83	0	0	High	No	No	No
4f	448.47	7	1	110.94	9	1.25	1.53	0	0	High	No	No	No
4g	416.43	6	1	101.71	8	1.47	1.38	0	0	High	No	No	No
4h	406.39	7	2	121.94	7	0.60	0.53	0	0	High	No	No	No
4i	393.40	6	1	106.64	7	0.14	0.16	0	0	High	No	No	No
4j	380.36	7	1	114.85	7	-0.10	0.41	0	0	High	No	No	No

BBB: Blood-Brain Barrier; GI: Gastrointestinal; HBA: Hydrogen-Bond Acceptors; HBD: Hydrogen-Bond Donors; MLogP: Octanol/Water Partition Coefficient; MW: Molecular Weight; TPSA: Topological Polar Surface Area; WLogP: Lipophilicity.

New derivatives of [1,4]-benzoxazin-3-one, linked to a 1,2,3-triazole core, have been efficiently synthesized with high yields using the Cu(I)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) method. These compounds were characterized through various spectroscopic techniques, including IR, ¹H NMR, ¹³C NMR, DEPT-135, and elemental analysis. Molecular docking studies revealed that all synthesized molecules (4a-4j) exhibited significant potential, with activities including anti-inflammatory, antibacterial, antioxidant, anticancer, and antidiabetic properties. 5 ns molecular dynamics (MD) simulations of the most promising complexes, 4a-3W2S as well as 4b-3DK9, demonstrated overall stability with fluctuations indicating conformational flexibility and adaptation while maintaining ligand-protein interactions. ADME analysis confirmed that all synthetic molecules (4a-4j) exhibited favorable pharmacological properties, adhering to Lipinski and Veber's rules. These results highlight the potential of these derivatives for future pharmaceutical and chemical applications, with further in vitro and in vivo studies planned to evaluate therapeutic efficacy and explore other uses.

Author Contributions

Conceptualization, O.M.N., A.S.A. and M.C.; methodology, M.B., A.I., A.E.D. and B.E.; software, D.A., A.F. and N.H.; validation, M.B., M.E., F.C. and M.C.; formal analysis, S.L., A.E.D., and B.E.; investigation, S.L., A.I. and A.E.D.; resources, A.I., M.E., A.E.D., and A.F.; data curation, S.L., D.A. and N.H.; writing—original draft preparation, D.A., A.F. and N.H.; writing—review and editing, M.B., M.E. and O.M.N.; visualization, M.B., O.M.N., A.S.A.; supervision, F.C. and M.C.; project administration, M.B., M.E. and A.S.A.; funding acquisition, O.M.N. and A.S.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research is funded by Researchers Supporting Project number (RSP2023R132), King Saud University, Riyadh, Saudi Arabia. The Faculty of Sciences and Technology at Sultan Moulay Slimane University provided support for the analyses and the supply of chemicals.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We want to express our sincere gratitude to all the individuals who have been of help during the project’s realization. Special thanks to the Moroccan National Center for Technical and Scientific Research (CNRST) of Morocco for its encouragement and for providing access to the technical resources of the UATRS Division. We would like to extend our sincere appreciation to the Researchers Supporting Project number (RSP2023R132), King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflicts of interest.

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Schemes 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

SupportinginformationSR.docx
scheme1.png
Scheme 1. Synthesis of 2-(4-[([1,4]-benzoxazin-3-on-4-yl)methyl]-1H-1,2,3-triazol-1-yl)-N'-(benzylidene) acetohydrazide derivatives 4.
scheme2.png
Scheme2. Structures of ligand molecules (4a-4j), used for molecular docking

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Newly synthesized 1,2,3-triazoles based on [1,4]-benzoxazin- 3-one: In silico evaluation of anti-inflammatory, antibacterial, antioxidant, anticancer, and antidiabetic properties, along with molecular dynamics simulation and ADME analysis

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Abstract

Figures

Introduction

Materials and Methods

General

Ligand Preparation

Preparation of Protein

Molecular Dynamic Simulations

ADME Studies

Results and Discussion

Antidiabetic activity

Anti-cancer activity

Antioxidant activity

Antibacterial activity

Anti-inflammatory

Molecular dynamic simulation

Results of ADME Analysis

Conclusions

Declarations

References

Schemes

Additional Declarations

Supplementary Files

Status:

Version 1