Isatin sulfonamide analogs containing a Michael addition acceptor: A new class of caspase 3/7 inhibitors

Article abstract of DOI:10.1021/jm070506t

A series of isatin sulfonamide analogs having a Michael acceptor were prepared and their potencies for inhibiting caspase-1, -3, -6, -7, and -8 were evaluated. These compounds have nanomolar potency for inhibiting the executioner caspases, caspase-3 and c

Full text of DOI:10.1021/jm070506t

J. Med. Chem. 2007, 50, 3751-3755
3751
Isatin Sulfonamide Analogs Containing a Michael Addition Acceptor: A New Class of Caspase
3/7 Inhibitors
Wenhua Chu,† Justin Rothfuss,† Andre´ d’Avignon,‡ Chenbo Zeng,† Dong Zhou,† Richard S. Hotchkiss,§ and Robert H. Mach*,†
DiVision of Radiological Sciences, Washington UniVersity School of Medicine, 510 South Kingshighway BouleVard, St. Louis, Missouri 63110,
Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130, Department of Anesthesiology, Washington UniVersity School of
Medicine, 510 South Kingshighway BouleVard, St. Louis, Missouri 63110
ReceiVed May 1, 2007
A series of isatin sulfonamide analogs having a Michael acceptor were prepared and their potencies for
inhibiting caspase-1, -3, -6, -7, and -8 were evaluated. These compounds have nanomolar potency for inhibiting
the executioner caspases, caspase-3 and caspase-7, and have a low potency for inhibiting caspase-1, caspase-
6, and caspase-8. The inhibition mechanism was investigated through NMR studies of the reaction between
11d and benzylmercaptan as a model for Cys-285 in the active site of caspase-3.
Introduction
Apoptosis, or programmed cell death, is a conserved process
that is mediated by the activation of a series of cysteine aspartyl-
specific proteases called caspases. Apoptosis plays an important
role in a wide variety of normal cellular processes including
fetal development, tissue homeostasis, and maintenance of the
immune system.1 However, abnormal apoptosis has been
observed in a large number of pathological conditions, including
ischemia-reperfusion injury (stroke and myocardial infarction),
cardiomyopathy, neurodegeneration (Alzheimer’s disease, Par-
kinson’s disease, Huntington’s disease, and ALS), sepsis, type
I diabetes, and allograft rejection.2-6 Therefore, the development
of drugs that can halt the process of apoptosis has been an active
area of research in the pharmaceutical industry.2,7 In addition,
Figure 1.
the benefits of many drugs, especially antitumor drugs, can be
attributed to their activation of the apoptotic process.8-13
Therefore, the development of a noninvasive imaging procedure
that can study the process of apoptosis in a variety of disease
states and monitor the ability of a drug to either induce or halt
apoptosis would be of tremendous value to the research and
clinical community.
a diverse range of caspase inhibitors have been developed over
the years.15 There are two different classes of caspase inhibitors,
reversible and irreversible, depending on the interaction of the
“warhead” group with Cys-285 in the active site of the enzyme.
Although many irreversible inhibitors with a Michael acceptor
group have been reported as cysteine protease inhibitors, only
a few compounds with a Michael acceptor group have been
reported as caspase-3 inhibitors. Ekici et al. have reported that
aza-peptide Michael acceptors with an aza-Asp residue at P1,
for example, Cbz-Asp-Glu-Val-AAsp-CHdCH-COOBzl, 1
(Figure 1), are potent inhibitors of caspase-3.16,17
Recently, a number of isatin-based inhibitors of caspase-3
and caspase-7 have been reported.18,19 One compound, (S)-(+)-
5-[1-(2-methoxymethyl-pyrrolidine)sulfonyl]isatin, 2 (Figure 1),
has been shown to reduce tissue damage in an isolated rabbit
heart model of ischemic injury.20 Our research interest is
centered around the development of nonpeptide small molecule
inhibitors of executioner caspases for use as therapeutic drugs
and radiotracer imaging of apoptosis. We have reported that
the replacement of the 2-methoxymethyl group in 2 with a
phenoxymethyl or 2-pyridin-3-yl-oxymethyl moiety, the re-
placement of the pyrrolidine ring with an azetidine ring, and
the substitution of the isatin nitrogen atom with an alkyl group,
resulted in a dramatic improvement in potency for inhibiting
caspase-3 activity.21 One isatin sulfonamide analog, 1-[4-(2-
fluoroethoxy)-benzyl]-5-(2-phenoxymethyl-pyrrolidine-1-sulfo-
nyl)-1H-indole-2,3-dione, 3 (WC-II-89, Figure 1), has been
labeled with 18F and is currently being examined as a potential
radiotracer for imaging caspase-3 activation in tissues undergo-
The caspase family of cysteine proteases has two different
classes of caspases involved in apoptosis, the initiator caspases
and the executioner caspases.14 The initiator caspases, which
include caspase-2, -8, -9, and -10, are located at the top of the
signaling cascade; their primary function is to activate the
executioner caspases, caspase-3, -6, and -7. The executioner
caspases are responsible for the physiological (e.g., cleavage
of the DNA repair enzyme poly(ADP-ribose) polymerase-1,
nuclear laminins, and cytoskeleton proteins) and morphological
changes (DNA strand breaks, nuclear membrane damage, and
membrane blebbing) that occur in apoptosis.2 A third class of
caspases, caspases-1, -4, -5, and -13, are involved in cytokine
maturation and are not believed to play an active role in
apoptosis.
Because of their roles in inflammation and apoptosis, the
caspase proteases have received enormous research interest, and
* To whom correspondence should be addressed. Tel.: 314-362-8538.
Fax: 314-362-0039. E-mail: rhmach@mir.wustl.edu.
† Division of Radiological Sciences, Washington University School of
Medicine.
‡ Department of Chemistry, Washington University.
§ Department of Anesthesiology, Washington University School of
Medicine.
10.1021/jm070506t CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/23/2007

