Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors G. M. Kamal B. Gunaherath a,  , Marilyn T. Marron a , E. M. Kithsiri Wijeratne a , Luke Whitesell b,⇑ , A. A. Leslie Gunatilaka a,⇑ a Southwest Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, AZ 85706, United States b Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, United States article info Article history: Received 18 April 2013 Revised 13 June 2013 Accepted 19 June 2013 Available online 27 June 2013 Keywords: Novobiocin analogues Heat shock protein 90 (HSP90) Synthesis Structure–activity relationship Biological activity abstract Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibi- tion of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of lucifer- ase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90- binding agents, but with greater potency than NB. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Heat shock protein 90 (HSP90) is an abundant molecular chap- erone that is essential for the post-translational folding and confor- mational maintenance of over 150 client proteins. 1 Many of these HSP90 client proteins are directly associated with the cancer phe- notype including the tumor suppressor p53, receptor-linked tyro- sine kinases such as HER2, steroid receptors, and a wide range of other mediators of intracellular signal transduction. As a conse- quence of constitutive activation of the heat-shock response, levels of HSP90 are frequently elevated in highly malignant tumors. 2 Be- cause HSP90 stabilizes many of the oncoproteins required for can- cer survival and growth, inhibition of HSP90 by compounds such as geldanamycin (an N-terminus binding agent) can impair the growth and survival of diverse tumor types. In contrast to the many HSP90 N-terminal inhibitors undergoing clinical evaluation, 3 C-terminal inhibitors have only recently attracted attention as po- tential anticancer agents. Novobiocin (NB, 1a) is a well-known nat- ural product inhibitor of bacterial DNA gyrase. Consisting of a coumarin core, an acyl substituent and a noviose sugar moiety, it has been demonstrated to bind the C-terminus of HSP90 at a puta- tive ATP-binding site. 4 However, a very high concentration (700 lM) of NB is required to impair HSP90 chaperone function and induce degradation of client proteins such as HER2 in human cancer cells. 5 As part of our continuing search for natural prod- uct-based HSP90 inhibitors, 6 we prepared twenty seven NB ana- logues from NB (1a) and coumermycin (CA, 2) and evaluated their activity using a suite of biological assays selected to monitor different aspects of HSP90 function. Herein we report synthetic methods and structure-activity relationships (SARs) for these com- pounds. The biological activities reported include global effects on proliferation and cell cycle progression as well as more specific 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.06.042 Abbreviations: DAPI, 4 0 ,6-diamidino-2-phenylindole; DCC, dicyclohexylcarbodi- imide; EDC, N-(3-dimethylaminopropyl)-N 0 -ethyl-carbodiimide; FITC, fluorescein isothiocyanate; NBT/BCIP, nitro-blue tetrazolium chloride and 5-bromo-4-chloro- 3 0 -indolyphosphate p-toluidine salt; PBA, phosphate buffered saline with bovine albumin; PFA, formaldehyde in phosphate buffered saline with bovine albumin; PMSF, phenylmethylsulfonyl fluoride; 4-PP, 4-pyrrolidinopyridine; TNES, a buffer ⇑ Corresponding authors. Tel.: +1 520 621 9932; fax: +1 520 621 8378 (A.A.L.G.); tel.: +1 617 452 3542; fax: +1 617 258 7228 (L.W.). E-mail addresses: whitesell@wi.mit.edu (L. Whitesell), leslieg@cals.arizona.edu (A.A. Leslie Gunatilaka).   Present address: Department of Chemistry, The Open University of Sri Lanka, P.O. Box 21, Nugegoda, Sri Lanka. Bioorganic & Medicinal Chemistry 21 (2013) 5118–5129 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc