Unsaturated genistein disaccharide glycoside as a novel agent affecting microtubules Aleksandra Rusin a , Agnieszka Gogler a , Magdalena Głowala-Kosin ´ ska a , Daria Bochenek a,  , Aleksandra Gruca a , Grzegorz Grynkiewicz b , Jadwiga Zawisza c , Wiesław Szeja c , Zdzisław Krawczyk a,c, * a Department of Tumor Biology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch Gliwice, Wybrze _ ze AK 15, 44-100 Gliwice, Poland b Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland c Department of Organic, Bioorganic Chemistry and Biotechnology, Silesian Technical University, Krzywoustego 4, 44-100 Gliwice, Poland article info Article history: Received 7 May 2009 Revised 14 July 2009 Accepted 16 July 2009 Available online 22 July 2009 Keywords: Genistein glycoside Microtubule interfering drugs abstract Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a lead compound in drug design. While multiple molecular targets for genistein have been identified, so far neither for this isoflavonoid nor for its natural or synthetic derivatives disruption of microtubules and mitotic spindles has been reported. Here we describe such properties of the synthetic glycosidic deriva- tive of genistein significantly more cytotoxic than genistein, 7-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyr- anosyl)-(1?4)-(6-O-acetyl-hex-2-ene-a-D-erythro-pyranosyl)genistein, shortly named G21. We found that G21 causes significant mitotic delay, frequent appearance of multipolar spindles, and alteration of the interphase microtubule array. Ó 2009 Published by Elsevier Ltd. Epidemiological and experimental studies place genistein among chemopreventive agents and a complementary drugs in a treatment of cardiovascular diseases, postmenopausal syndromes, and cancer. 1,2 At lower concentrations, essentially not exceeding 20 lM, this isoflavonoid may act as a selective estrogen receptor modulator (SERM), with higher affinity to ERb than ERa, while at higher doses genistein reveals antiproliferative activity. Although multiple molecular targets have been identified or suggested, the major mechanism by which genistein impedes growth of cancer- ous cells are thought to be: the inhibition of the activity of tyrosine kinases, topoisomerase II, and transcription factor NF-jB, as well as affecting activities which control cell cycle. 3 These observations inspired several groups to synthesize vari- ous derivatives of genistein with the intention to either improve biochemical and pharmacokinetic characteristics of the parent drug or to obtain compounds containing essential elements of the parent substance but having novel properties and/or affecting novel molecular targets. 4–10 So far none of the novel derivatives revealed better inhibitory action on tyrosine kinase activity than genistein 11,12 while some approaches targeting genistein to tyro- sine kinases through conjugation of the isoflavone with EGF or spe- cific antibodies gave promising results. 13,14 Much effort has been put into the design and synthesis of new genistein derivatives with improved anticancer activity that could affect proliferation of cancerous cells by mechanisms other than inhibition of tyrosine kinases and chemical glycosylation seems a viable option for such purpose. 15 The genistein derivatives, glyco- sides of 2,3-unsaturated sugars, were found to inhibit proliferation of multiple cancer cell lines at a concentration significantly (5–10 times) lower than a parent compound, what makes them attractive objects, both for pharmacological studies and as lead compounds for further modifications. Of multiple novel derivatives, the most promising obtained so far is: 7-O-(2,3,4,6-tetra-O-acetyl-b-D-galac- topyranosyl)-(1?4)-(6-O-acetyl-hex-2-ene-a-D-erythropyranosyl) genistein, shortly named G21. 16,17 In the present report we describe the results of our investiga- tion which demonstrate, that the synthetic genistein glycoside, G21 has the potential to affect microtubule dynamics, leading to malformation of mitotic spindles. To our knowledge, the G21 is the first derivative of genistein exhibiting features of a mitotic spindle poison. We also describe the simplified method of regio- and stereoselective synthesis of this compound. This novel genistein derivative was earlier obtained in a multi- step procedure based on palladium catalyzed exchange of unsatu- rated anomeric carbonate esters, 16,18 which resulted in the mixture of 7-, 4 0 - regio- and a,b-stereoisomers and meticulous chromatog- raphy was required in order to isolate the desired compound. Here, we describe a novel approach to synthesis of G21 which resulted in high regio- and stereoselectivity of glycosylation of genistein agly- con, in keeping with observation that usually the reactivity of the phenolic hydroxyl group at 7-OH is higher than that of other posi- tions in flavonoids. 19–21 Both the anomeric leaving group and the method of its activation have been changed. As a part of the 0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.bmcl.2009.07.089 * Corresponding author. Tel.: +48 322789757; fax: +48 32 278 9840. E-mail addresses: krawczyk@io.gliwice.pl, arusin@io.gliwice.pl (Z. Krawczyk).   Present address: ETH Zürich, Institut f. Biochemie, Schafmattstr. 18, 8093 Zürich, Switzerland. Bioorganic & Medicinal Chemistry Letters 19 (2009) 4939–4943 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl