Aril ve Aroil Hidrazon İçeren Heterosiklik Fenolik Bileşiklerin Antioksidan Potansiyelinin İncelenmesi: İn Vitro ve Teorik Araştırmalar

Öz

Bu çalışma, çeşitli heterosiklik fenollerin radikal süpürücü ve indirgeme potansiyellerinin deneysel ve hesaplamalı yaklaşımların birlikte kullanıldığı kapsamlı bir değerlendirmesini sunmaktadır. Radikal süpürücü ve indirgeme potansiyelleri DPPH ve CUPRAC deneyleri ile belirlenmiş; etki mekanizmasını aydınlatmak amacıyla moleküler kenetleme, MM/PBSA enerji değerlendirmeleri ve moleküler dinamik simülasyonlarından elde edilen atomistik düzeydeki bulgularla desteklenmiştir. Test edilen bileşikler arasında, üç fenolik hidroksil grubu taşıyan bileşik 5 üstün antioksidan performans göstermiş ve TEAC değerleri CUPRAC için 7,85, DPPH için ise 3,60 olarak elde edilmiştir. Hesaplamalı sonuçlar deneysel verilerle tam uyum göstermiş ve bileşik 5’in en güçlü bağlanma afinitelerine sahip olduğunu ortaya koymuştur; özellikle MM/PBSA bağlanma serbest enerjileri 2C9V ve 1GTA için sırasıyla -86,19 kcal/mol ve -24,08 kcal/mol olarak belirlenmiştir. Öngörülen bağlanma modunun kararlılığını daha ileri düzeyde doğrulamak amacıyla 2C9V-5 kompleksi için 100 ns’lik bir MD simülasyonu gerçekleştirilmiş ve kompleksin simülasyon süresi boyunca dinamik ve yapısal kararlılığını koruduğu doğrulanmıştır. Toplu olarak değerlendirildiğinde, bu bulgular bileşik 5’i umut verici bir öncü antioksidan aday olarak tanımlamakta ve rasyonel antioksidan keşfi için deneysel ve hesaplamalı yaklaşımların birlikte kullanılmasının etkinliğini ortaya koymaktadır.

Anahtar Kelimeler

• Heterosiklik bileşikler
• Fenoller
• CUPRAC
• DPPH
• İn siliko etkileşimler

Exploring the Antioxidant Potential of Heterocyclic Phenolic Compounds with Aryl and Aroyl Hydrazones: In Vitro and Theoretical Investigations

Öz

Abstract: This study presents a comprehensive evaluation of the radical scavenging and reducing potentials of various heterocyclic phenols through a combined experimental and computational approach. The radical scavenging and reducing potentials were determined through DPPH and CUPRAC experiments, which were further supported by atomistic insights from molecular docking, MM/PBSA energy assessments, and molecular dynamic simulations to clarify the mechanism of action. Among the tested compounds, compound 5, bearing three phenolic hydroxyl groups, displayed superior antioxidant performance, yielding TEAC values of 7.85 (CUPRAC) and 3.60 (DPPH). The computational results were in full agreement with the experimental data, showing that compound 5 attained the strongest binding affinities; specifically, its MM/PBSA binding free energies were determined as -86.19 kcal/mol and -24.08 kcal/mol for 2C9V and 1GTA, respectively. To further validate the stability of the predicted binding mode, a 100 ns MD simulation was conducted for the 2C9V-5 complex, confirming its dynamic and structural stability throughout the simulation. Collectively, these findings identify compound 5 as a promising lead antioxidant and demonstrate the effectiveness of combining experimental and computational approaches for rational antioxidant discovery.