3752 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Brief Articles
Scheme 1a
a Reagents: (a) TFA, CH2Cl2; (b) 5-sulfonylisatin chloride, Et3N; (c) NaH, DMF; (d) R-C6H4-CH2Br; (e) malononitrile, MeOH.
ing apoptosis.22 On the basis of our result showing isatin
Table 1
derivatives as potent inhibitors of caspase-3/7, here we report a
new series of isatin derivatives containing a Michael acceptor
as caspase-3/7 inhibitors. We refer to these new compounds as
isatin Michael acceptor (IMAa) caspase-3/7 inhibitors.
IC50 (nM)
casp-6
#
casp-1
casp-3
casp-7
casp-8 Log P
9d >15 000
10b >15 000
10c >15 000
9.85 ( 0.9 8900 ( 424 34.8 ( 1.4
>50 000 4.82
>50 000 2.25
>50 000 3.76
3.9 ( 0.6
3.6 ( 0.5
9550 ( 354 11.7 ( 1.0
5025 ( 318 6.6 ( 0.1
Results and Discussion
8
1830 ( 128 272 ( 24.7 407 ( 15
11a 2825 ( 248 119.3 ( 4.0 698 ( 94
11b 6220 ( 1250 27.8 ( 2.5 918 ( 151 51.7 ( 6.2
11c 2300 ( 250 31.8 ( 6.2 744 ( 48
11d 5700 ( 850 20.1 ( 1.3 840 ( 125 92.2 ( 11.8 >50 000 4.28
1585 ( 163 >50 000 1.07
785 ( 276
>50 000 1.71
>50 000 3.50
The synthesis of 5-(2-phenoxymethyl-pyrrolidine-1-sulfonyl)-
isatin and its IMA analogs are shown in Scheme 1. The isatin
analogs 6, 9a-c,21 and 9d were reacted with malononitrile in
methanol to give the IMA analogs, 8 and 11a-d, respectively.
The 5-(2-pyridin-3-yl-oxymethyl)pyrrolidine-1-sulfonyl)isatin
analogs 10a, 10c, and 10d were prepared by using the same
sequence of reactions described in the synthesis of 9d (Scheme
1). For the compound 10b, the isatin nitrogen of 7 was first
alkylated with (4-bromomethyl-phenoxy)-tert-butyl-diphenyl-
silane, then the protecting group tert-butyl-diphenyl-silane was
removed with n-Bu4NF in THF to afford 10b. The IMA analogs
of the 5-(2-pyridin-3-yl-oxymethyl)pyrrolidine-1-sulfonyl)isatin,
12a-d, were prepared with the same methods of 11a-d.
The IC50 values from the enzyme assays are summarized in
Table 1. The results show that the phenoxymethyl and pyridin-
3-yl-oxymethyl isatin analogs, 9d, 10b, and 10c, are potent and
selective inhibitors for caspase-3/7 relative to caspases-1, -6,
and -8. The IMA analogs of phenoxymethyl isatin compounds
8 and 11a, where the isatin nitrogen of the indol ring is not
alkylated or instead possesses a methyl group, have low potency
for caspase-3 and -7 inhibition; these IC50 values are 272 and
119.3 nM for caspase-3 and 1585 and 785 nM for caspase-7.
When the isatin nitrogen of the indol ring was alkylated with
126.0 ( 19.3 >50 000 2.77
12a 3250 ( 450 7.6 ( 1.1
12b 2720 ( 580 7.8 ( 1.9
823 ( 86
650 ( 22
515 ( 77
610 ( 113 29.6 ( 1.4
3700 ( 390 23.5 ( 3.5
32.8 ( 4.9
28.3 ( 5.4
26.3 ( 0.8
>50 000 2.45
>50 000 1.77
>50 000 3.22
>50 000 2.36
>50 000 4.19
12c 3400 ( 0
5.1 ( 0.7
12d 3900 ( 530 7.8 ( 1.5
3a >50 000
9.7 ( 1.3
a Reference 22.
an aromatic group, the potency of IMA analogs 11b, 11c, and
11d improved drastically for caspase-3/7, with IC50 values of
27.8, 31.8, and 20.1 nM for caspase-3 and 51.7, 126.0, and 92.2
nM for caspase-7, while retaining their high selectivity. Also,
all of these compounds have less activity for inhibition of
caspase-1 (IC50: 2300-6200 nM), caspase-6 (IC50 744-926
nM), and caspase-8 (IC50 > 50 000 nM) upon addition of the
aromatic group. Similarly, the IMA analogs of pyridin-3-yl-
oxymethyl isatin, 12a, 12b, 12c, and 11d, are potent and
selective inhibitors for caspase-3 (IC50: 7.6, 7.8, 5.1, and 7.8
nM) and caspase-7 (IC50: 32.8, 28.6, 26.3, and 15.1 nM) and
show weak inhibition of caspase-1 (IC50: 2700-3200 nM),
caspase-6 (IC50: 515-770 nM), and caspase-8 (IC50: >50 000
nM). The IMA analogs of pyridin-3-yl-oxymethyl isatin also
display improved potency for inhibiting caspases-3/7 than the
corresponding IMA analogs of phenoxymethyl isatin (Table 1,
a Abbreviation: IMA, isatin Michael acceptor.

