**Single Crystal X-Ray Structure for the Disordered Two Independent Molecules of Novel Isoflavone: Synthesis, Hirshfeld Surface Analysis, Inhibition and Docking Studies on IKK**β **of 3-(2,3-dihydrobenzo [b][1,4]dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one**

**Soon Young Shin <sup>1</sup> , Young Han Lee <sup>1</sup> , Yoongho Lim <sup>2</sup> , Ha Jin Lee <sup>3</sup> , Ji Hye Lee <sup>4</sup> , Miri Yoo <sup>4</sup> , Seunghyun Ahn 4,\* and Dongsoo Koh 4,\***


Received: 14 September 2020; Accepted: 6 October 2020; Published: 9 October 2020

**Abstract:** The structure of the isoflavone compound, 3-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)-6,7-dimethoxy-4H-chromen-4-one (**5**), was elucidated by 2D-NMR spectra, mass spectrum and single crystal X-ray crystallography. Compound **5**, C19H16O6, was crystallized in the monoclinic space group *P*21/c with the cell parameters; a = 12.0654(5) Å, b =11.0666(5) Å, c = 23.9550(11) Å, β = 101.3757(16)◦ , V = 3135.7(2) Å<sup>3</sup> , and Z = 8. The asymmetric unit of compound **5** consists of two independent molecules **5I** and **5II**. Both molecules exhibit the disorder of each methylene group present in their 1,4-dioxane rings with relative occupancies of 0.599(10) (**5I**) and 0.812(9) (**5II**) for the major component **A**, and 0.401(10) (**5I**) and 0.188(9) (**5II**) for the minor component **B**, respectively. Each independent molecule revealed remarkable discrepancies in bond lengths, bond angles and dihedral angles in the disordered regions of 1,4-dioxane rings. The common feature of the molecules **5I** and **5II** are a chromone ring and a benzodioxin ring, which are more tilted towards each other in **5I** than in **5II**. An additional difference between the molecules is seen in the relative disposition of two methoxy substituents. In the crystal, the molecule **5II** forms inversion dimers which are linked into chains along an *a*-axis direction by intermolecular C–H· · · O interactions. Additional C–H· · · O hydrogen bonds connected the molecules **5I** and **5II** each other to form a three-dimensional network. Hirshfeld surface analysis evaluated the relative intermolecular interactions which contribute to each crystal structure **5I** and **5II**. Western blot analysis demonstrated that compound **5** inhibited the TNFα-induced phosphorylation of IKKα/β, resulting in attenuating further downstream NF-κB signaling. A molecular docking study predicted the possible binding of compound **5** to the active site of IKKβ. Compound **5** showed an inhibitory effect on the clonogenicity of HCT116 human colon cancer cells. These results suggest that compound **5** can be used as a platform for the development of an anti-cancer agent targeting IKKα/β.

**Keywords:** disordered crystal structure; hydrogen bonding; in silico docking; Hirshfeld surface; NF-κB signaling; IKKβ inhibitor
