Exploration of Binding Affinities of a 3β,6β-Diacetoxy-5α-cholestan-5-ol with Human Serum Albumin: Insights from Synthesis, Characterization, Crystal Structure, Antioxidant and Molecular Docking

The present study describes the synthesis, characterization, and in vitro molecular interactions of a steroid 3β,6β-diacetoxy-5α-cholestan-5-ol. Through conventional and solid-state methods, a cholestane derivative was successfully synthesized, and a variety of analytical techniques were employed to confirm its identity, including high-resolution mass spectrometry (HRMS), Fourier transforms infrared (FT-IR), nuclear magnetic resonance (NMR), elemental analysis, and X-ray single-crystal diffraction. Optimizing the geometry of the steroid was undertaken using density functional theory (DFT), and the results showed great concordance with the data from the experiments. Fluorescence spectral methods and ultraviolet–vis absorption titration were employed to study the in vitro molecular interaction of the steroid regarding human serum albumin (HSA). The Stern-Volmer, modified Stern-Volmer, and thermodynamic parameters’ findings showed that steroids had a significant binding affinity to HSA and were further investigated by molecular docking studies to understand the participation of active amino acids in forming non-bonding interactions with steroids. Fluorescence studies have shown that compound 3 interacts with human serum albumin (HSA) through a static quenching mechanism. The binding affinity of compound 3 for HSA was found to be 3.18 × 104 mol−1, and the Gibbs free energy change (ΔG) for the binding reaction was −9.86 kcal mol−1 at 298 K. This indicates that the binding of compound 3 to HSA is thermodynamically favorable. The thermodynamic parameters as well as the binding score obtained from molecular docking at various Sudlow’s sites was −8.2, −8.5, and −8.6 kcal/mol for Sites I, II, and III, respectively, supporting the system’s spontaneity. Aside from its structural properties, the steroid demonstrated noteworthy antioxidant activity, as evidenced by its IC50 value of 58.5 μM, which is comparable to that of ascorbic acid. The findings presented here contribute to a better understanding of the pharmacodynamics of steroids.


Introduction
Cholestane derivatives are important steroid molecules that have several biological functions in the human body. Cholesterol, the primary precursor for cholestane derivatives, is an essential component of cell membranes and is required for the synthesis of hormones and bile acids [1]. Some cholestane derivatives have demonstrated positive effects in the treatment of different diseases [1][2][3][4][5]. However, it is worth noting that elevated blood cholesterol levels could potentially heighten the risk of heart-related illnesses [6][7][8]. Statins stand out among the extensively researched cholesterol derivatives [3,9], given their remarkable efficacy in lowering cholesterol levels in individuals with hypercholesterolemia [10,11] as well as their application in various other treatments [12]. The mechanism of action of statins involves the inhibition of HMG-CoA reductase [10], an enzyme responsible for cholesterol synthesis in the liver. By reducing cholesterol in the blood, statins can reduce the risk of X-ray analysis of the synthesized steroid compound confirmed its structure and stereochemistry. 3β,6β-diacetoxy-5-hydroxy-5α-cholestane (3) crystallizes in the space group C2 monoclinic crystal system. The unit cell contained four asymmetric molecules, each with similar geometric parameters. The dimensions of the unit cell are a = 31.634(3), b = 9.9420 (11), and c = 9.7325 (11). The steroid backbone adopts a chair conformation in the A, B, and C rings [35]. Ring A and ring B contain acetoxy groups at C3 and C6, respectively, while there is a hydroxyl group at C5 at the α position, which connects the junction of the two rings. The anisotropic displacement tensor of the terminal atoms of the 3β-acetoxy group, present at the 3-position of the cholestane skeleton, is strongly anisotropic, indicating that these atoms have a large amplitude of vibration perpendicular to the mean plane of the group, as has been reported in the literature [36]. The five-membered D ring is in an envelope conformation with C13 at the flap. The A/B and C/D ring in the steroid core exhibit trans junctions, which means that the C-C bonds connecting these rings are oriented in a trans configuration with the two carbon atoms on opposite sides of the ring. All of the carbon atoms in the lateral alkyl chain are in a straight line because the chain is fully extended. Steroids are characterized by the presence of trans junctions and a fully extended lateral alkyl chain. These characteristics affect how steroids interact with other molecules, which is crucial for the biological activity of steroids. The average bond lengths are: C(sp3)-C(sp3) = 1.  (Table 1). These interactions are similar in distance, 2.077 Å, and enrich the stability of the crystal structure by keeping the molecules in a regular array, as shown in packing Figure 1. Some bond distances, especially the acetyl group at C3, were found to be slightly altered. This may be because the crystal quality was not standard quality for diffraction analysis. The endocyclic and torsional bond angles in the steroid skeleton are shown in Table 1. These angles were determined by X-ray crystallography and compared with values obtained by density functional theory (DFT) calculations. The experimental result closely resembles the predicted result, indicating that the synthesized steroid compound's structure is accurate ( Figure 1). array, as shown in packing Figure 1. Some bond distances, especially the acetyl group at C3, were found to be slightly altered. This may be because the crystal quality was not standard quality for diffraction analysis. The endocyclic and torsional bond angles in the steroid skeleton are shown in Table 1. These angles were determined by X-ray crystallography and compared with values obtained by density functional theory (DFT) calculations. The experimental result closely resembles the predicted result, indicating that the synthesized steroid compound's structure is accurate ( Figure 1). Computational methods are increasingly used to study heterocyclic molecules. DFT can predict structures, energetics, and electronic properties. FMOs play a key role in reactivity, optical properties, and biological activity. Reduced density gradients (RDGs) are a measure of the electron density in a molecule. They are significant because they can be used to identify regions of high electron density, which are often associated with chemical bonds. RDGs can also be used to study the interactions between molecules. To study the Computational methods are increasingly used to study heterocyclic molecules. DFT can predict structures, energetics, and electronic properties. FMOs play a key role in reactivity, optical properties, and biological activity. Reduced density gradients (RDGs) are a measure of the electron density in a molecule. They are significant because they can be used to identify regions of high electron density, which are often associated with chemical bonds. RDGs can also be used to study the interactions between molecules. To study the electronic structure of the steroid molecule (3), a Gaussian software package was utilized to conduct calculations employing density functional theory (DFT). The initial coordinate source for theoretical calculations was the Crystallographic Information File (CIF). Density functional theory (DFT) was employed to optimize the molecular structure of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane, utilizing the B3LYP functional and the 6-311++G(d,p) basis set. The structures obtained from the DFT calculations are shown in Figure 1a along with the corresponding numbering scheme. The calculated values by DFT calculation were matched with the values achieved by X-ray diffraction analysis. The results demonstrate a correlation between the two sets of data, showing agreement between the electronic structure determined by DFT, the experimental results from X-ray diffraction (Table 1), and the literature [36]. Certain bond lengths and torsional angles, however, showed deviations because the DFT calculations were performed in the gas phase, whereas X-ray diffraction experiments are typically performed on solid-state samples. Furthermore, the molecule's crystal must be capable of producing accurate diffraction patterns.
The crystal packing of the molecules reveals that they exist as O-H···O hydrogenbonded dimers. The hydrogen bonding occurs between the hydroxyl group at C5 of one molecule and the carbonyl O atom of the acetoxy moiety of the other molecule. The O-H···O hydrogen bond distances are 2.81 Å. In addition to the hydrogen bonds, there are also three short intramolecular distances: H30B···O4 2.56 Å, H6···O3 2.65 Å, and H30A···O4 2.56 Å. These short distances suggest that there is a significant interaction between the polar hydrogen and the carbonyl O atoms. The interaction between these atoms helps stabilize the molecule by keeping the atoms in close proximity. This interaction also helps determine the structure of the molecule by affecting the orientation of the atoms (Table 2). The six-membered rings in a molecule (3) adopt conformations that closely resemble a chair form. This is supported by the puckering parameters determined by Cremer and Pople (1975) [37]. The puckering parameters for ring A are Q = 0.584 Å, θ = 3.6 • , and ϕ = 212 • . For ring B, the parameters are Q = 0.549 Å, θ = 104 • , and ϕ = 173.6 • . And for ring C, the values are Q = 0.576 Å, θ = 176.7 • , and ϕ = 216 • . The D-ring exhibits a twisted conformation around the C13-C14 bond with the puckering parameters Q = 0.452 Å and ϕ = 113.6 • , as shown in Table 3. All rings in the molecule are in a trans-fused configuration. The acetoxy group at C3 is positioned 3β with respect to ring A, while the substituents at ring B are in the 5α and 6β orientations, respectively. Table 3. Puckering Parameters of Steroid (3) with Five and Three Six-Membered Rings in its Skeleton.

