Separation of Cdc25 dual speci®city phosphatase inhibition and DNA cleaving activities in a focused library of analogs of the antitumor antibiotic Dnacin Peter Wipf, a,p Corey R. Hopkins, a Eleanor O. Phillips b and John S. Lazo b,p a Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA b Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA Received 4 April 2002; accepted 1 June 2002 Abstract ÐBiological evaluation of 96 analogs and synthetic intermediates of the naphthyridinomycin-type antitumor antibiotic Dnacin led to the identi®cation of several low-micromolar inhibitors of dual speci®city phosphatases, speci®cally Cdc25A 1 , Cdc25B 2 , and VHR, as well as the tyrosine phosphatase PTP1B. While the parent Dnacins are potent DNA cleavage agents, most of the analog structures, even those that retained signi®cant phosphatase inhibitory activities, did not lead to plasmid DNA cleavage. Thus, the DNA-targeting and the phosphatase- inhibitory activities of Dnacins can be assigned to different pharmacophores. q 2002 Elsevier Science Ltd. All rights reserved. 1. Introduction Dual speci®city phosphatases were initially discovered in viral strains and represent an exciting new ®eld of research for the design of mechanism-based anticancer agents. 1 Both phosphoserine, phosphothreonine, and phosphotyrosine protein substrates are cleaved by DSPases. Mechanistically, they appear to be quite closely related to PTPases and are inhibited by vanadate but not by the standard PSTPase inhibitors. 2,3 In humans, a family of related cyclin- dependent kinases (CDKs) that regulate progression through each phase of the cell division cycle are activated by Cdc25 phosphatases that remove inhibitory phosphate from tyrosine and threonine residues. 4 There is a strong link between overexpression of Cdc25 DSPases and onco- genic transformation, particularly in human breast cancer. 5 In addition to synthetic compounds, 1 several natural products, including coscinosulfate, 6 nocardione, 7 and dysidiolide, 8 have been found to inhibit Cdc25 dual speci®city phosphatases at micromolar levels (Fig. 1). The ®rst natural products with documented Cdc25 inhibitory effects were Dnacin A 1 and B 1 , two naphthyridinomycin- type antitumor antibiotics isolated from actinomycete, strain No. C-14482 (N-1001) by researchers from Takeda, Ltd. (Fig. 2). 9 Cdc25B DSPase activity was inhibited non- competitively with median inhibitory concentration (IC 50 ) values of 141 and 64 mM, respectively. 10 These alkaloids showed potent bactericidal activity against repair-de®cient E. coli strains, such as recA, recB, and polA strains. In addition, DNA-cleaving activity in the presence of reducing agents was detected, and this activity was suppressed by scavengers for oxygen free radicals and an iron-speci®c chelator, desferrioxamine E. Therefore, the primary cellular target of Dnacin B 1 appeared to be DNA through the genera- tion of superoxide radicals. 11,12 Natural products continue to be major sources for the development of new drugs. Recent analyses of the number and sources of anticancer and antiinfective agents indicate that over 60% of the approved drugs and pre-NDA candi- dates are of natural origin. 13 While in standard discovery combinatorial libraries the compound-to-hit ratio is shifting from roughly 100,000:1 to 400,000 new compounds to 1 hit, estimates for natural product compound-to-hit ratios vary from 2000:1 to 11,000 extracts for a new drug. 14 Natural products therefore appear to have a clear evolutionary advantage in drug discovery, and in recent years, an expan- sion of the structural diversity pool by preparation of libraries of natural products or natural product-like molecules has become a major focus of combinatorial chemistry. 15 2. Results and discussion As part of our synthetic studies toward Dnacins and naphthyridinomycin-type alkaloids, 16,17 we have prepared and assayed a focused library of analogs which include intermediates of our synthetic approach toward these natural products. Our goal was to investigate if distinct structural Tetrahedron 58 (2002) 6367±6372 Pergamon TETRAHEDRON 0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S0040-4020(02)00636-1 Keywords: Dnacin; dual speci®city phosphatase inhibitors; Cdc25; VHR; tyrosine phosphatase; PTP1B; DNA cleavage; natural product library; SAR. p Corresponding authors. Tel.: 11-412-624-8606; fax: 11-412-624-0787; e-mail: pwipf@pitt.edu