## 2 Materials and Methods  
**Ligand Design:** The hydroxy group at positions 3 and/or 6 of the \(\alpha\)-mangostin structure was conjugated to glycine to create the ligands, as shown in Figure 1. The chemical structures of conjugates of \(\alpha\)-mangostin were graphically drawn using Marvinsketch (Chemaxon(r), Budapest, Hungary) [24]. All sketched conjugates were then accurately refined by geometric optimization, utilizing the MMFF94 force field in Avogadro [25]. **The Prediction of Pharmacokine and Toxicological Profiles:** The toxicity, distribution, and absorption profiles of \(\alpha\)-mangostin and its conjugates were predicted using the PreADMET webserver. Pharmacokine properties such as Caco-2 permeability, human intestine absorption, blood-brain barrier penetration, and protein plasma binding were evaluated. Caco-2 permeability pertains to a compound's capacity to pass through a monolayer of Caco-2 cells, which act as an intestinal epithelial model representing the degree to which a compound can be absorbed into the human intestine [26]. Humanintestine absorption refers to the proportion of a medicine taken orally that is absorbed into the bloodstream through the intestinal wall [27]. Blood-brain barrier penetration is the ability of a compound to pass across the blood-brain barrier and reach the central nervous system [28]. Plasma binding protein indicates the degree to which a drug binds to plasma proteins such as albumin [29]. To forecast toxicity parameters, three tests were employed: the Ames test, a frequently used assay for determining the mutagenic potential of chemical substances. The Ames test is based on multiple strains of the bacterium _Salmonella typhimurium_ that have mutations in genes involved in histidine production. These strains cannot grow in a histidine-free media until they undergo a mutation that restores their capacity to synthesize histidine [30]. The Ames test determines the ability of a chemical substance to cause such alterations. The carcinoma test in rodents (rat and mice) was also employed. Rats and mice metabolize chemicals differently, resulting in differences in toxicity profiles and responses to toxicants. Additionally, rats have a longer lifespan and bigger body size than mice, which might influence the duration and scale of toxicity studies [31; 32]. **Pharmacophore Modeling:** The 3D ligand-based pharmacophore model was constructed using LigandScout 4.4.3 (Inte:Ligand GmbH(r), Vienna, Austria) [33]. The model databases were collected from the Database of Useful Decoys. To verify the robustness of the model in capturing the 3D ligand-based interaction features, a set of 20,818 decoys and a set of 627 actives were screened during the validation process. Upon validation, the established 3D ligand-based pharmacophore model was used to match features with conjugates of \(\alpha\)-mangostin. Pharmacophore fit scores were computed to determine the extent of fit with the pharmacophore models [34]. **Molecular Docking:** The molecular structure of ER\(\alpha\) complexed with 4-hydroxytamoxifen was obtained from the Protein Data Bank using PDB ID 3 ERT [35]. Models for docking were prepared using AutoDockTools 1.5.6 (Scripps Research, La Jolla, CA, USA) [36]. Before molecular docking, all the ligands were created by adding hydrogen atoms and merging them with nonpolar hydrogen atoms, adding Gasteiger charges, defining rotatable bonds, and then saving in PDBQT format [37]. The Gasteiger charge, which was utilized on the ligands for a potent postprediction, performed well in all ligand sizes and was  
Figure 1: Modified \(\alpha\)-mangostin. (**a**) \(\alpha\)-Mangostin, R1 and R2: OH; (**b**) Am1 Gly, R1: L-glycine; (**c**) Am2 Gly, R2: L-glycine; (**d**) Am3 Gly, R1 and R2: L-glycine. particularly successful on ligands with great flexibility [38]. Hydrogens were added to the macromolecule, Kollman charges were applied, and the charged macromolecule was saved in PDBQT format [39]. Kollman charges are calculated using quantum mechanics and then adjusted empirically.