*3.4. In Silico Docking with IKK*β

The docking calculations were carried out using the protein structure of IKKβ (The Protein Data Bank code; 4KIK.pdb). Using the Sybyl program, the apo-protein of IKKβ was obtained by removing the original ligand **K252a** contained in 4KIK.pdb. The original ligand K252a was again docked to the apo-protein, to confirm that the flexible docking procedure worked well. Through a flexible docking procedure repeated 30 times, 30 complexes between apo-protein and **K252a** were obtained. Their binding energy ranged from −28.44 to −10.24 kcal/mol and their binding poses were good to be comparable with 4KIK.pdb.The binding pocket of IKKβ was determined using the LigPlot software as previously reported [48]. They are composed of 16 residues; 14 residues, namely Leu21, Gly22, Thr23, Val25, Ala42, Lys44, Glu61, Val74, Met96, Tyr98, Glu149, Asn150, Ile165, and Asp166 are involved in hydrophobic interactions, and two residues, Glu97 and Cys99 are involved in hydrogen bonds. Using the three-dimensional structure of compound **5** obtained in this study, docking with apo-protein was performed in the same way as the original ligand. The binding energy generated by the 30 iterations ranged from −15.92 to −13.09 kcal/mol. The interactions between IKKβ and compound **5**

*3.3. Hirshfeld Surface analysis of Compound 5*

interactions between the molecules [47].

were analyzed using the LigPlot progam. Six residues including Thr23, Val29, Glu61, Met65, Met96, and Ile165 showed the hydrophobic interactions with the ligand and three residues including Gly27, Lys44, and Asp166 formed hydrogen bonds (H bonds) with the ligand (Figure 9A). The binding pocket of compound **5** resided in IKKβ was visualized using the PyMOL program (PyMOL Molecular Graphics System, version 1.0r1, Schrödinger, LLC, Portland, OR, USA). Isoflavone compound **5** in IKKβ exhibited a slightly different binding pattern from those of the original ligand **K252a**. However, both isoflavone **5** and original ligand **K252a** have been shown to bind well at the active site of the IKKβ protein (Figure 9B). **Figure 6.** (**A**) Hirshfeld surfaces of molecule **5I** mapped with dnorm, shape index and curvedness. (**B**) Hirshfeld surfaces of molecule **5II** mapped with dnorm, shape index and curvedness. According to two-dimensional fingerprint plots analysis, the dominant interaction in each molecule **5I** and **5II** originates from H···H contacts, which are the major contributors of 43.5% and 42.5% to the total Hirshfeld surface, respectively. The contribution from the O···H/H···O contacts of 25.1% and 29.1% of each molecule **5I** and **5II** is represented by a pair of sharp spikes that are characteristic of hydrogen-bonding interactions. Other meaningful interactions include C···H/H···C with contributions of 17.8% and 16.7% from **5I** and **5II**, respectively (Figure 7A–H).

*Crystals* **2020**, *10*, x FOR PEER REVIEW 9 of 15

In order to quantify the intermolecular interactions in the crystals of the titled compound **5**, a Hirshfeld surface (HS) analysis was carried out. Based on the Hirshfeld analysis on all conformers, two independent molecules (**5I** and **5II**) showed different dnorm, shape index (SI) and curvedness, however, each set of conformers **A** and **B** revealed the same Hirshfeld analysis results [46]. The 3D Hirshfeld surfaces of two independent molecules (**5I** and **5II**) were illustrated in Figure 6A,B, which maps dnorm, shape index and curvedness. The deep red spots on the dnorm Hirshfeld surfaces of each molecule represent the close contact interactions, which are mainly responsible for the significant intermolecular C–H···O interactions. Shape index and curvedness can also be used to identify the characteristic packing modes. The shape indexes of **5I** and **5II** show red concave regions on the surface around the acceptor atoms and blue regions around the donor H atoms. The maps of curvedness for **5I** and **5II** show no flat surface patches representing that there are no stacking

**Figure 7.** Two-dimensional fingerprint plots of the most important intermolecular contacts in each molecule **I** and **II**. For **I**: full (**A**) and resolved into O···H (**B**), H···H (**C**), C···H (**D**). For **II**: full (**E**) and resolved into O···H (**F**), H···H (**G**), C···H (**H**). **Figure 7.** Two-dimensional fingerprint plots of the most important intermolecular contacts in each molecule **I** and **II**. For **I**: full (**A**) and resolved into O···H (**B**), H···H (**C**), C···H (**D**). For **II**: full (**E**) and resolved into O···H (**F**), H···H (**G**), C···H (**H**).

The overall contribution to the total Hirshfeld surface is illustrated in Figure 8.

**Figure 8.** Summary of the overall detailed intermolecular interactions and their contribution to each crystal structure **I** and **II**. For **I**; H···H ; 43.5%, O···H; 25.1%, O···C; 6.4%, C···H; 17.8%, C···C; 5.7%, O···O; 1.5%, For **II**; H···H ; 42.5%, O···H; 29.1%, O···C; 5.6%, C···H; 16.3%, C···C; 5.7%, O···O; 0.8%. crystal structure **I** and **II**. For **I**; H···H; 43.5%, O···H; 25.1%, O···C; 6.4%, C···H; 17.8%, C···C; 5.7%, O···O; 1.5%, For **II**; H···H; 42.5%, O···H; 29.1%, O···C; 5.6%, C···H; 16.3%, C···C; 5.7%, O···O; 0.8%.