Anahtar Kelimeler

• Heterocyclic compounds
• Phenols
• CUPRAC
• DPPH
• In silico interactions

Kaynakça

1. [1] Birben, E., Sahiner, U. M., Sackesen, C., Erzurum, S., Kalayci, O. 2012. Oxidative Stress and Antioxidant Defense. World Allergy Organization Journal, 5(1), 9–19.
2. [2] Selvaraj, N. R., Nandan, D., Nair, B. G., Nair, V. A., Venugopal, P., Aradhya, R. 2025. Oxidative Stress and Redox Imbalance: Common Mechanisms in Cancer Stem Cells and Neurodegenerative Diseases. Cells, 14(7), 511.
3. [3] Zhou, N., Bao, W. Q., Zhang, C., Jiang, M. L., Liang, T. L., Ma, G. Y., Liu, L., Pan, H. D., Li, R. Z. 2025. Immunometabolism and oxidative stress: roles and therapeutic strategies in cancer and aging. NPJ Aging, 11, 59.
4. [4] Forni, C., Facchiano, F., Bartoli, M., Pieretti, S., Facchiano, A., D’Arcangelo, D., Norelli, S., Valle, G., Nisini, R., Beninati, S., Tabolacci, C., Jadeja, R. N. 2019. Beneficial Role of Phytochemicals on Oxidative Stress and Age-Related Diseases. BioMed Research International, 2019, 1–16.
5. [5] Gulcin, İ. 2025. Antioxidants: a comprehensive review. Archives of Toxicology, 99, 1893–1997.
6. [6] Muscolo, A., Mariateresa, O., Giulio, T., Mariateresa, R. 2024. Oxidative Stress: The Role of Antioxidant Phytochemicals in the Prevention and Treatment of Diseases. International Journal of Molecular Sciences, 25(6), 3264.
7. [7] Zeb, A. 2020. Concept, mechanism, and applications of phenolic antioxidants in foods. Journal of Food Biochemistry, 44, e13394.
8. [8] Leopoldini, M., Marino, T., Russo, N., Toscano, M. 2004. Antioxidant Properties of Phenolic Compounds: H-Atom versus Electron Transfer Mechanism. Journal of Physical Chemistry A, 108, 4916–4922.