Cholestane-Skeleton
Molecules 2023, 28, x FOR PEER REVIEW 6 of 26  (9) 2.881 (9) 173 (12) x, y, 1−z The six-membered rings in a molecule (3) adopt conformations that closely resemble a chair form. This is supported by the puckering parameters determined by Cremer and Pople (1975) [37]. The puckering parameters for ring A are Q = 0.584 Å, θ = 3.6°, and φ = 212°. For ring B, the parameters are Q = 0.549 Å, θ = 104°, and φ = 173.6°. And for ring C, the values are Q = 0.576 Å, θ = 176.7°, and φ = 216°. The D-ring exhibits a twisted conformation around the C13-C14 bond with the puckering parameters Q = 0.452 Å and φ = 113.6°, as shown in Table 3. All rings in the molecule are in a trans-fused configuration. The acetoxy group at C3 is positioned 3β with respect to ring A, while the substituents at ring B are in the 5α and 6β orientations, respectively.

Reduced Density Gradients and Frontier Molecular Orbitals (FMOs)
Apart from the Hirshfeld surfaces mentioned in Section 2.4, the color-filled reduced density gradient (RDG) of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane was also examined in real space using electron density analysis. This study was performed to further understand the influence of intermolecular and intramolecular interactions within the crystal structure. As shown in Figure 2, different color regions were analyzed to better understand these interactions. The surfaces are color-coded based on the magnitude of sign(λ2)р on a scale of blue-green-red. Blue signifies powerful attractive forces, green represents weak attractive interactions, and a strong non-bonded overlap is represented by red in this color scheme. The strength of interactions can be visually assessed by examining the scatter plot and filled isosurface plots. Denser points in the scatter diagram ( Figure 2b) indicate a higher electron density and stronger interactions. A dense peak and scattered dots of sign(λ2)р within the purview of −0.02∼0 a.u in the provided figure indicate the presence of interactions within the molecule with varying intensities, ranging from weak to strong van der Waals interactions (−0.02 to −0.01 a.u.). The green peak observed only in this region confirms the absence of intramolecular hydrogen bonding within the molecule. Furthermore, the predominance of van der Waals interactions suggests that they are the main force in the system. Positive values of sign(λ2)р indicate the presence of steric effects, resulting in red regions observed in the molecule.

Reduced Density Gradients and Frontier Molecular Orbitals (FMOs)
Apart from the Hirshfeld surfaces mentioned in Section 2.4, the color-filled reduced density gradient (RDG) of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane was also examined in real space using electron density analysis. This study was performed to further understand the influence of intermolecular and intramolecular interactions within the crystal structure. As shown in Figure 2, different color regions were analyzed to better understand these interactions. The surfaces are color-coded based on the magnitude of sign(λ 2 )p on a scale of blue-green-red. Blue signifies powerful attractive forces, green represents weak attractive interactions, and a strong non-bonded overlap is represented by red in this color scheme. The strength of interactions can be visually assessed by examining the scatter plot and filled isosurface plots. Denser points in the scatter diagram ( Figure 2b) indicate a higher electron density and stronger interactions. A dense peak and scattered dots of sign(λ 2 )p within the purview of −0.02∼0 a.u in the provided figure indicate the presence of interactions within the molecule with varying intensities, ranging from weak to strong van der Waals interactions (−0.02 to −0.01 a.u.). The green peak observed only in this region confirms the absence of intramolecular hydrogen bonding within the molecule. Furthermore, the predominance of van der Waals interactions suggests that they are the main force in the system. Positive values of sign(λ 2 )p indicate the presence of steric effects, resulting in red regions observed in the molecule. The presented data on the frontier molecular orbitals (FMOs) of compound (3) are significant, as they reveal key insights into its reactivity, stability, and receptor binding affinity. The molecule's HOMO and LUMO distribution indicates important binding regions for receptor interactions. Molecular properties are determined by the properties of the frontier molecular orbitals (FMOs), which are the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMOs). The low-energy LUMO of the acceptor molecule and the higher HOMO value of the donor molecule are both necessary for a molecule to be able to grant electrons to a proper acceptor [38,39]. Molecule reactivity is contingent upon the energy difference between the HOMO and the LUMO, a measurement of the energy disparity between the HOMO and LUMO. A larger HOMO-LUMO gap, which electrons find challenging in transitioning between distant energy levels, indicates lower reactivity. A larger HOMO-LUMO gap means that it is chemically hard because it is difficult to modify it by adding or removing electrons [40]. The spatial arrangement of the HOMO and the LUMO is critical for the recognition of receptor binding sites in ligands (molecules). Figure 3 illustrates that the HOMO and LUMO of 3β,6βdiacetoxy-5-hydroxy-5α-cholestane (3) are mainly distributed on the hydroxyl and acetoxy groups and the steroid ring. This suggests that these specific regions of the ligand are more likely to bind to the receptor, suggesting their importance in receptor interactions. The HOMO and LUMO plots of compound (3) are shown in Figure 2, with positive phases highlighted in red and negative phases highlighted in green. The energies HOMO and LUMO are 7.38 and 0.44 eV, respectively, resulting in a 6.94 eV HOMO-LUMO gap. This significant gap reflects the robustness and stability of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane (3), which corresponds to the characteristics of its skeletal structure. The atomic orbitals (AOs) associated with the oxygen atoms of the hydroxy and acetoxy groups located at the 5, 3, and 6-positions of the steroid skeleton (-O-H and -C=O in Figure 1) contribute to the formation of a mixed n-and p-type HOMO and a p-LUMO* when the HOMO and LUMO orbitals of compound (3) are surface-analyzed. Consequently, due to the aforementioned interactions, a delocalization effect was noticed in the acetoxy groups when switching from HOMO to LUMO. This means that the electron density spreads out and becomes more distributed over the acetoxy groups in LUMO orbitals compared to The presented data on the frontier molecular orbitals (FMOs) of compound (3) are significant, as they reveal key insights into its reactivity, stability, and receptor binding affinity. The molecule's HOMO and LUMO distribution indicates important binding regions for receptor interactions. Molecular properties are determined by the properties of the frontier molecular orbitals (FMOs), which are the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMOs). The low-energy LUMO of the acceptor molecule and the higher HOMO value of the donor molecule are both necessary for a molecule to be able to grant electrons to a proper acceptor [38,39]. Molecule reactivity is contingent upon the energy difference between the HOMO and the LUMO, a measurement of the energy disparity between the HOMO and LUMO. A larger HOMO-LUMO gap, which electrons find challenging in transitioning between distant energy levels, indicates lower reactivity. A larger HOMO-LUMO gap means that it is chemically hard because it is difficult to modify it by adding or removing electrons [40]. The spatial arrangement of the HOMO and the LUMO is critical for the recognition of receptor binding sites in ligands (molecules). Figure 3 illustrates that the HOMO and LUMO of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane (3) are mainly distributed on the hydroxyl and acetoxy groups and the steroid ring. This suggests that these specific regions of the ligand are more likely to bind to the receptor, suggesting their importance in receptor interactions. The HOMO and LUMO plots of compound (3) are shown in Figure 2, with positive phases highlighted in red and negative phases highlighted in green. The energies HOMO and LUMO are 7.38 and 0.44 eV, respectively, resulting in a 6.94 eV HOMO-LUMO gap. This significant gap reflects the robustness and stability of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane (3), which corresponds to the characteristics of its skeletal structure. The atomic orbitals (AOs) associated with the oxygen atoms of the hydroxy and acetoxy groups located at the 5, 3, and 6-positions of the steroid skeleton (-O-H and -C=O in Figure 1) contribute to the formation of a mixed n-and p-type HOMO and a p-LUMO* when the HOMO and LUMO orbitals of compound (3) are surface-analyzed. Consequently, due to the aforementioned interactions, a delocalization effect was noticed in the acetoxy groups when switching from HOMO to LUMO. This means that the electron density spreads out and becomes more distributed over the acetoxy groups in LUMO orbitals compared to HOMO orbitals. A relatively large HOMO-LUMO gap indicates that the compound is chemically hard, which can enhance its stability and receptor binding ability. The delocalized nature of the orbitals may also contribute to these properties. This information can be used to guide the design and optimization of the compound for specific applications, such as receptor binding or drug delivery. HOMO orbitals. A relatively large HOMO-LUMO gap indicates that the compound is chemically hard, which can enhance its stability and receptor binding ability. The delocalized nature of the orbitals may also contribute to these properties. This information can be used to guide the design and optimization of the compound for specific applications, such as receptor binding or drug delivery.

