Parallel synthesis and biological evaluation of 5,6,7,8-tetrahydrobenzothieno[2,3-d]pyrimidin-4(3H)-one cytotoxic agents selective for p21-deficient cells Lee D. Jennings, a, * Scott L. Kincaid, a Yanong D. Wang, b Girija Krishnamurthy, c Carl F. Beyer, d John P. McGinnis, d Miriam Miranda, d Carolyn M. Discafani d and Sridhar K. Rabindran d a Department of Exploratory Chemistry, Wyeth Research, Pearl River, NY 10965, USA b Department of Medicinal Chemistry, Wyeth Research, Pearl River, NY 10965, USA c Department of Biophysics and Enzymology, Wyeth Research, Pearl River, NY 10965, USA d Department of Oncology Research, Wyeth Research, Pearl River, NY 10965, USA Received 21 June 2005; revised 18 July 2005; accepted 25 July 2005 Available online 6 September 2005 Abstract—A novel series of inhibitors of cancer cell proliferation, selective against p21 cell cycle checkpoint-disrupted cells vs. cells with intact p21 checkpoint, were identified by high-throughput screening. Optimization of both ends of the lead molecule to improve potency, using parallel synthesis and iterative design, is described. The 2-(1,4-dibenzodioxane)-substituted derivative 14 was iden- tified as a highly selective and potent agent displaying an IC 50 of 91 nM in the p21-deficient cell line. Ó 2005 Elsevier Ltd. All rights reserved. Cell cycle checkpoints are signal transduction pathways, which ensure that each step in the cell division cycle, has been successfully completed before the onset of the next phase. 1 Loss of checkpoint control is a hallmark of tu- mor cells, as it is thought to increase the mutation rate, allowing a more rapid progression of cells to the tumor- igenic state. 1,2 However, inactivation of these check- points results in aberrant responses to cellular damage. This failure of checkpoint responses in malignant cells can be exploited in cancer drug discovery. Identification of compounds that selectively kill checkpoint-deficient cells can be expected to preferentially target tumor cells, while sparing normal cells. 3,4 The p53 tumor suppressor gene is a major regulator of the G1/S-phase checkpoint and one of the most com- monly mutated genes in human cancer. 5,6 p21, a down- stream effector of p53, inhibits the cyclin-dependent kinases and arrests cell cycle progression in response to environmental insults causing DNA damage 7 or micro- tubule perturbation. 8 Disruption of this checkpoint by deletion of the p21 protein leads to failure of the cell to arrest, leading to endoreduplication, and finally, to apoptosis. 9,10 p21-deficient cells show increased sensitiv- ity, compared to isogenic p21-proficient parental cells, to a variety of clinically used antineoplastic drugs, validat- ing the role of checkpoints in chemosensitivity. We have prepared p21-deficient cells from human colon carcinoma cell line HCT116 by targeted deletion of the p21 gene. 9 HCT116, the parental cell lines from which the p21À/À cells were derived, is one of the few colon cancer cell lines with an apparently intact p21 check- point. We anticipate that a search for compounds that preferentially kill the p21-deficient cells, compared to the p21-proficient cells, will enable us to identify com- pounds that are not simply toxic, but have selectivity to- ward cells defective in checkpoint control. Targeting these cells should improve the likelihood of obtaining lead molecules that preferentially inhibit the growth of tumor cells rather than normal cells, a requirement for an anticancer agent. Furthermore, since loss of p21 checkpoint is a hallmark of cancer (primarily through 0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2005.07.072 Keywords: Inhibitors of cell cycle progression; Parallel synthesis; Tubulin binding. * Corresponding author. Tel.: +1 845 602 3618; fax: +1 845 602 3045; e-mail: jenninl@wyeth.com Bioorganic & Medicinal Chemistry Letters 15 (2005) 4731–4735