Multifaceted analysis of 3β-chloro-5α-cholestane-6-one cyanoacetic acid hydrazone: From green solid-state synthesis and structural elucidation to computational modeling and human serum albumin interactions

Modifications of cholestane derivatives, including the incorporation of functional groups or alterations in stereochemistry, lead to a diverse range of biological effects and functions. Therefore, this study presents the synthesis, structural elucidation, and molecular interactions of steroid, 3β-chloro-5α-cholestane-6-one cyanoacetic acid hydrazone (CCHC) (3) studied in vitro. Cholestane derivatives were successfully synthesized using traditional and solid-state techniques, achieving 50% and 75% yields, respectively. Their molecular structures were confirmed by multiple analytical techniques, such as Fourier transform infrared (FTIR) spectroscopy, high-resolution mass spectrometry (HRMS), nuclear magnetic resonance (NMR) spectroscopy, and elemental analysis. Additionally, structural comparisons with previously reported X-ray single-crystal diffraction data further supported their characterization. Although the CCHC has been previously reported via X-ray crystallography, this study provides comprehensive spectroscopic and theoretical characterization. Density functional theory (DFT) was used to optimize the geometry of the synthesized CCHC (3) in both gaseous and solvent phases, with results that were in good agreement with the experimental results. A significant amount of solid-state packing is controlled by noncovalent interactions, including N···H, C···H, and H···H, contacts, though H···O and Cl···H interactions contribute approximately 7.4% and 7.8%, respectively. Reduced density gradient (RDG) analysis also suggests strong intramolecular interactions in the lattice. Fluorescence spectroscopy, circular dichroism (CD), and UV-vis absorption titrations were employed to investigate the in vitro binding behavior of the steroid with human serum albumin (HSA). CCHC-HSA binding affinity was determined by Stern-Volmer and modified Stern-Volmer analyses, as well as by thermodynamic parameters. The role of specific amino acid residues in the non-bonding interactions with molecule 3 was studied using molecular docking and normal mode analysis (NMA) dynamics simulations. Fluorescence analysis has revealed that CCHC engages with HSA via a static quenching mechanism. The interaction exhibits a binding affinity of 3.79 × 10⁴ M⁻1 at 298 K. The interaction between CCHC and HSA was found to be thermodynamically favorable, as indicated by the Gibbs free energy change (ΔG) values of -6.24, -5.50, and -6.43 kcal mol⁻1 at temperatures ranging from 298 to 310 K. Complementary molecular docking studies yielded a binding score of -8.322 kcal mol⁻1, further corroborating the spontaneity of the interaction. Ligand- and receptor-ligand pharmacophore-based modeling of CCHC (3) with HSA revealed hydrogen bonding and hydrophobic interactions, revealing its binding capabilities and potential drug transport and pharmacokinetic uses. The ComplexMoGAPI study validated the environmental sustainability of the developed technology, establishing it as a feasible option for routine tests. The elevated greenness score highlights its suitability for eco-friendly uses in freshly formulated products. These results enhance our understanding of the pharmacodynamic behavior of steroid-like compounds in biological systems.