Synthesis and in Vitro Evaluation of Sulfonamide Isatin Michael Acceptors as Small Molecule Inhibitors of Caspase-6 Wenhua Chu, Justin Rothfuss, Yunxiang Chu, Dong Zhou, and Robert H. Mach* Department of Radiology, Washington UniVersity School of Medicine, 510 S. Kingshighway BouleVard, St. Louis, Missouri 63110 ReceiVed February 3, 2009 Abstract: A key step in the onset of Huntington’s disease is the caspase-6 mediated cleavage of the protein huntingtin into toxic fragments. Therefore, the inhibition of caspase-6 has been identified as a target for therapeutic drug development for the treatment of this disease. In this study, a series of isatin sulfonamide Michael acceptors having a high nanomolar potency for inhibiting caspase-6 and increased selectivity for caspase-6 versus caspase-3 inhibition is reported. Apoptosis, the process by which a cell undergoes “pro- grammed cell death,” is thought to play a significant role in neurodegenerative diseases of the central nervous system (CNS a ). 1,2 Although apoptosis is crucial for normal tissue homeostasis, it may lead directly to the onset of Alzheimer’s disease and other neurological disorders if abnormal cell death occurs. 3 Apoptosis describes a coordinated sequence of mor- phological events that result in the activation of a cell’s inherent suicide program, which ultimately leads to its systematic destruction. Most importantly, evidence shows that the activation of a family of cysteine proteases, known as caspases (cysteinyl aspartate-specific proteases), is closely related to the apoptotic sequence of cell destruction. In other words, a caspase cascade sits at a critical point in the apoptotic process by receiving the signal for the initiation of the cell death process. Once the signal is received, caspases then trigger a variety of functional protein cleavages that result in the systematic disassembly of the cell. 4 There are two different classes of caspases involved in apoptosis, initiator and effector caspases, which are defined by their roles in apoptosis. Initiator caspases (caspase-2, -8, -9, and -10) usually trigger activation of caspase cascades, which in turn activate the execution phase of apoptosis. These initiator caspases later activate effector caspases (caspase-3, -6, -7) to cleave key functional proteins. 5,6 An excess of newly cleaved protein fragments often leads to the death of the cell. Activation of caspase-3 and caspase-6 has been identified as a critical component of apoptosis in neurons, especially in Alzheimer’s disease (AD) and Huntington’s disease (HD). 7-12 Gervais et al. reported that caspase-3 is the predominant caspase involved in the amyloid- precursor protein (APP) cleavage, consistent with its marked elevation in dying neurons of AD brains and colocalization of its APP cleavage product with amyloid- in senile plaques. 13 HD is a progressive neurodegenerative disorder caused by polyglutamine expansion in the N-terminus of the protein huntingtin, which is an important caspase substrate. Caspase-3 cleaves huntingtin at positions 513 and 530, while caspase-6 cleaves the protein at position 586. Production of toxic fragments through the cleavage of huntingtin by caspases is a key event in the development of the HD. 14,15 Graham et al. reported that mice expressing mutant huntingtin, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration; these caspase-6-resistant mutant huntingtin mice are protected against neurotoxicity induced by multiple stressors including N-methyl-D-aspartic acid, quinolinic acid, and staurosporine. Therefore, specifically preventing proteolysis at the capase-6 consensus sequence at amino acid 586 of mutant huntingtin can prevent the development of behavioral, motor, and neuropatho- logical features in murine models of HD. 16 Furthermore, although increased caspase-6 activity may correlate with aging in the absence of AD, it is always associated with clinical and pathological features of confirmed AD cases. As a result of the potential role of caspases and apoptosis in neurodegenerative diseases, specific caspase inhibitors have gained ample attention from pharmaceutical and biotechnological sectors as a potential target for drug discovery. Caspase inhibitors can bind in reversible, irreversible, or bimodal manners, depending on the reactivity of the electro- phile. 17,18 Although several caspase-3 inhibitors have been reported, only a few caspase-6 inhibitors have been developed. Current inhibitors of caspase-6 are mostly synthesized from peptides, 19 such as Ac-Val-Glu-Ile-Asp-CHO (1), and no small molecule caspase-6 inhibitors have been reported. Although peptide inhibitors may decrease caspase-6 activity in vitro, they do not efficiently cross the blood-brain barrier (BBB) and enter the brain. Therefore, they do not function well when tested in vivo, since amino acids and peptides usually require transport mechanisms to enter the brain. The lack of CNS penetration is a major challenge for targeting caspases in the brain for the treatment of neurodegenerative disease, and the development of nonpeptidic inhibitors capable of crossing the BBB by passive diffusion may overcome this limitation. Isatin sulfonamide analogues have been reported as selec- tive non-peptide reversible inhibitors of caspase-3, and the selectivity for caspase-3 is determined by the presence of the L-phenoxymethylpyrrolidine (Figure 1) or L-phenoxym- ethylazetidine ring. 20-24 We recently reported that the isatin sulfonamide analogues having a Michael acceptor (isatin Michael acceptor or IMA) have nanomolar potency for inhibiting the executioner caspases, caspase-3, and caspase-7 (e.g., 3, Figure 1). It is interesting to note that all the IMA analogues have an increased inhibition potency of roughly 10-fold for caspase-6 when compared to their complementary isatin analogues. 25 In the strategic development of nonpep- tidic caspase-6 inhibitors, replacing the L-phenoxymethylpyr- rolidine ring in 3 with other nitrogen heterocycles may reduce their selectivity for caspase-3 and increase the selectivity for caspase-6 in IMA analogues. Here we report a new series of isatin derivatives containing a Michael acceptor as selective caspase-6 inhibitors. The syntheses of sulfonamide isatin and its IMA analogs are shown in Scheme 1. Piperidine was coupled with 5-chlorosul- fonylisatin, 4, in tetrahydrofuran using triethylamine as an acid scavenger to afford the sulfonamide intermediate, 5. The isatin nitrogen of 5 was alkylated by treatment of 5 with sodium hydride in dimethylsulfone at 0 °C followed by addition of an * To whom correspondence should be addressed. Phone: 314-362-8538. Fax: 314-362-8555. E-mail: rhmach@mir.wustl.edu. a Abbreviations: AD, Alzheimer’s disease; HD, Huntington’s disease; APP, amyloid- precursor protein; BBB, blood-brain barrier; CNS, central nervous system; IMA, isatin Michael acceptor. J. Med. Chem. 2009, 52, 2188–2191 2188 10.1021/jm900135r CCC: $40.75  2009 American Chemical Society Published on Web 03/30/2009