Novel amidino substituted 2-phenylbenzothiazoles: Synthesis, antitumor evaluation in vitro and acute toxicity testing in vivo Livio Racané b , Marijeta Kralj c , Lidija Šuman c , Ranko Stojkovic ´ c , Vesna Tralic ´ -Kulenovic ´ b , Grace Karminski-Zamola a, * a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulic ´ev trg 20, PO Box 177, HR-10000 Zagreb, Croatia b Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb, baruna Filipovic ´a 10000 Zagreb, Croatia c Division of Molecular Medicine, Ru - der Boškovic ´ Institute, Bijenic ˇka cesta 54, PO Box 180, HR-10000 Zagreb, Croatia article info Article history: Received 22 July 2009 Revised 17 December 2009 Accepted 21 December 2009 Available online 29 December 2009 Keywords: Nitro-amidino-, amino-amidino- and diamidino-substituted 2- phenylbenzothiazoles Antitumor activity ‘in vitro’ Toxicity testing ‘in vivo’ abstract The efficient synthesis of new bis-substituted nitro-amidino, amino-amidino (10a, 10b–13a, 13b) and previously prepared diamidino 2-phenyl-benzothiazoles (9a, 9b) is described. The compounds 11a and 11b were prepared by recently developed methodology of the key precursors in zwitterionic form 8a and 8b with 4-nitrobenzoylchloride in a very good yield (70%). All compounds except diamidino- substituted 2-phenylbenzothiazole 9a show exceptionally prominent tumor cell-growth inhibitory activity and cytotoxicity, whereby the special selectivity of amino-amidine 2-phenylbenzothiazole 12a towards MCF-7 and H 460 cells makes this compound a prospective lead compound that should be further evaluated in animal models. All in vivo tested compounds (12a, 12b, 13a and 13b) are absorbed from mice gastrointestinal system. LD 50 are between 67.33 and 696.2 mg/kg body weight (OECD/EPA toxicity categories 2–3). Ó 2010 Published by Elsevier Ltd. 1. Introduction In the last two decades, many heterocyclic compounds from the benzothiazole series were synthesized and their biological and phar- macological activity investigated. They were studied extensively for their antiallergic, 1 anti-inflammatory, 1,2 antitumor 3–7 and analge- sic 8,9 activity. Considering the mechanism of action, it was shown that benzothiazole derivatives act as tyrosine kinase 10–13 and topo- isomerase I and II inhibitors. 14,15 Therefore, various benzothiazole compounds are further of considerable interest for their diverse pharmaceutical uses. It was reported recently about a novel series of optically active 2-aminobenzothiazole derivatives and their in vitro cytotoxicity against mouse Ehrlich Ascites Carcinoma (EAC) and two human cancer cell lines MCF-7 and HeLa. In alkaline comet assay some compounds showed dose-dependent DNA damaging activity. 16 Newly prepared 2-acetyl-3-(6-methoxyben- zothiazo)-2-yl-amino-acrylonitrile showed significant antiprolifer- ative activity and is a potent inducer of programed cell death in leu- kemia cells. 17 The most recent article describing the synthesis of corresponding aminophenyl- substituted benzothiazoles with different adenine mimic which resulted in the identification of promising scaffolds, giving rise to the inhibition of different kinases with the IC 50 values in the nanomolar range. 18 Our previously obtained results showed that antiproliferative activity of cyano, amidino 19 and amino 20 substituted 2-phen- ylbenzothiazole derivatives strongly depends on the position of the substituent on 2-phenylbenzothiazole skeleton, as well as on the type of amidino substituent attached. We found that, in a series of unsubstituted, N-isopropyl substituted, as well as 2-imidazolinyl mono and bisamidino derivatives of 2-phenylbenzothiazole, N-iso- propyl substituted amidine posses less pronounced antiprolifera- tive activity on tested tumor cells. In relation with the above considerations, we designed and effi- ciently synthesized new nitro-amidino, amino-amidino and diami- dino-substituted 2-phenylbenzothiazole derivatives and tested their antitumor activity in vitro and determinate acute oral toxicity in mice of the most active compounds. 2. Results and discussion 2.1. Chemistry An efficient synthesis of different amidino substituted 2-phen- ylbenzothiazole derivatives was carried out by reactions outlined in Scheme 1. Bis-nitrile and nitro-nitrile 5–7 derivatives were prepared by condensation reaction of cyano or nitro-substituted 2-aminobenz- othiole 1 or 2 21 with commercially available 4-cyano or 4-nitro- benzoylchloride 3 and 4 (77–84%), respectively and were used as 0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.bmc.2009.12.054 * Corresponding author. Tel.: +385 14597297; fax: +385 14597224. E-mail address: gzamola@fkit.hr (G. Karminski-Zamola). Bioorganic & Medicinal Chemistry 18 (2010) 1038–1044 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc