vic-Dioxime Hydrazone Ligand as a Novel RAS/RAF/MEK/ERK Pathway Inhibitor in Colorectal Cancer: Synthesis, Characterization and In Silico Evaluation

Abstract

It is aimed to perform an evaluation of a hydrazone derivative of the vic-dioxime ligand by examining its structural properties, bioactivity via molecular docking and absorption, distribution, metabolism, excretion (ADME) analysis, and stability using molecular dynamics (MD). With this respect, the innovative design, synthesis, and advanced in silico analysis of a novel vic-dioxime hydrazone ligand, (1Z,2E)-2-(hydroxyimino)-N'-[(1Z)-3,4,5-trimethoxyphenyl)methylene]ethanehydroximohydrazide (LH2) was explored. The compound was characterized through a series of spectroscopic techniques, including ¹H NMR, ¹³C NMR, FT-IR, and UV-Vis spectroscopy. In silico studies were performed to evaluate the ligand's binding affinities to major proteins of RAS/RAF/MEK/ERK signaling pathway through molecular docking. ADME profiling was conducted to assess the ligand's pharmacokinetic properties. Additionally, MD analysis was performed to evaluate the binding stability of the target-compound complex through RMSD, RMSF, RG, hydrogen bonding and MMPBSA free energy calculations. As a result of molecular docking analysis, the compound was found to have the highest binding energy value to RAS among the other target proteins RAF and ERK. In addition, it was obtained that LH2 complies with the conditions required to satisfy Lipinski's rule of five. Moreover, ERK-ligand complex exhibited the highest stability among the tested targets according to the MD simulation results. According to the results of the investigation, in silico anti-cancer activity was revealed based on molecular docking analysis and it was determined that novel vic-dioxime hydrazone ligand could act as a targeted therapeutic RAS/RAF/MEK/ERK pathway inhibitor in colorectal cancer.

Keywords

• vic-Dioxime
• Hydrazone Derivatives
• In Silico
• Colorectal Cancer
• RAS/RAF/MEK/ERK Pathway

References

1. Alam, M., Nami, S. A. A., Husain, A., Lee, D. U., & Park, S. (2013). Synthesis, characterization, x-ray diffraction, antimicrobial and in vitro cytotoxicity studies of 7a-Aza-B-homostigmast-5-eno [7a, 7-d] tetrazole. Comptes Rendus Chimie, 16(3), 201-206. https://doi.org/10.1016/j.crci.2012.12.018
2. Azmal, M., Paul, J. K., Prima, F. S., Talukder, O. F., & Ghosh, A. (2024). An in silico molecular docking and simulation study to identify potential anticancer phytochemicals targeting the RAS signaling pathway. PLOS ONE, 19(9), e0310637. https://doi.org/10.1371/journal.pone.0310637
3. Becke, A. D. (1993). Density-functional thermochemistry. III. The role of exact exchange. Journal of Chemical Physics, 98(7), 5648-5652. https://doi.org/10.1063/1.464913
4. Chen, D., Chen, H., Huang, Y., Zhang, Y., Zhou, J., & Xu, Y. (2023). The underlying regulatory mechanisms of colorectal carcinoma by combining vitexin and aspirin: Based on systems biology, molecular docking, molecular dynamics simulation, and in vitro study. Frontiers in Endocrinology, 14, 114104. https://doi.org/10.3389/fendo.2023.1147132
5. Crişan O, Marc G, Nastasă, C, Oniga S, Vlase L, Pırnau A & Oniga O. (2023). Synthesis and in silico approaches of new symmetric Bis-Thiazolidine-2,4-Diones as Ras and Raf oncoproteins inhibitors. Farmacia, 71(2), 254-263. https://doi.org/10.31925/farmacia.2023.2.4
6. Daina, A., Michielin, O., & Zoete, V. (2017). SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports, 7, 42717. https://doi.org/10.1038/srep42717
7. Göktürk, T., Topkaya, C., Zengin, T., Hökelek, T., Çağlar, U., & Güp, R. (2023). Nickel(II) complex of p-tolylglyoxime: Structural investigations, ADMET predictions, in-silico DNA and HSA binding assessments. Journal of Coordination Chemistry, 76(1-20). https://doi.org/10.1080/00958972.2025.2493712
8. Joshi, R., Gaikwad, H., Soge, B., Alshammari, A., Albekairi, N. A., Kabra, A., Yashwante, U., Kolte, B., Lokhande, P., & Meshram, R. J. (2025). Exploring pyrazolines as potential inhibitors of NSP3-macrodomain of SARS-CoV-2: synthesis and in silico analysis. Scientific Reports, 15(1), 767. https://doi.org/10.1038/s41598-024-81711-5

