Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Interaction of C20-substituted derivative of pregnenolone acetate with copper (II) leads to ROS generation, DNA cleavage and apoptosis in cervical cancer cells: Therapeutic potential of copper chelation for cancer treatment Atif Zafar a , Swarnendra Singh b , Sabahuddin Ahmad c , Saman Khan a , Mohammad Imran Siddiqi c , Imrana Naseem a, ⁎ a Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, Uttar Pradesh, India b Department of Dermatology and Venereology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India c Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India ARTICLEINFO Keywords: Pregnenolone acetate Tetrazole Copper Redox cycling Apoptosis DNA damage ABSTRACT Cervical cancer is a leading cause of cancer-related deaths among women in developing countries. Therefore, development of new chemotherapeutic agents is required. Unlike normal cells, cancer cells contain elevated copper levels which play an integral role in angiogenesis. Thus, targeting copper via copper-specifc chelators in cancer cells can serve as efective anticancer strategy. In this work, a copper chelator pregnenolone acetate nucleus-based tetrazole derivative (ligand-L) was synthesized and characterized by elemental analysis, ESI-MS, 1 HNMRand 13 CNMR.DNAbindingabilityofligand-LwasstudiedusingUV–Visand fuorescence spectroscopy. Fluorescence spectroscopy studies reveal that quenching constant of ligand-L-DNA and ligand-L-Cu(II) were foundtobe7.4×10 3 M −1 and8.8×10 3 M −1 , respectively. In vitro toxicity of ligand-L was studied on human cervical cancer C33A cancer cells. Results showed that ligand-L exhibit signifcant cytotoxic activity against cervical cancer C33A cells with IC 50 value 5.0 ± 1.8 µM. Further, it was found that ligand-L cytotoxicity is due to redox cycling of copper to generate ROS which leads to DNA damage and apoptosis. In conclusion, this is the report where we synthesized pregnenolone acetate-based tetrazole derivative against C33A cells that targets cellular copper to induce pro-oxidant death in cancer cells. These fndings will provide signifcant insights into the development of new chemical molecules with better copper chelating and pro-oxidant properties against cancer cells. 1. Introduction Cervicalcanceristhethirdmostcommoncancerthatafectswomen worldwide with an estimated 530,000 new cases and 275,000 deaths each year in developing countries, out of which India represents one- quarter of the cervix cancer burden globally [1]. The pathological progression of cervical tumorigenesis is associated with high-risk Human Papilloma Virus (HPV) types [2]. Traditional therapeutic ap- proaches for cervical cancer include surgery, radiotherapy and che- motherapy [3]. However, platinum–based chemotherapy against cer- vicalcancerexhibitsdrugresistanceandserioussideefects [4,5].Thus, there is a need to identify and synthesize new chemotherapeutic agents against cervical cancer. In continuation of our pursuit to design and synthesize new anti- tumor agents against cervical cancer, it is important to note that cancer cells exhibit altered metabolism with elevated copper levels as com- pared to normal cells [6–8]. Copper is an important redox active metal ion associated with DNA bases, particularly with guanine [9]. Elevated copperincancerplaysanintegralroleinangiogenesisbyfunctioningas a co-factor of several pro-angiogenic molecules such as VEGF, angio- genin and basic fbroblast growth factor (bFGF) [10,11]. Therefore, development of copper-specifc chelators provides a selective approach to target tumor cells. The cytotoxicty of copper complexes towards cancer cells thus arises from their high redox activity to produce re- active oxygen species (ROS) [12]. ROS generation by ligand-copper complexes further leads to metal-driven oxidative DNA damage and cell death [13]. Steroids are a class of naturally occurring organic compounds which exhibit diverse biological activities [14–17]. Steroid-based therapeutics has attracted considerable interest due to various advantages such as https://doi.org/10.1016/j.bioorg.2019.03.031 Received 6 December 2018; Received in revised form 26 February 2019; Accepted 14 March 2019 ⁎ Corresponding author. E-mail address: imrananaseem2009@gmail.com (I. Naseem). Bioorganic Chemistry 87 (2019) 276–290 Available online 19 March 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved. T