DOI: 10.1002/ejic.201501361 Full Paper Bioactive Complexes Ru II (η 6 -p-cymene) Complexes of Bioactive 1,2-Benzothiazines: Protein Binding vs. Antitumor Activity Adnan Ashraf, [a,b] Mario Kubanik, [a] Farhana Aman, [a,b] Hannah Holtkamp, [a] Tilo Söhnel, [a] Stephen M. F. Jamieson, [c] Muhammad Hanif* [a,d] Waseeq Ahmad Siddiqui,* [b] and Christian G. Hartinger* [a] Dedicated to Professor Peter J. Sadler on the occasion of his 70th birthday Abstract: 1,2-Benzothiazine-3-carboxamide 1,1-dioxide deriva- tives such as meloxicam are known to display numerous phar- macological activities. We prepared a series of 4-hydroxy-2- alkyl-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide li- gands 1a–f and their Ru(η 6 -p-cymene) complexes 2a–f, inspired by synergistic effects observed with other bioactive ligands co- ordinated to metal centres. The molecular structures of 1a, 2a, and 2b were determined by X-ray diffraction analyses. The sta- bility of the metal complexes was characterized in DMSO and DMSO/H 2 O on the basis of 1 H NMR spectroscopy and their pro- Introduction Ruthenium complexes have attracted increasing attention as new anticancer agents in the last several years. The Ru III drug candidates NAMI-A {imidazolium trans-[tetrachlorido(DMSO)- (1H-imidazole)ruthenate(III)]} and KP1019 {indazolium trans- [tetrachloridobis(1H-indazole)ruthenate(III)]} or its more soluble analogue NKP-1339 {sodium trans-[tetrachloridobis(1H-indazole)- ruthenate(III)]} are currently in human clinical phase II trials or only recently have been taken out of such trials. [1] Both NAMI- A and KP1019 are structurally related but behave distinctly dif- ferently in in vitro and in vivo assays. KP1019 has been found to be active against colorectal cancer cells and has activity su- perior to 5-fluorouracil, one of the mainstream drugs for this cancer type. Alternatively, NAMI-A has been found to effectively inhibit tumor metastasis but does not appear to act against the primary tumor. [2] [a] School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand E-mail: c.hartinger@auckland.ac.nz m.hanif@auckland.ac.nz http://hartinger.wordpress.fos.auckland.ac.nz/ [b] Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan E-mail: waseeq786@gmail.com [c] Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand [d] Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan Supporting information and ORCID(s) from the author(s) for this article are available on the WWW under http://dx.doi.org/10.1002/ejic.201501361. Eur. J. Inorg. Chem. 2016, 1376–1382 © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1376 tein binding capabilities were studied using mass spectrometry. In vitro cytotoxicities of the Ru complexes were determined against human colorectal carcinoma (HCT116), non-small cell lung carcinoma (NCI-H460) and cervical carcinoma (SiHa) cell lines. The low levels of biological activity observed for these Ru complexes were put into context by considering their chemical reactivity with proteins. The binding of proteins resulted in cleavage of the benzothiazine backbone when the complex was present in concentrations equimolar with respect to pro- tein. Recently, interest in anticancer Ru II (arene) complexes has sig- nificantly grown. These complexes are highly versatile with re- gards to the modulation of their pharmacological properties. The arene ligands employed to enhance the stability of Ru II complexes, also present the opportunity to balance the lipo- philic/hydrophilic character of the resulting metal compounds. Furthermore, variations of the co-ligands impact the reactivity and biological activities of these Ru II -based compounds. For in- stance, RAPTA-C [Ru(η 6 -p-cymene)(PTA)Cl 2 ] (PTA = 1,3,5-triaza- 7-phosphatricyclodecane) and RAED-C [Ru(η 6 -p-cym- ene)(en)Cl]PF 6 (en = 1,2-ethylenediamine) are two lead struc- tures with contrasting biological activities and modes of ac- tion. [3] RAED-C preferentially binds to DNA whereas RAPTA-C targets proteins. [4] In terms of their anticancer activity, RAED-C and its analogues are active against primary tumors both in vitro and in vivo. [5] In contrast, RAPTA-C is marginally cytotoxic against cancer cells but effectively inhibits metastasis in vivo. These realizations demonstrate that small variations in the li- gand system can dramatically alter reactivities with cellular tar- gets, thus impacting biological activities. [4] As a consequence, a variety of ligand systems have been conjugated to the Ru(ar- ene) moiety. [3a,6] A current approach to design new anticancer candidates is to combine bioactive ligands with Ru(arene) pharmacophores. This yields multifunctional compounds due to synergistic ef- fects between both the ligand and the metal centre. Several examples of this strategy include, but are not limited to: anti- cancer Ru(arene) of quinolones (e.g., ofloxacin, nalidixic acid), [7] lapachol, [8] flavonols [9] and hydroxypyr(id)ones. [6b,10] These het-