PAPER www.rsc.org/pps | Photochemical & Photobiological Sciences Photoreactivity of indirubin derivatives David Olivier, a Marie-Anne Poincelot, a Samuel Douillard, a Carine Lefevre, a Julien Moureau, a Yoan Ferandin, b Karima Bettayeb, b Zhijian Xiao, c Prokopios Magiatis, d Leandros Skaltsounis, d Laurent Meijer b and Thierry Patrice* a Received 24th July 2007, Accepted 2nd January 2008 First published as an Advance Article on the web 22nd January 2008 DOI: 10.1039/b711261k Twenty-nine analogs of indirubin, an isomer of indigo, have been synthesized to optimize its promising kinase inhibitory scaffold. These compounds being also pigmented, have been tested for their photoreactivity. Absorption maxima were between 485 nm and 560 nm. Addition of fetal calf serum induced fluorescence and time dependent absorption modifications. Appropriate illumination induced Reactive Oxygen Species (ROS) production for nineteen compounds out of twenty-nine. The relationship between fluorescence and ROS production is discussed. Six compounds showed an important toxicity on F98 cells, a murine glioma cell line. Three of these were found to be also phototoxic, as four other non-toxic compounds. All but onze phototoxic compounds were detected as ROS producers by in vitro tests. Photoreactivity assessment is important to anticipate adverse reactions for compounds that might be clinically developed. The experimental assay was found to be the only way to evaluate the photoreactivity of this family of compounds since no predictive criteria on structures could be found. Combining the vascular tumor growth inhibition induced by kinase inhibitors with the massive local blood flow arrest following photodynamic treatment may be an efficient anti-cancer strategy. These data could orientate further syntheses of either non-photoreactive compounds or compounds displaying both kinase inhibitory activity and strong phototoxicity. Introduction Indirubins are isomers of indigo. These bis-indoles can be gener- ated from various plants, molluscs (the Murex species producing the famous Tyrean purple), mammalian urine, and various bacterial sources. Indigo and indirubin are heavily pigmented compounds derived from the dimerization of isatin and indoxyl, two precursors found either free or conjugated to carbohydrates in the indigo-producing plants which have constituted the main source of blue dye for thousands of years. 1 A book reviewing most cultural, chemical, biological and pharmacological aspects of indirubins has been recently published. 2 Indirubin is the active ingredient of a traditional Chinese medicinal recipe (Danggui Longhui Wan) used to treat various diseases including chronic myelocytic leukemia. 3,4 Indirubin and its analogs (collectively referred to as indirubins) were also among some of the early cyclin-dependent kinases (CDKs) inhibitors to be discovered. 3,5 CDKs exert essential functions in regulating the cell division cycle and are involved in cancers. 6 They play essential functions in the nervous system and are involved in various neurodegenerative diseases such as Alzheimer’s, 7,8 Parkinson’s 9 and Nieman–Pick’s diseases, 10 ischemia 11 or traumatic brain injury. 12 They are also a HGRL, Laboratoire de Photobiologie des Cancers, Laser Department, 44093, Nantes, France. E-mail: patrice.laserdpt@wanadoo.fr; Fax: +33 2- 40-16–59-35; Tel: +33 2-40-16–56-75 or +33 2-40-16-53-37 b CNRS, Station Biologique, Cell Cycle Group, BP 74, Roscoff cedex, France c Institute of Hematology, 288 Nanjing Road, Tianjin, 300020, China d University of Athens, Laboratory of Pharmacognosy, Department of Pharmacy and Natural Products Chemistry, Faculty of Pharmacy, Panepis- timiopolis Zografou, Athens, 15771, Greece involved in inflammation 13 and insulin secretion by pancreatic cells. 14 Apart from CDKs, indirubins were found to target glycogen synthase kinase-3 (GSK-3), glycogen phosphorylase b and the aryl hydrocarbon receptor which mediates the effects of many xenobiotics such as dioxin. 15,16 Indirubins are thus extensively studied for their pharmacological properties as a new promising family of anticancer and neuroprotective agents. 2,17 As these substances are likely to absorb light between 400 nm and 600 nm, it was of interest to assess their potential photore- activity after light exposure. The purpose of this work was to study fluorescence, Reactive Oxygen Species (ROS) production by 29 indirubins and their phototoxicity in cultured cells with the aim of firstly, evaluating the potential phototoxic risk with these heavily pigmented compounds and, secondly, evaluating the potential efficacy of indirubins as photosensitizers. Materials and methods In this study, we have investigated 29 indirubins for their pho- todynamic properties. Their structures are shown in Fig. 1. We performed three kinds of assays for photodynamic application: absorption and fluorescence measurements in the presence or absence of Fetal Calf Serum (FCS), ROS production and finally cell toxicity and phototoxicity. All these assays are routinely used in our laboratory and have been described elsewhere. 18 We chose the F98 murine glioma cell line as it is both chemo- and radio- resistant and likely to give solid tumors when grafted in rat brains. 19 Using a model of same cellular origin allows a better comparison between in vitro and in vivo results. The effect of FCS was analyzed as by experience we knew that it sometimes significantly modifies 328 | Photochem. Photobiol. Sci., 2008, 7, 328–336 This journal is © The Royal Society of Chemistry and Owner Societies 2008