Hirshfeld Surface Analysis
The 3D Hirshfeld surface analysis is crucial for understanding the stability of organic crystals. It quantitatively identifies hydrogen-hydrogen (H-H) interactions as the most significant contributors, comprising 83.8% of the total interactions in the present molecule 3. Specific intra-and intermolecular interactions, which are regarded as the crystal's support structure, control the stability of organic crystals. Therefore, a crucial component of crystal engineering is the qualitative and quantitative measurement of these interactions. An effective method for assessing the intramolecular and intermolecular interactions that take place during crystal packing is the 3D Hirshfeld surface analysis. Based on a Hirshfeld surface study, two-dimensional fingerprint plots were created using CrystalExplorer 21.5.
The Hirshfeld surface of steroid (3) was generated with an isovalue of 0.5 using the electron density and mapped onto the dnorm descriptor. The color scheme used ranges from red (shorter distances than the sum of van der Waals radii) to white and blue (longer distances than the sum of van der Waals radii). The 3D mapping was carried out in the range of −0.5293 to 2.1132 dnorm values, where negative values are shown in red, positive values are shown in blue, and zero values are shown in white. These colors represent intermolecular contacts that are short, long, or weak. Intermolecular forces were depicted as crimson circular marks on the Hirshfeld surface. The presence of red circle spots on the pentacyclic ring of steroid (3) in Figure 4a,b indicates intermolecular interactions with a second steroid molecule in a reciprocal manner. Figure 4c-f show a 2D fingerprint plot of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane, which reveals two distinct spikes that display various interactions between neighboring molecules in the crystal lattice. Reciprocal hy-

Hirshfeld Surface Analysis
The 3D Hirshfeld surface analysis is crucial for understanding the stability of organic crystals. It quantitatively identifies hydrogen-hydrogen (H-H) interactions as the most significant contributors, comprising 83.8% of the total interactions in the present molecule 3. Specific intra-and intermolecular interactions, which are regarded as the crystal's support structure, control the stability of organic crystals. Therefore, a crucial component of crystal engineering is the qualitative and quantitative measurement of these interactions. An effective method for assessing the intramolecular and intermolecular interactions that take place during crystal packing is the 3D Hirshfeld surface analysis. Based on a Hirshfeld surface study, two-dimensional fingerprint plots were created using CrystalExplorer 21.5.
The Hirshfeld surface of steroid (3) was generated with an isovalue of 0.5 using the electron density and mapped onto the dnorm descriptor. The color scheme used ranges from red (shorter distances than the sum of van der Waals radii) to white and blue (longer distances than the sum of van der Waals radii). The 3D mapping was carried out in the range of −0.5293 to 2.1132 dnorm values, where negative values are shown in red, positive values are shown in blue, and zero values are shown in white. These colors represent intermolecular contacts that are short, long, or weak. Intermolecular forces were depicted as crimson circular marks on the Hirshfeld surface. The presence of red circle spots on the pentacyclic ring of steroid (3) in Figure 4a,b indicates intermolecular interactions with a second steroid molecule in a reciprocal manner. Figure 4c-f show a 2D fingerprint plot of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane, which reveals two distinct spikes that display various interactions between neighboring molecules in the crystal lattice. Reciprocal hydrogen-hydrogen (H-H), oxygen-hydrogen (O-H), and carbon-hydrogen (C-H) intermolecular interactions appear in the 2D fingerprint plots as single, double, and unidentified spikes, accounting for 83.8%, 14.4%, and 1.0% of the total plot, respectively. Figure 4d, in particular, shows that H-H interactions are important in maintaining the stability of the crystal structure, as evidenced by their central location among the scattered points. These H-H contacts contribute a significant 83.8% of the total Hirshfeld surfaces, as shown in Figure 4. These findings offer valuable insights in designing new crystals with enhanced properties and predicting crystal packing. The technique also provides qualitative information about the short range and relatively strong nature of H-H interactions, influencing the crystal's properties. Overall, this analysis aids in comprehending the relationship between the crystal structure and stability, enabling improved crystal engineering. lecular interactions appear in the 2D fingerprint plots as single, double, and unident spikes, accounting for 83.8%, 14.4%, and 1.0% of the total plot, respectively. Figure 4 particular, shows that H-H interactions are important in maintaining the stability o crystal structure, as evidenced by their central location among the scattered points. T H-H contacts contribute a significant 83.8% of the total Hirshfeld surfaces, as show Figure 4. These findings offer valuable insights in designing new crystals with enha properties and predicting crystal packing. The technique also provides qualitative i mation about the short range and relatively strong nature of H-H interactions, influen the crystal's properties. Overall, this analysis aids in comprehending the relationshi tween the crystal structure and stability, enabling improved crystal engineering.

Human Serum Albumin (HSA)-Steroid Binding Studies
HSA-steroid binding studies investigate the interplays involving human serum bumin and steroids in order to better understand their binding mechanisms. HSA f tions as a steroid carrier protein, influencing their bioavailability and pharmacokin Various spectroscopic tools including UV-vis absorption spectroscopy and fluoresc spectroscopy have been employed to investigate the binding interactions between lig and HSA, allowing for the molecular evaluation of their biological activities. These have improved researchers' understanding of the molecular mechanisms underlying oid biology and their engagements with human serum albumin (HSA). Moreover, approaches can be used to identify the participating amino acid residues in steroid b ing and judge the binding strength between steroids and HSA.

UV-vis Absorption Analysis
UV-visible absorption is a highly responsive method used to observe and stud formation of protein-ligand complexes and gain insight into how complex formatio fects protein structural integrity [41]. The procedure is mainly due to the enhanced sorption of UV-vis radiation caused by samples and protein molecules. Tryptophan rosine, and phenylalanine constitute the primary sources of aromatic moieties tha

Human Serum Albumin (HSA)-Steroid Binding Studies
HSA-steroid binding studies investigate the interplays involving human serum albumin and steroids in order to better understand their binding mechanisms. HSA functions as a steroid carrier protein, influencing their bioavailability and pharmacokinetics. Various spectroscopic tools including UV-vis absorption spectroscopy and fluorescence spectroscopy have been employed to investigate the binding interactions between ligands and HSA, allowing for the molecular evaluation of their biological activities. These tools have improved researchers' understanding of the molecular mechanisms underlying steroid biology and their engagements with human serum albumin (HSA). Moreover, these approaches can be used to identify the participating amino acid residues in steroid binding and judge the binding strength between steroids and HSA.