9. [9] Platzer, M., Kiese, S., Tybussek, T., Herfellner, T., Schneider, F., Schweiggert-Weisz, U., Eisner, P. 2022. Radical Scavenging Mechanisms of Phenolic Compounds: A Quantitative Structure-Property Relationship (QSPR) Study. Frontiers in Nutrition, 9, 882458.
10. [10] Chen, J., Yang, J., Ma, L., Li, J., Shahzad, N., Kim, C. K. 2020. Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Scientific Reports, 10, 2611.
11. [11] Ali, H. M., Abo-Shady, A., Sharaf Eldeen, H. A., Soror, H. A., Shousha, W. G., Abdel-Barry, O. A., Saleh, A. M. 2013. Structural features, kinetics and SAR study of radical scavenging and antioxidant activities of phenolic and anilinic compounds. Chemical Central Journal, 7, 53.
12. [12] Chen, Z., Świsłocka, R., Choińska, R., Marszałek, K., Dąbrowska, A., Lewandowski, W., Lewandowska, H. 2024. Exploring the Correlation Between the Molecular Structure and Biological Activities of Metal–Phenolic Compound Complexes: Research and Description of the Role of Metal Ions in Improving the Antioxidant Activities of Phenolic Compounds. International Journal of Molecular Sciences, 25, 11775.
13. [13] Dilek, Ö. 2025. Investigation of antioxidant activities of novel fluorine-based azo compounds combined with in silico molecular docking and in vitro CUPRAC method. Journal of the Indian Chemical Society, 102, 101513.
14. [14] Dilek, Ö., Yeşil, T. A., Tilki, T., Dede, B. 2025. Design, synthesis, and quantum chemical calculations of triazole-based aroylhydrazone molecules: Dual assessment of antioxidant properties via in vitro and in silico approaches. Journal of Molecular Structure, 1322, 140502.
15. [15] Welsch, M. E., Snyder, S. A., Stockwell, B. R. 2010. Privileged scaffolds for library design and drug discovery. Current Opinion in Chemical Biology, 14, 347–361.
16. [16] Pisoschi, A. M., Pop, A. 2015. The role of antioxidants in the chemistry of oxidative stress: A review. European Journal of Medicinal Chemistry, 97, 55–74.
17. [17] Blois, M. S. 1958. Antioxidant Determinations by the Use of a Stable Free Radical. Nature, 181, 1199–1200.
18. [18] Brand-Williams, W., Cuvelier, M. E., Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity. LWT - Food Science and Technology, 28, 25–30.
19. [19] Apak, R., Güçlü, K., Özyürek, M., Karademir, S. E. 2004. Novel Total Antioxidant Capacity Index for Dietary Polyphenols and Vitamins C and E, Using Their Cupric Ion Reducing Capability in the Presence of Neocuproine: CUPRAC Method. Journal of Agricultural and Food Chemistry, 52, 7970–7981.
20. [20] Kitchen, D. B., Decornez, H., Furr, J. R., Bajorath, J. 2004. Docking and scoring in virtual screening for drug discovery: methods and applications. Nature Reviews Drug Discovery, 3, 935–949.
21. [21] Leopoldini, M., Russo, N., Toscano, M. 2011. The molecular basis of working mechanism of natural polyphenolic antioxidants. Food Chemistry, 125, 288–306.
22. [22] Ulutürk, M., Karabacak Atay, Ç., Tilki, T., Dede, B. 2025. Structural properties, molecular modeling studies and antioxidant activities of novel benzoic acid-based azo molecules: A combined in vitro and in silico study. Journal of Molecular Structure, 1344, 142990.
23. [23] Sies, H., Jones, D. P. 2020. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nature Reviews Molecular Cell Biology, 21, 363–383.
24. [24] Forman, H. J., Ursini, F., Maiorino, M. 2014. An overview of mechanisms of redox signaling. Journal of Molecular and Cellular Cardiology, 73, 2–9.
25. [25] Dilek, Ö., Dükel, M., Zarzour, F., Karabacak Atay, Ç., Tilki, T. 2025. Structure–activity optimization of Deferasirox–derived aroyl hydrazones: Synthesis, DFT characterization, and mechanistic insights into selective anticancer activity against colon and breast cancer. Bioorganic Chemistry, 165, 109061.
26. [26] Hollingsworth, S. A., Dror, R. O. 2018. Molecular Dynamics Simulation for All. Neuron, 99, 1129–1143.
27. [27] De Vivo, M., Masetti, M., Bottegoni, G., Cavalli, A. 2016. Role of Molecular Dynamics and Related Methods in Drug Discovery. Journal of Medicinal Chemistry, 59, 4035–4061.
28. [28] Dilek, Ö. 2025. Comprehensive study of imidazole-based hydrazones: From design, synthesis, and characterization to in vitro and in silico antiproliferative activity analysis. Turkish Journal of Chemistry, 49, 419–438.
29. [29] Yeşil, T. A. 2025. New Azo‐Azomethine Compounds: Comprehensive Evaluation of In Silico Biological Activities, ADMEt Profiling, and In Vitro Antioxidant Properties. ChemistrySelect, 10, e202405875.
30. [30] Apak, R., Özyürek, M., Güçlü, K., Çapanoğlu, E. 2016. Antioxidant Activity/Capacity Measurement. 1. Classification, Physicochemical Principles, Mechanisms, and Electron Transfer (ET)-Based Assays. Journal of Agricultural and Food Chemistry, 64, 997–1027.
