Photochemical & Photobiological Sciences RSCPublishing ARTICLE This journal is © The Royal Society of Chemistry 2014 Photochem. Photobiol. Sci ., 2014, 00, 1-10 | 1 Cite this: DOI: 10.1039/x0xx00000x Received 00th July 2014, Accepted 00th January 2012 DOI: 10.1039/x0xx00000x www.rsc.org/ A New Antitumoral Heteroarylaminothieno[3,2-b] pyridine Derivative: Incorporation in Liposomes and Interaction with Proteins Monitored by Fluorescence C. N. C. Costa a , A. C. L. Hortelão a , J. M. F. Ramos a , A. D. S. Oliveira a , R. C. Calhelha b , M.-J. R. P. Queiroz b , P. J. G. Coutinho a , E.M.S. Castanheira a † a Centro de Física, Universidade do Minho (CFUM), Campus de Gualtar, 4710-057 Braga, Portugal. b Centro de Química, Universidade do Minho (CQ/UM), Campus de Gualtar, 4710-057 Braga, Portugal. † Corresponding author; Phone: +351 253604321; Fax: +351 253604061; ecoutinho@fisica.uminho.pt The fluorescence properties of the new potent antitumoral methyl 3-amino-6-(benzo[d]thiazol- 2-ylamino)thieno[3,2-b]pyridine-2-carboxylate in solution and when encapsulated in several different nanoliposome formulations were investigated. The compound exhibits very reasonable fluorescence quantum yields and a solvent sensitive emission in several polar and non-polar media, despite not being fluorescent in protic solvents. Fluorescence anisotropy measurements of the compound incorporated in liposomes revealed that this thienopyridine derivative can be carried in the hydrophobic region of the lipid membrane. Liposome formulations including this antitumor compound are nanometric in size, with a diameter lower than 130 nm and generally low polydispersity, being promising for future drug delivery developments. The interaction of the compound with bovine serum albumin (BSA) and the multidrug resistance protein MDR1 was monitored by FRET, the compound acting as energy acceptor. It was observed a lower interaction with MDR1 protein than with the native form of BSA, which is an important result regarding applications of this antitumoral drug. Introduction Thienopyridine derivatives have attracted much attention as they exhibit important biological activity, namely as antitumoral agents, 1-5 nonreceptor Src kinase inhibitors, 6 receptor tyrosine kinase 7 and vascular endothelial growth factor (VEGFR-2) inhibitors, 8-9 the latter related to angiogenesis and metastasis. The methyl 3-amino-6-(benzo[d]thiazol-2-ylamino)- thieno[3,2-b]pyridine-2-carboxylate (1) (Figure 1) was previously synthetized by us by palladium-catalysed C-N Buchwald-Hartwig coupling. 2 Compound 1 showed to be the most potent antitumor compound among a series of synthesized methyl 3-amino-6-[(hetero)arylamino]thieno[3,2-b]pyridine-2- carboxylates, presenting very low growth inhibitory concentration values (GI 50 ) between 3.5 and 6.9 μM, when tested in vitro (with a 24h incubation time and using DMSO as solvent) against several human tumour cell lines, MCF-7 (breast adenocarcinoma), A375-C5 (melanoma) and NCI-H460 (non-small cell lung cancer). 2 Due to this potent antitumor activity of compound 1, which is the most active of a series of analogues, 2 its behaviour in liposomes and when interacting with both plasma and membrane proteins is of particular interest, considering future developments using this thienopyridine derivative as an antitumoral drug. N S NH 2 CO 2 CH 3 N H N S Compound 1 Fig. 1 Structure of the methyl 3-amino-6-(benzo[d]thiazol-2- ylamino)thieno[3,2-b]pyridine-2-carboxylate (1). The multidrug resistance is one of the major gaps of many forms of chemotherapy, which can be associated with an overexpression of drug pumps. These pumps are usually membrane proteins and the study of drug interaction with such proteins may be useful for reversing drug resistance in cancer. 10 Multidrug transport proteins can actively remove toxic compounds and are of fundamental importance in the resistance to both antibiotics and anticancer drugs. 11,12 The interaction with human and bovine serum albumins (HSA and BSA, respectively) has also been extensively studied