UV-Vis Absorption Analysis
UV-visible absorption is a highly responsive method used to observe and study the formation of protein-ligand complexes and gain insight into how complex formation affects protein structural integrity [41]. The procedure is mainly due to the enhanced absorption of UV-vis radiation caused by samples and protein molecules. Tryptophan, tyrosine, and phenylalanine constitute the primary sources of aromatic moieties that are present and absorb light. The maximum wavelength of human serum albumin (HSA) is 280 nm, which is characteristic of the three aromatic residues. Particularly responsive to alterations in the microenvironment, tryptophan absorbs primarily at 280 nm. In general, modifications to the aromatic residues' microenvironment that follow ligand binding offer insights into the phenomenon of binding. Figure 5 displays the HSA absorption spectra in the presence and absence of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane. The peak in absorbance observed at 278 nm is focused on the microenvironment surrounding the tryptophan and tyrosine residues of HSA. The magnitude of the peaks decreased as the various concentrations of compound 3 were introduced to the constant amount of HSA. Overall, the peak of the magnitude confirms that HSA causes significant structural changes when it interacts with steroids. modifications to the aromatic residues' microenvironment that follow ligand binding offer insights into the phenomenon of binding. Figure 5 displays the HSA absorption spectra in the presence and absence of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane. The peak in absorbance observed at 278 nm is focused on the microenvironment surrounding the tryptophan and tyrosine residues of HSA. The magnitude of the peaks decreased as the various concentrations of compound 3 were introduced to the constant amount of HSA. Overall, the peak of the magnitude confirms that HSA causes significant structural changes when it interacts with steroids. To check for interference from the buffer solution, a control was performed with increasing concentrations of the compound ranging from 5 to 50 μM, in the absence of HSA (Figure 5b). The results showed that the buffer solution had a very low absorbance with a flat spectrum at all wavelengths, so it did not interfere with the measurement of the absorbance of other substances. Additionally, no significant change was observed in the UVvis spectrum of the control as the concentration of steroidal compound 3 was increased. Finally, the wavelength of the compound in the UV-vis spectrum (Figure 5c) was found to be approximately 212 nm, which is outside of the wavelength range of HSA, so there was no interference in the results. To check for interference from the buffer solution, a control was performed with increasing concentrations of the compound ranging from 5 to 50 µM, in the absence of HSA (Figure 5b). The results showed that the buffer solution had a very low absorbance with a flat spectrum at all wavelengths, so it did not interfere with the measurement of the absorbance of other substances. Additionally, no significant change was observed in the UV-vis spectrum of the control as the concentration of steroidal compound 3 was increased. Finally, the wavelength of the compound in the UV-vis spectrum (Figure 5c) was found to be approximately 212 nm, which is outside of the wavelength range of HSA, so there was no interference in the results.

Fluorescence Studies
Fluorescence spectroscopy has demonstrated its utility as a valuable scientific tool in the investigation of a variety of effects on biomacromolecules, including protein-drug interactions, conformational changes, and the mechanism of quenching in protein-drug interactions [42]. Three amino acids-tryptophan, tyrosine, and phenylalanine-are authorized to regulate the fluorescence of proteins. Tryptophan is the most important fluorophore and fluoresces most strongly in subdomain IIA of the active cavity of HSA. Tyrosine and phenylalanine fluoresce as well, but much more weakly than tryptophan does. Tyrosine fluorescence can be quenched by ionization or by proximity to other amino acids, such as amino groups, carboxyl groups, or tryptophan. In this study, the binding mechanism and formation of complexes involving HSA were explored by titrating it against 3β,6β-diacetoxy-5-hydroxy-5α-cholestane. The concentration of HSA was kept constant at 5 µM, while the concentration of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane was varied from 5 to 50 µM. The presence and absence of the steroid were compared by obtaining HSA spectra, as shown in Figure 6a. The findings indicated that HSA exhibits a fluorescence emission peak at approximately 338 nm. The peak suppression increased as the steroid concentration increased, indicating the formation of a complex that engaged with HSA. The intensity of the fluorescence of HSA dropped by about 41% at the highest steroid dose (50 µM), while the wavelength (λem) and peak morphology remained unaffected. This suggests that steroid 3 binds to HSA in a non-covalent manner. 3β,6βdiacetoxy-5-hydroxy-5α-cholestane (steroid) induced a slow fluorescence quenching in HSA. The quenching of HSA fluorescence by the drug was concentration-dependent, with more prominent quenching observed at 50 µM. The mechanism of fluorescence quenching can take place statically or dynamically. A stable complex between the protein and the quencher results in the quenching of static fluorescence. Various studies consistently demonstrate an inverse relationship between the quenching constant (Kq) and temperature. This relationship is attributed to the fact that the frequency of diffusion decreases with increasing temperature. Dynamic fluorescence quenching occurs due to transient collisions between the quencher and protein. As the frequency of collisions rises, the quenching rate constant (Kq) also escalates in a temperature-dependent manner. Additionally, without actual ligand binding, dynamic fluorescence quenching can take place, which means that HSA's shape and function are unaffected, providing consistency with the literature. The mechanism of fluorescence quenching was determined using the Stern-Volmer equation establishing a mathematical relationship between the quencher concentration and protein fluorescence quenching. The Stern-Volmer plot demonstrating the interaction of HSA with steroid (3) is shown in Figure 6b. The Stern-Volmer quenching constant (Ksv) can be used to estimate the quenching efficiency of a drug. A higher Ksv value indicates that the drug is better at quenching the fluorescence of a fluorophore (any macromolecule or protein). The Ksv coefficient of the present steroid 3 was determined to be 1.45 × 10 4 M −1 . These values indicated that, with a certain amount of the quencher added, the fluorescence intensity decreases by 1%. The bimolecular quenching constant, Kq, can be calculated from the quenching constant, Ksv, and the average time spent in the excited state, τ0, using the following Equation (1): where Ksv is the Stern-Volmer constant and τ0 is the average lifetime of the protein in the absence of a quencher. For biopolymers, the value of τ0 is usually 10 −9 s. The Stern-Volmer equation was used to estimate the rate constant at which HSA and steroid (3) bind, also known as the bimolecular quenching rate constant (Kq). The value of Kq was found to be approximately 1.45 × 10 12 M −1 s −1 . This finding supports the existence of a static quenching process that contributes to the formation of a stable steroid-HSA complex in its resting state. The bimolecular quenching constant, Kq, is necessary to comprehend how HSA and drugs interact. The interaction between HSA and steroid (3) at ambient temperature was studied using a linear regression plot of F0/F against [Q]. The KSV and Kq values were determined to be in the order of 10 4 . This finding suggests that the quenching mechanism between HSA and 3β,6β-diacetoxy-5-hydroxy-5α-cholestane likely began with a static quenching mechanism, as documented in the literature [43]. Additionally, employing a modified Stern-Volmer equation relies on a double logarithmic graph plotting log (F0 F)/F vs. log [Q], the binding constant (Kb) and binding sites (n) on HSA can be ascertained. The slope of the resulting line indicates the number of binding sites (n), while the intercept (ordinate of the origin, shown in Figure 6c) corresponds to the logarithmic value of K b (logK b ). Fluorescence quenching experiments yielded the HSA-steroid (3) binding and thermodynamic parameters, which are also shown in Table 4. In general, drugs with higher binding constants exhibit stronger binding to receptors than drugs with lower binding constants. The regression equation produced a slope close to one, indicating that, under specific conditions, a single binding site with a high affinity for steroids on HSA was found. The steroid 3 displayed a binding constant of 3.18 × 10 4 M −1 , ascertained through experimentation. This high value indicates that 3β,6β-diacetoxy-5-hydroxy-5α-cholestane binds well to HSA. In addition, the calculated Gibbs free energy (∆G o ) for the binding of HSA to steroids is −9.86 kcal/mol, and these findings show that the binding process happens spontaneously in nature. The molecular docking resulted in a binding score of −8.6 kcal/mol, suggesting a significant interaction between HSA and steroid 3 because it falls within the range of binding affinities deemed biologically relevant. The experimental and predicted binding fractions agree with each other, further supporting the existence of this interaction. to one, indicating that, under specific conditions, a single binding site with a high affinity for steroids on HSA was found. The steroid 3 displayed a binding constant of 3.18 × 10 4 M −1 , ascertained through experimentation. This high value indicates that 3β,6β-diacetoxy-5-hydroxy-5α-cholestane binds well to HSA. In addition, the calculated Gibbs free energy (ΔGo) for the binding of HSA to steroids is −9.86 kcal/mol, and these findings show that the binding process happens spontaneously in nature. The molecular docking resulted in a binding score of −8.6 kcal/mol, suggesting a significant interaction between HSA and steroid 3 because it falls within the range of binding affinities deemed biologically relevant. The experimental and predicted binding fractions agree with each other, further supporting the existence of this interaction.  The effect of temperature on the HSA-Stern-Volmer quenching constants with compound (3) was investigated. The studies were performed at 298, 308, and 318 K. As shown in Table 4 and Figure 6b, the K SV values decreased as the temperature increased. This indicates that the quenching mechanism is static, as the interaction between HSA and compound (3) is not affected by the increased thermal energy. Similarly, the bimolecular quenching constant (Kq) decreased as the temperature increased from 298 to 318 K, which is consistent with a static quenching mechanism. Ligands bind to proteins through non-covalent interactions, including hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic interactions [43]. The dimensions and signs suggested by Ross and Subramanian are employed for the computation of the thermodynamic parameters (∆H and ∆S) connected to these interactions [44]. The binding forces are controlled by the thermodynamic variables ∆H and ∆S in protein-ligand binding. While ∆S denotes changes in disorder, ∆H represents the reaction's heat production or absorption. Positive ∆S denotes increased disorder, and negative ∆H denotes heat release, both of which favor binding. The sum of ∆H and ∆S determines the driving force of the binding, ∆G, with a negative value indicating favorable binding. The following Equations (2)-(4) are used to calculate the thermodynamic parameters: In this context, ∆G • represents the alteration in free energy, ∆H • indicates a change in enthalpy, ∆S • denotes the change in entropy, R stands for the gas constant (8.314 J/mol/K), T represents the temperature (in Kelvin), and K is the constant.
The slope and intercept of the van't Hoff plot can be used to calculate the enthalpy change (∆H • ) and entropy change (∆S • ) of a binding reaction, respectively [45]. The values for ∆H • , ∆S • , and the binding free energy (∆G • ) are shown in Table 5. A negative ∆H • indicates that the binding process is exothermic and favors the interaction, indicating that the hydrophobic and hydrogen bond interactions between the drug and human serum albumin are exothermic, releasing heat upon formation. A positive ∆S • indicates that there is more disorder during binding, which is advantageous because it suggests that the interactions between the drug and human serum albumin are not very strong, allowing them to bind in more disordered conformations. The presence of a negative ∆H • and positive ∆S • indicates that hydrogen bonds and hydrophobic interactions are the main binding forces between the drug and human serum albumin. These interactions, despite being weak, are advantageous because they dissipate heat and permit more disordered conformations. Analyzing the data in Table 6 shows that the magnitude of the negative values decreases with an increasing temperature. This observation suggests that as the temperature increases, the spontaneity of the reaction decreases.

Molecular Docking Analysis
HSA has three major binding sites referred to as subdomains I, II, and III. These subdomains are allosterically coupled, meaning that the binding of a ligand to one subdomain can affect the binding of ligands to the other subdomains The binding intensity of a compound can vary at different binding sites, such as sites I, II, and III [46]. The best target site in the protein can be predicted based on the binding score obtained from various binding sites in a docking analysis. Molecular docking is a useful computational method for investigating the interactions between macromolecules (like proteins) and small molecules, ligands, or drugs. It plays a crucial role in the drug discovery and development processes by analyzing and forecasting the binding affinities and modes of ligands to target proteins. A popular piece of software called AutoDock Vina uses an algorithm to explore a ligand's conformational space and forecast the binding pose and affinities it will have for a target protein. By accounting for electrostatic interactions, hydrogen bonds, and van der Waals forces, it evaluates the energy of the interaction between the ligand and the protein. The human serum albumin (HSA) molecule is composed of three structurally related domains: domain I, which contains amino acid residues 1-195, domain II, which contains amino acid residues 196-383, and domain III, which contains amino acid residues 385-585, as shown in Figure 7a. 3β,6β-diacetoxy-5-hydroxy-5α-cholestan, a steroidal molecule (Figure 7b), was individually docked with the active pockets of HSA in domains I, II, and III to examine its binding preferences (Figure 7c). The docked pose of HSA with the steroid (3) in binding sites I, II, and III with the highest binding energy (negative) is shown in Figure 8 (binding sites I, II, and III). 3β,6βdiacetoxy-5-hydroxy-5α-cholestan showed the highest binding energy at three different sites within the HSA. Specifically, the energy of site I was determined to be −8.2 kcal/mol, while it was −8.5 kcal/mol for site II and −8.6 kcal/ mol for site III. In the docking model, the presence of actively participating amino acids was observed, contributing to nonbonding interactions such as hydrogen bonding and van der Waals forces at different sites within HSA. To further investigate these interactions, specific docking poses were chosen to analyze non-bonded interactions between steroids and active amino acid residues responsible for forming the active pocket of HSA. At binding site 1, steroid (3) had a binding score of −8.2 kcal/mol. In this model, the oxygen atom of the acetoxy group forms a hydrogen bond with LYS199 of HSA. In addition, several amino acids including ASP451, TRP214, ARG222, GLU153, SER192, and ILE290 were found to surround steroids (2), resulting in van der Waals interactions. This information is depicted in Figure 8. Additionally, salt bridges and hydrophobic interactions between various amino acids and various steroid atoms at particular distances were seen ( Table 6). The amino acids LEU115, TYR138, ILE142, TYR161, PHE165, ARG186, HIS146, ARG186, and LYS190 were involved in these interactions. These interactions, along with the hydrophobic interactions and salt bridges including alkyl and pi-alky, collectively contributed to the proper orientation and stabilization of the ligand within the active pocket. The docked pose of HSA with the steroid (3) in binding sites I, II, and III with the highest binding energy (negative) is shown in Figure 8 (binding sites I, II, and III). 3β,6βdiacetoxy-5-hydroxy-5α-cholestan showed the highest binding energy at three different sites within the HSA. Specifically, the energy of site I was determined to be −8.2 kcal/mol, while it was −8.5 kcal/mol for site II and −8.6 kcal/mol for site III. In the docking model, the presence of actively participating amino acids was observed, contributing to nonbonding interactions such as hydrogen bonding and van der Waals forces at different sites within HSA. To further investigate these interactions, specific docking poses were chosen to analyze non-bonded interactions between steroids and active amino acid residues responsible for forming the active pocket of HSA. At binding site 1, steroid (3) had a binding score of −8.2 kcal/mol. In this model, the oxygen atom of the acetoxy group forms a hydrogen bond with LYS199 of HSA. In addition, several amino acids including ASP451, TRP214, ARG222, GLU153, SER192, and ILE290 were found to surround steroids (2), resulting in van der Waals interactions. This information is depicted in Figure 8. Additionally, salt bridges and hydrophobic interactions between various amino acids and various steroid atoms at particular distances were seen ( Table 6). The amino acids LEU115, TYR138, ILE142, TYR161, PHE165, ARG186, HIS146, ARG186, and LYS190 were involved in these interactions. These interactions, along with the hydrophobic interactions and salt bridges including alkyl and pi-alky, collectively contributed to the proper orientation and stabilization of the ligand within the active pocket.
As a result, the close interactions between the ligand and the active amino acid residues yielded a high docking score. The details of these interactions can be found in Table 6.
At binding site II, the best-docked pose of the steroid (3)-HSA obtained by various interactions was found to have the highest binding score of −8.5 kcal/mol. An analysis of Figure 8 (binding site II) shows that the oxygen atom and keto-oxygen atom of the acetoxy group at the 6-position of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane (3) form conventional hydrogen bonds with the amino acids TYR411 and ARG410, respectively. These hydrogen bonds play an important role in close interactions with amino acids present at the active sites of the protein. van der Waals forces were also observed, surrounding the molecule with different types of amino acids, including LEU430, ARG348, LEU460, LEU407, PHE403, ASN391, SER489, PHE488, LEU491, and GLU450. These amino acids, which form nonbonding interactions, assist the molecule and protein in close host-guest interactions. In addition to these interactions, some amino acids, including VAL344, LEU387, PRO384, ILE388, MET446, ALA449, LEU457, ARG453, and LEU453, participate in the formation of alkyl and pi-alkyl interactions of the steroid (3) skeleton, as shown in the figure. Hydrophobic amino acids, such as TRP214, ARG218, LEU219, PHE223, LEU234, LEU238, LEU238, LEU260, ILE264, ILE290, and ALA291, are buried inside the protein such that they are shielded from water. These hydrophobic amino acids make a protein fold stable even during interactions with different atoms of a molecule at specific distances, as shown in Table 6. It was discovered that LYS195 of these amino acids played a role in building a salt bridge with the carboxylate of steroid (3)  As a result, the close interactions between the ligand and the active amino acid residues yielded a high docking score. The details of these interactions can be found in Table 6.
At binding site II, the best-docked pose of the steroid (3)-HSA obtained by various interactions was found to have the highest binding score of −8.5 kcal/mol. An analysis of Figure 8 (binding site II) shows that the oxygen atom and keto-oxygen atom of the acetoxy group at the 6-position of 3β,6β-diacetoxy-5-hydroxy-5α-cholestane (3) form conventional hydrogen bonds with the amino acids TYR411 and ARG410, respectively. These hydrogen bonds play an important role in close interactions with amino acids present at the active sites of the protein. van der Waals forces were also observed, surrounding the molecule with different types of amino acids, including LEU430, ARG348, LEU460, LEU407, PHE403, ASN391, SER489, PHE488, LEU491, and GLU450. These amino acids, which form non-bonding interactions, assist the molecule and protein in close host-guest interactions. The best docking model for the steroid (3)-HSA had a binding score of −8.6 kcal/mol, according to the docking simulation. This suggests a strong interaction between site III and the steroid. The OH group of the steroid and the amino acid LEU115 most likely form a hydrogen bond, which accounts for the strong interaction. The steroid's binding to the site is probably going to be stabilized by this hydrogen bond. In addition to hydrogen bond formation, other non-bonding forces were observed in the best docking model. At binding site III, specific amino acids, namely, VAL116, LYS190, ILE142, MET123, and ARG146, were observed in close proximity to the steroid molecule. These amino acids interacted with each other through van der Waals forces, which are weak attractions between molecules or atoms. ARG186 was also involved in the formation of a carbon-hydrogen bond, indicating that it interacted with a carbon atom in the steroid molecule.
Furthermore, ARG117, TYR138, PHE165, TYR161, and ARG114 displayed alkyl and pi-alkyl forces with the skeleton of the steroid, indicating their contribution to the complex's stabilization, as depicted in Figure 8. As shown in Table 6, several hydrophobic  amino acids, including VAL344, LEU387, ILE388, LEU407, LEU430, ALA499, GLU450,  LEU453, ARG485, and PHE488, interacted with different atoms of the steroidal skeleton to improve the docking model's stability with the protein's secondary structure. Furthermore, at binding site III, ARG410 and LYS414 formed salt bridges with the steroid's carboxylate group. This suggests that these amino acids are important for enhancing the stability of the protein-steroid complex. The combined effect of these non-bonding forces results in the overall interaction between the steroid molecule and site III. The strength of this interaction is most likely determined by the number and type of non-bonding forces present. HSA-steroid interactions were examined and compared to previously published interaction profiles for HSA-ligand interactions. The comparison includes various binding sites composed of specific amino acids, such as Asp, Leu, Val, Lys, Phe, Tyr, Ser, Asn, Ala, Glu, Gln, and Gly. Some of these amino acids were also found to be involved in HSA-steroid interactions. These findings are supported by studies conducted by Shen et al. (2017) [47], and Veeralakshmi et al. (2017) [48]. Following a review of the literature, it was observed that a significant number of amino acid residues surrounding drug molecules exhibited hydrophobic properties. This finding suggests that the presence of hydrophobic effects might contribute to the stabilization of complexes formed between drugs and HSA. Likewise, most of the amino acid residues neighboring compound 3 exhibited hydrophobic characteristics. This suggests that the stability of the steroid-HSA complex could be influenced by the hydrophobic effects as well. Molecular docking analyses give a useful framework for observing and investigating on a microscopic level. They enable the quantitative study of the binding affinity score, which can provide insights into ligand-protein interactions [47,48]. The affinity of a compound for binding can exhibit variability based on the structural characteristics of the compound itself, the particular amino acids engaged in the binding process, and the specific binding site involved. As illustrated in this study, compound 3 demonstrates a higher binding affinity towards subdomain IIA compared to its affinities towards subdomains I, II, and III.