31. [31] Apak, R., Güçlü, K., Özyürek, M., Çelik, S. E. 2008. Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay. Microchimica Acta, 160, 413–419.
32. [32] Yeşil, T. A. 2026. Polyphenols containing new azo–aroylhydrazone hybrids: theoretical insights and evaluation of antioxidant potential via CUPRAC and DPPH assays. Bioorganic Chemistry, 168, 109377.
33. [33] Erdoğan, Ü., Uğur, Ş. S. 2025. Chitosan-enriched milk thistle extract-loaded liposomes anchored on nonwoven cotton fabric with antioxidant, anti-aging and UV protective effects. International Journal of Biological Macromolecules, 304, 140963.
34. [34] Erdoğan, Ü., Onem, E., Tilahun, M. M., Soyocak, A., Ak, A., Arın, U. E., Erzurumlu, Y. 2025. Investigation of Antioxidant, Antibacterial, and Anticancer Activities, and Molecular Modeling Studies of Berberis crataegina Fruit Extract. Chemistry & Biodiversity, 22, e202402591.
35. [35] Hanwell, M. D., Curtis, D. E., Lonie, D. C., Vandermeersch, T., Zurek, E., Hutchison, G. R. 2012. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. Journal of Cheminformatics, 4, 17.
36. [36] Berman, H. M. 2000. The Protein Data Bank. Nucleic Acids Research, 28, 235–242.
37. [37] BIOVIA. 2021. Discovery Studio Visualizer, Version 21.1.0.20298. Dassault Systèmes, San Diego.
38. [38] Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., Ferrin, T. E. 2004. UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry, 25, 1605–1612.
39. [39] Yu, J., Zhou, Y., Tanaka, I., Yao, M. 2010. Roll: a new algorithm for the detection of protein pockets and cavities with a rolling probe sphere. Bioinformatics, 26, 46–52.
40. [40] Trott, O., Olson, A. J. 2010. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry, 31, 455–461.
41. [41] Krieger, E., Vriend, G. 2014. YASARA View—molecular graphics for all devices—from smartphones to workstations. Bioinformatics, 30, 2981–2982.
42. [42] Krieger, E., Nielsen, J. E., Spronk, C. A. E. M., Vriend, G. 2006. Fast empirical pKa prediction by Ewald summation. Journal of Molecular Graphics and Modelling, 25, 481–486.
43. [43] Krieger, E., Dunbrack, R. L., Hooft, R. W. W., Krieger, B. 2012. Assignment of Protonation States in Proteins and Ligands: Combining pKa Prediction with Hydrogen Bonding Network Optimization. Methods in Molecular Biology, 819, 405–421.
44. [44] Maier, J. A., Martinez, C., Kasavajhala, K., Wickstrom, L., Hauser, K. E., Simmerling, C. 2015. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. Journal of Chemical Theory and Computation, 11, 3696–3713.
45. [45] Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A., Case, D. A. 2004. Development and testing of a general amber force field. Journal of Computational Chemistry, 25, 1157–1174.
46. [46] Jakalian, A., Jack, D. B., Bayly, C. I. 2002. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation. Journal of Computational Chemistry, 23, 1623–1641.
47. [47] Krieger, E., Vriend, G. 2015. New ways to boost molecular dynamics simulations. Journal of Computational Chemistry, 36, 996–1007.
48. [48] Hornak, V., Abel, R., Okur, A., Strockbine, B., Roitberg, A., Simmerling, C. 2006. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins: Structure, Function, and Bioinformatics, 65, 712–725.
49. [49] Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., Pedersen, L. G. 1995. A smooth particle mesh Ewald method. Journal of Chemical Physics, 103, 8577–8593.
50. [50] Yang, H., Xue, X., Li, H., Tay-Chan, S. C., Ong, S. P., Tian, E. F. 2017. A new parameter to simultaneously assess antioxidant activity for multiple phenolic compounds present in food products. Food Chemistry, 229, 215–222.
51. [51] Sharma, R., Yang, Y., Sharma, A., Awasthi, S., Awasthi, Y. C. 2004. Antioxidant Role of Glutathione S-Transferases: Protection Against Oxidant Toxicity and Regulation of Stress-Mediated Apoptosis. Antioxidants & Redox Signaling, 6, 289–300.
52. [52] Alves, P. E. S., Preet, G., Dias, L., Oliveira, M., Silva, R., Castro, I., Silva, G., Júnior, J., Lima, N., Silva, D. H., Andrade, T., Jaspars, M., Feitosa, C. 2022. The Free Radical Scavenging Property of the Leaves, Branches, and Roots of Mansoa hirsuta DC: In Vitro Assessment, 3D Pharmacophore, and Molecular Docking Study. Molecules, 27, 6016.
53. [53] Strange, R. W., Antonyuk, S. V., Hough, M. A., Doucette, P. A., Valentine, J. S., Hasnain, S. S. 2006. Variable Metallation of Human Superoxide Dismutase: Atomic Resolution Crystal Structures of Cu–Zn, Zn–Zn and As-isolated Wild-type Enzymes. Journal of Molecular Biology, 356, 1152–1162.
54. [54] Che, M., Wang, R., Li, X., Wang, H. Y., Zheng, X. F. S. 2016. Expanding roles of superoxide dismutases in cell regulation and cancer. Drug Discovery Today, 21, 143–149.