Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Proliferation inhibition of novel diphenylamine derivatives Ladislav Janovec a, ⁎ , Jana Janočková b,c , Mária Matejová a , Eva Konkoľová b , Helena Paulíková d , Daniela Lichancová b , Lenka Júnošová d , Slávka Hamuľaková a , Ján Imrich a , Mária Kožurková b,c a Department of Organic Chemistry, P. J. Safarik University, Faculty of Science, Moyzesova 11, 04001 Kosice, Slovak Republic b Department of Biochemistry, P. J. Safarik University, Faculty of Science, Moyzesova 11, 04001 Kosice, Slovak Republic c Biomedical Research Center, University Hospital Hradec Kralove, Sokolovska 581, Hradec Kralove, Czech Republic d Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak Technical University, Radlinskeho 9, 81237 Bratislava, Slovak Republic ARTICLEINFO Keywords: Antiproliferative Diphenylamine DNA minor groove binder DNA binding study ABSTRACT Nonsteroidal anti-infammatory drugs (NSAIDs) are the most widely used drugs in the world but some NSAIDs such as diclofenac and tolfenamic acid display levels of cytotoxicity, an efect which has been attributed to the presence of diphenylamine contained in their structures. A novel series of diphenylamine derivatives were synthetised and evaluated for their cytotoxic activities and proliferation inhibition. The most active compounds in the cytotoxicity tests were derivative 6g with an IC 50 value of 2.5 ± 1.1 × 10 −6 M and derivative 6f with an IC 50 value of 6.0 ± 3.0 × 10 −6 M (L1210 cell line) after 48 h incubation. The results demonstrate that leukemic L1210 cells were much more sensitive to compounds 6f and 6g than the HEK293T cells (IC 50 = 35 × 10 −6 M for 6f and IC 50 > 50 × 10 −6 M for 6g) and NIH-3T3 (IC 50 > 50 × 10 −6 M for both derivatives). The IC 50 values show that these substances may selectively kill leukemic cells over non-cancer cells. Cell cycle analysis revealed that a primary trend of the diphenylamine derivatives was to arrest the cells in the G 1 -phase of the cell cycle within the frst 24 h. UV–visible, fuorescence spectroscopy and circular dichroism were used in order to study the binding mode of the novel compounds with DNA. The binding constants determined by UV–visible spec- troscopy were found to be in the range of 2.1–8.7 × 10 4 M −1 . We suggest that the observed trend for binding constant K is likely to be a result of diferent binding thermodynamics accompanying the formation of the complexes. 1. Introduction Nonsteroidal anti-infammatory drugs (NSAIDs) are the most widely used drugs in the world [1], but some NSAIDs such as diclofenac and tolfenamic acid display levels of cytotoxicity, an efect which has been attributed to the presence of diphenylamine contained in their struc- tures (Fig. 1). There is some structural resemblance between the di- phenylamine core and DNA minor groove binders, and therefore we synthesized a series of diphenylamine derivatives (6a–6g) in order to test their cytotoxic activity and their DNA binding ability (Fig. 1) [1]. The minor groove has a helical curvature which can also vary slightly depending on the sequence, so agents with unfused heterocyclic sys- tems which bind to the minor groove must therefore have a concave shape that complements that of the groove [2]. Small molecules which bind to genomic DNA and which are easily accessible to chromosomal DNA have proven to be efective anticancer therapeutic agents, and therefore researchers continue to be interested in designing pharma- cophores which are capable of interacting with DNA [3]. The modes through which this interaction occurs include intercalation between adjacent base pairs, and intrusion into the minor and major grooves. Intercalation and minor groove binding are the predominant DNA- binding modes of small ligands [4–6]. In this report, we present the results of our assays showing that diphenylamino carboxylic acid derivatives 6a–6g demonstrate inter- esting levels of antiproliferative activity with interaction with DNA through groove-binding. The IC 50 values obtained for the compounds also show that novel derivatives 6f and 6g may selectively kill leukemic cells over non-cancer cells. 2. Results and discussions 2.1. Chemistry Derivatives 6a–6g were synthesized using the following reaction pathway (Scheme 1) [7]. 4-Acetamido-2-chlorobenzoic acid (3) was prepared with a yield of 65% through the oxidation of 4-acetamido-2- https://doi.org/10.1016/j.bioorg.2018.10.063 Received 26 January 2018; Received in revised form 19 October 2018; Accepted 29 October 2018 ⁎ Corresponding author. E-mail address: ladislav.janovec@upjs.sk (L. Janovec). Bioorganic Chemistry 83 (2019) 487–499 Available online 02 November 2018 0045-2068/ © 2018 Elsevier Inc. All rights reserved. T