Synthesis of indirubin-N 0 -glycosides and their anti-proliferative activity against human cancer cell lines Stefanie Libnow, a Karen Methling, b Martin Hein, a Dirk Michalik, c Manuela Harms, b Kristian Wende, b Anke Flemming, a Martin Ko ¨ ckerling, a Helmut Reinke, a Patrick J. Bednarski, b Michael Lalk b and Peter Langer a,c, * a Institut fu ¨ r Chemie der Universita ¨ t Rostock, Albert-Einstein-Straße 3a, D-18059 Rostock, Germany b Institut fu ¨ r Pharmazie, Ernst-Moritz-Arndt-Universita ¨ t Greifswald, Friedrich-Ludwig-Jahn-Straße 17, D-17487 Greifswald, Germany c Leibniz Institut fu ¨ r Katalyse e.V. an der Universita ¨ t Rostock, Albert-Einstein-Straße 29a, D-18059 Rostock, Germany Received 2 January 2008; revised 27 March 2008; accepted 1 April 2008 Available online 6 April 2008 Abstract—The first indirubin-N 0 -glycosides were prepared based on reactions of isatin-N 0 -glycosides with indoxyls. The products show a significant anti-proliferative activity against various human cancer cell lines. Good results were observed for an indiru- bin-N 0 -mannoside which was shown to have medium to high anti-proliferative activity against all investigated cell lines. The highest activities and selectivities against the MCF-7 breast cancer cell line were observed for indirubin-N 0 -rhamnosides. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction In recent years, there has been a dramatic renewal of the interest in indirubin—the red isomer of the well-known dye indigo—due to the discovery of its great pharmaco- logical potential. 1 The pharmacological activity of indiru- bin has been known for a long time. In fact, it is the active ingredient of the traditional Chinese medicinal recipe Danggui Longhui Wan for the treatment of myelocytic leu- kemia. 2 Recently, Meijer and coworkers showed that indirubin derivatives selectively inhibit cyclin-dependent kinases (CDKs), which represent important components of cell cycles taking place in many human tumors, and thus have a great potential for the treatment of cancer. 3,4 The anti-proliferative effect of indirubin on human cancer cells is based on the inhibition of genes or proteins which regulate cell cycle progression. 5 The inhibition of GSK-3b and CDK5 by indirubin derivatives, important for treat- ment of Alzheimer’s disease, has also been studied. 6,7 Indirubin-3 0 -monoxime was shown to influence p27 Kip1 transcription and to inhibit C-Jun NH2-terminal kinase, which plays an important role in neuronal apoptosis. 8–10 In addition, it was reported that indirubin-3 0 -monoxime can selectively stop centrosome duplication in tumor cells without affecting normal cells. 4 The anti-proliferative activity of substituted indirubin derivatives against vari- ous cancer cell lines and CDK2 inhibitory activityhave re- cently been studied. 11 Some years ago, Laatsch et al. reported the isolation of N-glycosides of indigo. 12 In con- trast to pharmacologically inactive indigo, the indigo-N- glycosides (akashines) show a significant antiproliferative activity against various human cancer cell lines. Owing to the strong activity of the akashines, we set up a program directed towards the synthesis of N-glycosides of indigo (blue sugars) 13 and of indirubin (red sugars). 14 Herein, we report full details of the synthesis of indirubin-N 0 -gly- cosides. With respect to our preliminary communication in this field, 14 we studied the scope of our synthetic strat- egy and disclose, for the first time, the anti-proliferative activity of various indirubin-N 0 -glycosides on human cancer cells in vitro, which is considerably higher than the activity of the corresponding non-glycosylated indirubins. 2. Results and discussion 2.1. Chemistry Indirubins are available by reaction of a methanol solu- tion of indoxyl acetate with isatines under oxygen-free 0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2008.04.003 Keywords: Anti-proliferative activity; Carbohydrates; N-heterocycles; Indirubin. * Corresponding author. Tel.: +49 381 498 6410; fax: +49 381 498 6411; e-mail: peter.langer@uni-rostock.de Available online at www.sciencedirect.com Bioorganic & Medicinal Chemistry 16 (2008) 5570–5583