Crystal structure, molecular mechanics and In silico analyses of piperizine derivative against human mammary carcinoma cells inhibition.

In the present study, the single crystal of novel piperazine derivative4-(2, 3-chlorophenyl) piperazine-1-yl) (2-hydroxyphenyl) methanone (KDM) is grown by using the solvent evaporation method. The 3D structure of the molecule is confirmed by the single-crystal X-ray diffraction method. The study revealed that the molecular system is crystallized in the orthorhombic system with space group Pbca . The supramolecular crystal architecture establishes the stability of a compound via short contacts and halogen-hydrogen interactions. The Hirshfeld surface analysis were performed to evaluate the numerous intermolecular interactions based on the anisotropy of the topology. The Frontier molecular orbital (FMO) analysis and Molecular electrostatic potential (MEP) plots are investigated to understand the electronic structure properties of compounds using Density Functional Theory (DFT). In silico molecular docking, analysis is carried out to predict the best binding pose of the compound in the active site pocket of the BCl-XL/BAK protein-protein interface. Further, in vitro cytotoxicity studies against human breast cancer (MCF-7) cell lines of similarly designed piperazine-based derivatives showed prominent results. The results of the current study revealed that the compounds under investigation possess potential anti-cancer properties.


1.Introduction
Cancer treatment has remained dishearteningly low despite adopting multimodality therapy in the medical field.The 5-year survival rate typically ranges from 6% to 14% in males and 7% to 18% in females [1].Targeted medications are aimed at oncogenic driving mutations in adenocarcinoma treatments and treatment with radiation, surgery, and chemotherapy [2].Among the B-cell lymphoma (BCL) proteins' family, BCL-XL belongs to the family with anti-apoptotic function.It inhibits cell death by preventing the release of cytochrome c and apoptotic factors from the mitochondria into the cytoplasm.The release of apoptotic factors is a crucial step in initiating the cell death process.On the other hand, BCL-2-associated death promoter (BAK) is a pro-apoptotic that promotes cell death.When BAK is activated, it forms pores (channels) in the mitochondrial membrane, releasing cytochrome c with other apoptotic factors into the cytoplasm.The interaction between the BCL-XL and BAK is a critical aspect of the regulation of apoptosis, a process of programmed cell death that plays an significant role in tissue homeostasis maintainence and preventing the survival of damaged or abnormal cells [12].When BCL-XL and BAK interact, BAK's pro-apoptotic activity can be neutralized by preventing apoptosis and blocking the cytochrome's release from the mitochondria and other apoptotic factors.These released factors activate caspases, which are proteases that initiate cellular destruction during apoptosis.Hence, the balancing between pro-apoptotic and anti-apoptotic BCL family proteins is crucial for determining whether a cell will undergo apoptosis or survive.Disruption of this balance, such as increase of expression in anti-apoptotic proteins or reduce of expression of proapoptotic proteins contributing to cancer development and resistance to cancer treatments.A small molecule interaction with the interface of BCL-XL and BAK modulates the sensitive balance between cell survival and apoptosis.These interactions leads to implications for cellular function, tissue homeostasis, and disease processes, particularly in cancer therapy and the regulation of programmed cell death [13].Scientists and biologists are continually intrigued by molecules with small molecular masses because of their propensity to significantly influence the operation of macromolecules that make up living systems.Small-molecule modifies the protein function and provides the framework for chemically changing the state of disease and 'probes' in chemical genetics to analyze biological systems [3,4].Among heterocyclics, piperazine is a significant bioactive natural product.It is precious in drug discovery across therapeutic applications, including antitumor, antibacterial, antifungal, and anti-Alzheimer medications [5].Given this, we herein suggest a novel class of piperazine derivative for the inhibition of cancer cells and show its potential as a suitable candidate for drug development.