Antioxidant Potential Analysis
The ability of steroid 3 to reduce DPPH radicals was used to assess its antioxidant potential. The reduction potential of steroid 3 was measured by a decrease in the absorbance of DPPH radicals at 517 nm. The free radical scavenging activity of steroid 3 may be due to its ability to donate electrons to DPPH free radicals. This steroid has two acetoxy groups at the 3 and 6 positions and an OH group at the 5 position. These functional groups are either electron-rich or readily transfer hydrogen, and they can donate electrons to the unpaired electrons of the DPPH radical. This leads to the formation of hydrazine, a stable molecule with paired electrons. The findings indicated that steroid 3 could lower DPPH radicals, and the IC 50 value-the amount of steroid 3 needed to lower 50% of the DPPH radicals-was determined to be 58.5 µM. This is shown in Figure 9a,b, which represent the percentage of scavenging versus the concentration. This demonstrates the significant free radical scavenging activity of steroid 3. Although the IC 50 value of steroid 3 was notably higher than the standard (ascorbic acid IC 50 = 47.7 mM) [49], careful examination revealed that, in terms of the antioxidant study of samples, the IC 50 values of the sample and the standard were relatively close. This suggests that steroid 3, albeit to a slightly lesser extent, is also effective at scavenging free radicals. Scavenging free radicals is an important antioxidant activity, so this suggests that compound 3, a steroid, has the potential to be a promising candidate for the development of novel antioxidant therapies. free radical scavenging activity of steroid 3. Although the IC50 value of steroid 3 was no-tably higher than the standard (ascorbic acid IC50 = 47.7 mM) [49], careful examination revealed that, in terms of the antioxidant study of samples, the IC50 values of the sample and the standard were relatively close. This suggests that steroid 3, albeit to a slightly lesser extent, is also effective at scavenging free radicals. Scavenging free radicals is an important antioxidant activity, so this suggests that compound 3, a steroid, has the potential to be a promising candidate for the development of novel antioxidant therapies.

Materials and Method
Melting points are reported uncorrected. Infrared (IR) spectra were obtained by a Perkin Elmer 1600 FTIR spectrophotometer with KBr. Proton and carbon nuclear magnetic resonance ( 1 H & 13 C-NMR) spectra were recorded using a Varian VXR-300s instrument with Me4Si and CDCI3 as the internal standard. Chemical shifts are reported in parts per million (ppm), and the abbreviations are used to describe the signals observed (s, singlet; br, broad; m, multiplet centered at; d, doublet; t, triplet). When deuterium (D2O) was added, the O-H signals disappeared without significant alterations in other regions of the NMR spectra. Ultraviolet (UV) spectra were captured in chloroform using a Perkin Elmer 1800 FTIR instrument. To visualize spots and track the advancement of reactions, silica gel G-coated TLC plates were employed, along with iodine. Chromatograms were developed using mixtures of light petroleum-benzene and CHC13-acetone. Acetone was used to crystallize the product for further purification, and anhydrous sodium sulfate was employed as a desiccant to dry a solution.
Conventional procedure [50][51][52][53]: A mixture of cholestane-3β,5α,6β-triol (500 mg) (or 3βacetoxy-5α-cholestane-5, 6β-diol as an alternative), anhydrous pyridine (3 mL), and acetic anhydride (2 mL) was subjected to heating in a water bath for a duration of 3 h. Pouring the reaction mixture into 50 g of crushed ice allowed it to cool. The solid was washed thoroughly with water and diluted hydrochloric acid (5%, 2 × 20 mL) to remove any residual pyridine. Further washing with water was performed, then filtered under suction, followed by air-drying, resulting in a yellow solid with a 90% yield. The recrystallization of the solid product using acetone resulted in the formation of crystals, which were identified as 3β,6β-diacetoxy-5α-cholestan-5-ol. The melting point of the crystals was determined to be 167-168 °C, which closely aligns with the reported m.p. of 167 °C , as specified in the literature [54].

Materials and Method
Melting points are reported uncorrected. Infrared (IR) spectra were obtained by a Perkin Elmer 1600 FTIR spectrophotometer with KBr. Proton and carbon nuclear magnetic resonance ( 1 H & 13 C-NMR) spectra were recorded using a Varian VXR-300s instrument with Me4Si and CDCI 3 as the internal standard. Chemical shifts are reported in parts per million (ppm), and the abbreviations are used to describe the signals observed (s, singlet; br, broad; m, multiplet centered at; d, doublet; t, triplet). When deuterium (D 2 O) was added, the O-H signals disappeared without significant alterations in other regions of the NMR spectra. Ultraviolet (UV) spectra were captured in chloroform using a Perkin Elmer 1800 FTIR instrument. To visualize spots and track the advancement of reactions, silica gel G-coated TLC plates were employed, along with iodine. Chromatograms were developed using mixtures of light petroleum-benzene and CHC1 3 -acetone. Acetone was used to crystallize the product for further purification, and anhydrous sodium sulfate was employed as a desiccant to dry a solution.
Conventional procedure [50][51][52][53]: A mixture of cholestane-3β,5α,6β-triol (500 mg) (or 3βacetoxy-5α-cholestane-5, 6β-diol as an alternative), anhydrous pyridine (3 mL), and acetic anhydride (2 mL) was subjected to heating in a water bath for a duration of 3 h. Pouring the reaction mixture into 50 g of crushed ice allowed it to cool. The solid was washed thoroughly with water and diluted hydrochloric acid (5%, 2 × 20 mL) to remove any residual pyridine. Further washing with water was performed, then filtered under suction, followed by air-drying, resulting in a yellow solid with a 90% yield. The recrystallization of the solid product using acetone resulted in the formation of crystals, which were identified as 3β,6β-diacetoxy-5α-cholestan-5-ol. The melting point of the crystals was determined to be 167-168 • C, which closely aligns with the reported m.p. of 167 • C, as specified in the literature [54].

Single X-ray Crystallography and Computational Details
The diffraction data for crystals were obtained using the APEX2 diffractometer (Bruker-Noius, 2004). Subsequently, cell refinement and data reduction were performed using SAINT (Bruker, 2003) and the SAINT program, respectively. After making necessary corrections to the collected data, the title compound structure was determined using a direct method facilitated by the SHELXT-2015 software [55]. The structure was further Scheme 1. Synthesis of 3β,6β-diethoxy-5α-cholestane-5-ol (3). Either cholestane-3β,5α,6β-triol (1) or 3β-acetoxy-5α-cholestane-5,6β-diol (2) can be used as the reactant, and the synthesis can be carried out using either conventional or solid-state methods.
Physical, spectral, and analytical data of the synthesized steroid (3)

Single X-ray Crystallography and Computational Details
The diffraction data for crystals were obtained using the APEX2 diffractometer (Bruker-Noius, 2004). Subsequently, cell refinement and data reduction were performed using SAINT (Bruker, 2003) and the SAINT program, respectively. After making necessary corrections to the collected data, the title compound structure was determined using a direct method facilitated by the SHELXT-2015 software [55]. The structure was further refined using least squares with the help of the SHELXL-2015 software [56], which is included in the Olex2 package program [57]. The refinement process involves the anisotropic refinement of all non-hydrogen atoms. Subsequently, hydrogen atoms attached to non-carbon atoms were positioned based on the difference Fourier maps. The remaining hydrogen atoms were treated as rider atoms and refined using the isotropic displacement parameter. Using MERCURY software, diagrams and publication materials were produced [58].

Computational and Molecular Docking Studies
To examine the molecular structure of 3β,6β-diethoxy-5α-cholestan-5-ol, quantum chemical calculations were performed using the Gaussian 09 program [59]. The calculations were carried out in both vacuum and solvent phases using the 6-311G++(d,p) basis set and the B3LYP density functional [60,61]. The optimized structure is identified at the true minimum of the potential energy level, as confirmed by the actual value of the frequency derived at the same theoretical level. At the same theoretical level, the energy gaps of FMOs (HOMO and LUMO) of 3β,6β-diaoetoxy-5α-cholestane-5-ol are obtained. The effectiveness of long-range forces was proved using the Multiwfn program [62]. The calculation and analysis of a number of parameters related to these forces were made simpler by this application. Isosurface visualizations were also produced with the help of the VMD application to give the examined data a visual representation. To determine weak intermolecular interactions in the crystal, CrystalExplorer 21.5 [63] was used to study weak interactions between molecules in 3β,6β-diethoxy-5α-cholestan-5-ol crystals (3) creating Hirshfield surfaces and 2D fingerprints. Molecular docking is a method for predicting the extent to which small molecules bind to proteins. The Lamarckian Genetic Algorithm (LGA) operated in this study to perform an in-silico approach to the binding patterns of 3β, 6β-diacetoxy-5α-cholestan-5-ol with human serum albumin. The three-dimensional (3D) crystal structure of human serum albumin with PDB ID 1H9Z was used as a model of a receptor, and the crystallographic information file (CIF) of 3β, 6β-diacetoxy-5α-cholestan-5-ol was saved in PDF format and used as a ligand. Based on the reported literature regarding multiple active HSA sites [47], three different docking grid boxes were set up, each with a grid spacing of 0.375 and 60 × 60 × 60 points. For binding site I, the box centers were placed at x = 30.025, y = 9.580, z = 10.295; for binding site II, they were placed at x = 12.005, y = 8.737, z = 20.079; and for binding site III, they were placed at x = 33.049, y = 18.888, z = 35.8. The lowest energy structure docked at each active site was selected, and the Discovery Studio was employed to visualize the output and analyze hydrogen bonding and unbound interactions between the receptor and 3β,6β-diacetoxy-5α -cholestan-5-ol. Protein-ligand interaction profiles in multiple binding sites of the Human Serum Albumin (HSA) with steroid 3 were also obtained using the PLIP web tool [64].

