Structure–activity relationship study of novel necroptosis inhibitors Xin Teng, a Alexei Degterev, b Prakash Jagtap, a Xuechao Xing, a Sungwoon Choi, a Re ´gine Denu, a Junying Yuan b and Gregory D. Cuny a, * a Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, Brigham & WomenÕs Hospital and Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA b Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA Received 12 July 2005; accepted 29 July 2005 Available online 8 September 2005 Abstract—Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-a. 5-(1H-Indol-3-ylmethyl)-2- thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration. Ó 2005 Elsevier Ltd. All rights reserved. Cells die in several morphologically distinct ways. 1 One of these processes, called apoptosis, is characterized by a number of conserved and highly regulated steps includ- ing concomitant nucleus and cytoplasm condensation, DNA degradation, membrane blebbing, and caspase- mediated cleavage of various cellular proteins. Apopto- sis culminates in the formation of apoptotic bodies that are phagocytosed by adjacent cells including macro- phages in the periphery and microglial cells in the cen- tral nervous system (CNS). This efficient process prevents the accumulation of extracellular debris and consequently does not lead to an inflammatory response. Apoptosis is a genetically regulated process that is necessary both during development and for main- taining an organismÕs homeostasis. 2 However, under certain pathological conditions this process, which would normally be suppressed, is activated, leading to cell death and dysfunction. Many key cellular targets in this biochemical cascade have been identified and some of them serve as potential targets for therapeutic intervention, including caspases. 3 A second morphologically distinct way that cells die, called necrosis, is characterized by cell membrane and organelle disruption, cell swelling, mitochondria impair- ment, followed by cell lyses, which is accompanied by a host inflammatory response. 4 Unlike apoptosis, the underlying biochemical events in necrosis are not well understood. However, necrosis is known to play a prominent role in many pathological conditions, 5a including ischemia (i.e., stroke 5b and myocardial infarc- tion 5c ), trauma, and possibly some forms of neurodegen- eration. 5d Recent studies also suggest that apoptosis inhibition in many cases does not completely block cell death, but rather results in a change from an apoptotic to regulated caspase-independent cell death mechanisms with morphological features resembling necrosis. 6 One of these mechanisms, called necroptosis, has recently been described. 7 Identifying and preparing low molecu- lar weight molecules capable of inhibiting necroptosis can assist in elucidating its role in the patho-physiology of diseases and can provide lead compounds (i.e., necrostatins) for therapeutic development. 1 N H N H N O S Me 0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2005.07.077 Keywords: Necrosis; Necroptosis; Caspase-independent cell death; Stroke; SAR; Ischemic brain injury; Indoles; Hydantoin; Central nervous system. * Corresponding author. Tel.: +1 617 768 8640; fax: + 1 617 768 8606; e-mail: gcuny@rics.bwh.harvard.edu Bioorganic & Medicinal Chemistry Letters 15 (2005) 5039–5044