2188
J. Med. Chem. 2009, 52, 2188–2191
Caspase-3 cleaves huntingtin at positions 513 and 530, while
Synthesis and in Vitro Evaluation of
Sulfonamide Isatin Michael Acceptors as
Small Molecule Inhibitors of Caspase-6
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.
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.
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.
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
(CNSa).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.
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.
10.1021/jm900135r CCC: $40.75
2009 American Chemical Society
Published on Web 03/30/2009
Chu, Wenhua
