Research paper Spirotriazoline oxindoles: A novel chemical scaffold with in vitro anticancer properties Carlos J.A. Ribeiro, Rute C. Nunes, Joana D. Amaral, Lídia M. Gonçalves, Cecília M.P. Rodrigues, Rui Moreira, Maria M.M. Santos * Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal article info Article history: Received 1 August 2017 Received in revised form 7 September 2017 Accepted 19 September 2017 Available online 21 September 2017 Keywords: Spirooxindole Triazoline Cytotoxicity Anti-cancer agents Breast cancer abstract The design and synthesis of a library of twenty-six spirotriazoline oxindoles and their in vitro evaluation as potential anticancer agents is reported. The antiproliferative activity of the synthesized compounds was assessed against four different cancer cell lines (HCT-116 p53 (þ/þ) , HCT-116 p53 (/) , MCF-7, and MDA-MB-231). Four spirotriazoline oxindoles showed selectivity against the four cancer cell lines tested over the non-cancer derived HEK 293T cell line. To characterize the molecular mechanisms involved in compound antitumoral activity, two spirotriazoline oxindoles were selected for further studies. Both compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase and upregulated p53 steady-state levels, while decreasing its main inhibitor MDM2, in HCT-116 cells. Importantly, cytotoxic effects induced by spirotriazoline oxindoles occurred in cancer cells without eliciting cell death in non- malignant CCD-18Co human colon fibroblasts. In addition, four spirotriazoline oxindoles showed selectivity against the triple-negative breast cancer cell line MDA-MB-231 with IC 50 values of 3.5e6.7 mM. These results highlight the anticancer potential of spirotriazoline oxindoles, especially when dealing with aggressive and challenging triple-negative breast cancer. © 2017 Elsevier Masson SAS. All rights reserved. 1. Introduction It is well established that cancer represents a major public health issue worldwide. In 2012, it was estimated 14.1 million new cancer cases and 8.2 million deaths [1], and the World Health Or- ganization estimates a rise of 13.1 million cancer deaths until 2030. Specifically, breast cancer is the most common cancer in woman, and is the leading cause of cancer death in women aged 20e59 years in the USA [2]. In particular, a specific tumor subtype, known as triple-negative breast cancer (TNBC) is characterized as a more aggressive type of cancer with poorer prognostic. TNBCs are char- acterized by the lack of expression of estrogen and progesterone receptors, and absence of human epidermal growth factor receptor 2 overexpression, representing the hardest breast cancer to treat [3]. Although early detection and efficient therapies contributed to a decrease of cancer mortality in developed countries, overcoming drug intrinsic and acquired resistance represents a major challenge [4,5] and imposes a constant development of new molecules and the identification of novel targets, especially for TNBC patients [3]. Heterocycles possessing a spirooxindole framework are found in many natural products and medicinal agents with diverse biolog- ical activities [6e8]. The attractiveness of this type of scaffold in organic and medicinal chemistry is evident by the increasing number of publications on this topic over the last 10 years. This can be ascribed, at least partially, to the fact that the central spiro car- bon atom imposes a conformational restriction to the structure that can be beneficial for ligand-target binding, and thus potentially promoting an increase in potency and/or specificity [7]. Furthermore, the reduced molecular flexibility displayed by the spirooxindole scaffold can also potentially lead to better pharma- cokinetic properties [9]. Antiproliferative activity against cancer cell lines is one of the biological effects reported for spirooxindoles (Fig. 1) [10]. Specifically, CFI-400945 is a Polo-like kinase 4 (PLK4) inhibitor [11], compound ()-1 interferes with microtubule poly- merization and arrests mitosis [12], and compounds MI-77301 [13] and SM13 [14] modulate p53 activity. We have recently developed several small molecules showing potential anticancer activity [15e23], with a focus on novel five- membered ring spirooxindoles that act as p53 modulators (Fig. 2). Initial studies explored the spiroisoxazoline oxindole * Corresponding author. E-mail address: mariasantos@ff.ulisboa.pt (M.M.M. Santos). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech https://doi.org/10.1016/j.ejmech.2017.09.037 0223-5234/© 2017 Elsevier Masson SAS. All rights reserved. European Journal of Medicinal Chemistry 140 (2017) 494e509