HSA Sample Preparation
The concentration of human serum albumin (HSA) for a stock solution prepared by dissolving HSA in a 20 mM phosphate buffer solution at pH 7.4 was determined by measuring the absorbance of an HSA solution at 280 nm using a Perkin-Elmer-Lambda double beam UV-vis spectrophotometer. The extinction coefficient of HSA at 280 nm is 5.30. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed a single band, was used to confirm the purity of HSA. To prepare different concentrations of the steroid, a stock solution of 2 mM 3β,6β-diacetoxy-5α-cholestan-5-ol in dimethyl sulfoxide (DMSO) was prepared and diluted in phosphate buffer. Blanks were made using 20 mM PB at pH 7.4 and 298 K. The pH of each solution was determined using an Orion-401-plus pH meter and an Orion glass electrode. The absorption spectra of HSA-steroid complexes were measured at increasing concentrations of the compound (5-50 µM). The concentration of HSA was held constant at 5 µM in this HSA binding study.

UV-Vis Absorption Titration
At a temperature of 298 K, the UV-vis absorption spectrum of HSA was examined. Measurements were performed with and without various concentrations of 3β,6βdiaoetoxy-5α-cholestan-5-ol. The experiment was carried out using quartz cuvettes with a 1 cm path length and a Peltier temperature controller on a Perkin-Elmer-Lambda doublebeam UV-vis spectrophotometer. The concentration of human serum albumin was maintained at 5 micromolar in 20 mM sodium phosphate buffer, pH 7.4, and then treated with increasing concentrations of 3β,6β-diacetoxy-5α-cholestan-5-ol for titration. Appropriate values for negative controls were removed from HSA-3β,6β-diacetoxy-5α-cholestan-5-ol readings to eliminate sample absorbance.

Fluorescence Quenching Measurement
The intrinsic fluorescence of HSA was assessed using a Shimadzu fluorescence spectrophotometer RF-5301 equipped with a quartz cuvette and a 1 cm pathlength. The excitation wavelength was set at 295 nm, and emission spectra were recorded between 300 and 400 nm. The excitation and emission slit widths were both set to 5 nm. To explore fluorescence quenching experiments in more depth, The Stern-Volmer Equation (5) was used, as shown in references [65][66][67].
The effectiveness of a quencher's (Q) ability to quench fluorescence is gauged by the Stern-Volmer quenching constant (Ksv). It is determined by the average integrated fluorescence lifetime of the fluorophore (τ0), the molar concentration of the quencher, and the bimolecular rate constant (kq) of the quenching reaction. A tryptophan, τ0, equals roughly 10 −9 s. The modified Stern-Volmer Equation (6) was used to calculate the binding constants (Kb) and number of binding sites (n) involved in the interaction of HSA samples. The free energy change (∆G 0 ) of the process was determined by analyzing the fluorescence quenching data using Equation (6) and then plugging the results into Equation (7). These calculations facilitate the quantitative assessment of the binding constants (K b ) and number of binding sites (n) involved in the interaction of HSA samples.
Log (F 0 /F − 1) = log K b + n log [Q] (6) where F 0 and F represent the fluorescence intensity in the absence and presence of the quencher (sample), K b represents the binding constant, and n represents the number of binding sites, respectively. ∆G 0 = −RTlnK b (7) Using the above equation, the change in free energy (∆G • ) can be calculated from the binding constant (Kb) at temperature (T), where R is the universal gas constant (1.987 cal mol −1 K −1 ).

In Vitro Measurement of Antioxidant Properties Using the DPPH Radical Scavenging Assay
The DPPH assay is a common method for determining a substance's antioxidant capacity. It assesses a substance's ability to reduce the stable free radical DPPH [68,69]. The method works by observing the reduction in alcoholic DPPH solutions in the presence of antioxidants that donate hydrogen atoms or electrons. Measure the hydrogen or electrondonating ability of the compound by observing the bleaching of a purple methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) using spectrophotometry. The percent inhibition of DPPH is a reliable indicator of the antioxidant capacity of a sample. The larger the inhibition percentage, the stronger the antioxidant activity. The DPPH assay provides a simple and convenient method for evaluating the antioxidant potential of different compounds in vitro. Of note, this assay specifically assessed the compound's ability to lower DPPH and did not provide a comprehensive measure of its overall antioxidant activity. In this study, the antioxidant activity of steroid 3 was assessed using the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Using the conventional DPPH assay, the sample's capacity to scavenge free radicals was evaluated. For the UV measurements, a freshly made solution of DPPH in methanol (6 × 10 −5 M) was employed. Steroid 3 solutions were added to the DPPH solution in a 1:1 ratio and vortexed. The concentrations of the Steroid 3 solutions ranged from 6.25 to 100 µM. In other words, 1 mL of the steroid 3 solutions and 1 mL of the DPPH solution were combined. The reaction mixture was thoroughly vortexed and incubated for 30 min at room temperature in the dark. The standard used was ascorbic acid. At 517 nm, the inhibition percentage of the activity to scavenge DPPH radicals was determined using the following equation: A plot of the percentage of DPPH inhibition against the concentration of the sample solutions was used to estimate the IC50 values (the concentration needed to scavenge 50% of the free radical). In this case, A 0 represents DPPH absorbance in the absence of the steroid, and A represents DPPH absorbance in the presence of the steroid.

Statistical Analysis
The data were analyzed using the mean and standard deviation (SD). The number of individual experiments is indicated by n.

Conclusions
A cholestane derivative was synthesized using an eco-friendly approach. Its binding to human serum albumin (HSA) was studied using fluorescence spectroscopy, UV-vis spectroscopy, molecular docking, and density functional theory. The results showed spontaneous binding (∆G = −9.86 kcal/mol) supported by negative binding scores in molecular docking. The HSA-ligand system exhibited static quenching, suggesting conformational changes in the protein upon binding. The molecule's solid-state packing is governed by dominant H···H, C···H, and O···H interactions, while strong intramolecular interactions were found within the crystal structure through Reduced Density Gradients analysis. This is supported further by the frontier molecular orbital analysis, which revealed a large energy gap of 6.94 eV for the molecule. This indicates that the molecule is relatively stable and unlikely to decompose. The DPPH radical scavenging activity of the studied steroids was comparable to that of standard ascorbic acid. This indicates that the steroid may have antioxidant properties, but more research is needed to determine its mode of action and anticancer activity in vivo. Overall, our findings shed light on the mechanism of cholestane derivative binding to HSA as well as antioxidant analysis. The investigation into the interaction between cholesterol derivatives and human serum albumin (HSA) elucidates a fundamental framework conducive to the advancement of investigations in the domains of pharmaceutical delivery systems, agents for cholesterol reduction, cholesterol metabolism, and the integrity of peptide structures. These findings pave the way for potential future applications in research endeavors. These findings could also have implications for the development of new HSA-targeting therapeutic agents, as well as anticancer research.
The supplementary crystallographic data has been deposited at the Cambridge Crystallographic Data Center, CCDC No. 901851 and may be obtained free of charge from http://www.ccdc.cam.ac.uk (accessed on 10 July 2023).