2.Experimental and Computational Methods
The block-shaped single crystal KDM having dimensions 0.21⨯0.24⨯0.32cm was subjected to X-ray diffraction (XRD) using a Bruker D8 Diffractometer using Apex II CCD Detector equipped with monochromatic MoKα radiation ( Wavelength λ = 0.7103 Å).The structure was further solved by direct methods and refined using SHELXS and SHELXL, respectively [6,7].The geometrical figures were generated and visualized by PLATON and MERCURY software [8].The computational calculation was performed using the Gaussian 09 program [9] package at the B3LYP method level with a standard 6-311++ G(d, p) basis set.AutoDock Tools (ADT) (v1.5.7) [10] software package was employed to perform in silico molecular docking analysis.

Single Crystal Structure X-ray Analysis
The single crystal X-ray analysis unveiled the confirmation of 3D structure of the KDM in orthorhombic system with Pbca space group, respectively.The geometrical lattice parameters were found to be a=10.0827Å, b= 13.0922 Å, c= 24.6781 Å, α =β =γ =90°, Volume (V) = 3257.6Å 3 and Z= 8.The ORTEP view with a 50% probability of the KDM molecule is shown in Figure 1

Hirshfeld Surface Analysis
The packing modes investigations, intermolecular interaction, and molecular shape corresponding to the crystalline environment are explored using Hirshfeld surface analysis (Figure 2a).The information is obtained based on electron density distribution by mapping Hirshfeld surfaces with dnorm.The 2D fingerprint plots are generated by combining de and di distances with the help of Crystal Explorer 17.5 [11].The fingerprint plots resulted in different regions of the area with H...H (38.2%),Cl-H…H-Cl (22.2%),C-H…H-C (20.2%), and O-H…H-O (12.7%) interactions (Figure 2c).The most noticed interaction in comparison is the H...H interaction, and the least is N-H…H-N (1.1%) interaction.The shape index diagram depicts the π-π interaction in the molecule encircled in black (Figure 2b).

Frontier Molecular Orbital (FMO) and Molecular Electrostatic Potential (MEP) Analysis
The quantum chemical characteristics of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) show the donation and acceptance of electrons, respectively.The computations are carried out using basis level theory B3LYP/6-311++G (d, p).The physicochemical properties, such as kinetic stability, chemical hardness/softness, and reactivity of the molecule, are characterized by the HOMO-LUMO energy gap (Table 1).The HOMO and LUMO orbitals of the KDM molecule are shown in Figure 3.The energy gap is found to be 4.744eV.The electronegativity and electrophilicity of KDM are observed to be 2.831 and 3.672 eV, respectively.The MEP plot was employed to understand the molecule's predicted reactive sites, such as electrophilic or nucleophilic sites.An electron isosurface map with an electrostatic potential surface indicates the molecule's size, shape, charge density, and reactive sites.reported the design and synthesis of eighteen molecules and were further sent for cytotoxic studies against MCF-7 cell lines, which showed prominent anti-cancer activities [12].

Conclusion
The three-dimensional crystal structure of 4-(2, 3-chlorophenyl) piperazine-1-yl) (2-hydroxyphenyl) methanone (KDM) is determined using a single X-ray diffraction technique.The compound resulted in prominent hydrogen bond interactions in the experiment and DFT.Frontier molecular orbital (FMO) and molecular electrostatic potential (MEP) analyses of KDM were performed by DFT at the B3LYP/6311++G (d, p) basis set to understand the chemical reactive sites and global descriptor parameters of the compound.The KDM was firmly bound into the BCL-XL/BAK interface pocket, which deserves a potential candidate for cancer cell growth inhibition.
(a).The packing diagram of molecule along b-axis exhibited halogen mediated hydrogen bond interactions as shown in Figure1(b).The halogen-hydrogen interactions are obsereved among atoms CL8…H3 and Cl7…H10A with 2.7 and 2.9Å, respectively.The KDM is also stabilized by C-H…O type inermolecular interactions.

Figure 1 :
Figure 1: a) ORTEP drawn at 50% probability b) Packing diagram with halogen and hydrogen bond interactions along b-axis

Figure 4
depicts the resulting MEP map of the KDM compound.