9. Kaminsky, W., Jasinsky, J. P., Woudenberg, R., Goldberg, K. I., & West, D. X. (2002). Structural study of two N(4)-substituted thiosemicarbazones prepared from 1-phenyl-1,2-propanedione-2-oxime and their binuclear nickel(II) complexes. Journal of Molecular Structure, 608(2-3), 135-141. https://doi.org/10.1016/S0022-2860(01)00921-8
10. Kirschenbaum, L. J., Panda, R. K., Borish, E. T., & Mentasti, E. (1989). Vicinal dioximate complexes of silver(III) Inorganic Chemistry, 28(12), 3623-3627. https://doi.org/10.1021/ic00318a003
11. Lee, C., Yang, W., & Parr, R. G. (1988). Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Physical Review B, 37(2), 785-789. https://doi.org/10.1103/PhysRevB.37.785
12. Lipinski, C. A., Lombardo, F., Dominy, B. W., & Feeney, P. J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews, 46(1-3), 3-26. https://doi.org/10.1016/S0169-409X(96)00423-1
13. Mackerell, A. D., Jr, Feig, M., & Brooks, C. L. (2004). Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. Journal of Computational Chemistry, 25(11), 1400-1415. https://doi.org/10.1002/jcc.20065
14. Meshram, R. J., Bagul, K. T., Aouti, S. U., Shirsath, A. M., Duggal, H., & Gacche, R. N. (2021). Modeling and simulation study to identify threonine synthase as possible drug target in Leishmania major. Molecular Diversity, 25(3), 1679-1700. https://doi.org/10.1007/s11030-020-10129-8
15. Nouri Barkestani, M., Naserian, S., Khoddam, F., Shamdani, S., & Bambai, B. (2022). Optimization of IL-1RA structure to achieve a smaller protein with a higher affinity to its receptor. Scientific Reports, 12, 7483. https://doi.org/10.1038/s41598-022-11100-3
16. Onodera, K., Satou, K., & Hirota, H. (2007). Evaluations of molecular docking programs for virtual screening. Journal of Chemical Information and Modeling, 47(4), 1609-1618. https://doi.org/10.1021/ci7000378
17. Prior, I. A., Hood, F. E., & Hartley, J. L. (2020). The frequency of RAS mutations in cancer. Cancer Research, 80(14), 2969-2974. https://doi.org/10.1158/0008-5472.can-19-3682
18. Reddy, K. H., Prasad, N. B. L., & Reddy, T. S. (2003). Analytical properties of 1-phenyl-1,2-propanedione-2-oxime thiosemicarbazone: simultaneous spectrophotometric determination of copper(II) and nickel(II) in edible oils and seeds. Talanta, 59(3), 425-433. https://doi.org/10.1016/S0039-9140(02)00543-X
19. Sarıkavaklı, N., & Çakıcı, H. T. (2011). Synthesis and characterization of Co(II), Ni(II), Cu(II) and Zn(II) complexes of glyoxime hydrazone. Asian Journal of Chemistry, 23(3), 1321-1326.
20. Sarıkavaklı, N., & İrez, G. (2005). Synthesis and complex formation of some novel vic-dioxime derivatives of hydrazones. Turkish Journal of Chemistry, 29(1), 107-116.
21. Spivak, M., Stone, J. E., Ribeiro, J., Saam, J., Freddolino, L., Bernardi, R. C., & Tajkhorshid, E. (2023). VMD as a Platform for Interactive Small Molecule Preparation and Visualization in Quantum and Classical Simulations. Journal of Chemical Information and Modeling, 63(15), 4664-4678. https://doi.org/10.1021/acs.jcim.3c00658
22. Taş, E., Ulusoy, M., Güler, M., & Yılmaz, I. (2004). Synthesis, characterization and redox properties of a new vic-dioxime and its transition metal complexes. Transition Metal Chemistry, 29(2), 180-184. https://doi.org/10.1023/B:TMCH.0000019417.35877.b9
23. 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(2), 455-461. https://doi.org/10.1002/jcc.21334
24. Yaeger, R., & Corcoran, R. B. (2019). Targeting alterations in the RAF–MEK pathway. Cancer Discovery, 9(3), 329-341. https://doi.org/10.1158/2159-8290.cd-18-1321
25. Yang, X., Zheng, Y. T., & Rong, W. (2019). Sevoflurane induces apoptosis and inhibits the growth and motility of colon cancer in vitro and in vivo via inactivating Ras/Raf/MEK/ERK signaling. Life Sciences, 239, 116916. https://doi.org/10.1016/j.lfs.2019.116916
26. Zhu, J., Li, C., Yang, H., Guo, X., Huang, T., & Han, W. (2020). Computational Study on the Effect of Inactivating/Activating Mutations on the Inhibition of MEK1 by Trametinib. International Journal of Molecular Sciences, 21(6), 2167. https://doi.org/10.3390/ijms21062167
27. Zoete, V., Cuendet, M. A., Grosdidier, A., & Michielin, O. (2011). SwissParam: a fast force field generation tool for small organic molecules. Journal of Computational Chemistry, 32(11), 2359-2368. https://doi.org/10.1002/jcc.21816