Brief Articles
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3753
cycloheximide-treated rats relative to the untreated control.22
At this moment, it is not clear which is better, reversible or
irreversible caspase-3 inhibitors, for imaging apoptosis in vivo.
Nevertheless, it is our opinion that IMA-based radiotracers are
capable of producing similar if not better imaging results
compared to their isatin-based counterparts. Furthermore, the
Log P value of the IMA analogs are lower than the correspond-
ing isatin analogs value (e.g., 9d to 11d, Log P 4.82 to 4.28,
10b to 12b, 2.25 to 1.77, and 10c to 12c, 3.76 to 3.22,
respectively (Table 1)). This lower Log P value of the IMA
caspase-3 inhibitor increases the drug’s ability to penetrate the
cell in vivo and label the target.
Scheme 2
Conclusions
We have completed the synthesis and in vitro evaluation of
a series of IMA analogs having a high potency for inhibiting
caspase-3 and caspase-7 and a low potency for inhibiting
caspase-1, caspase-6, and caspase-8. The inhibition mechanism
was further investigated through the reaction of 11d and
benzylmercaptan. This reaction serves as a model for the
inhibition mechanism of the IMA inhibitors. The results of the
current study reveal a new class of nonpeptide-based caspase
inhibitors possessing a Michael acceptor, which may be useful
in assessing the role of executioner caspase inhibitors, minimiz-
ing tissue damage in diseases characterized by unregulated
apoptosis, and in vivo imaging of apoptosis.
11b, 11c, and 11d compare with 12a, 12b, and 12c, respec-
tively). It is interesting to note that all the IMA analogs have
an increased potency of roughly 10-fold for caspase-6 when
compared to their complementary isatin analogs (see Table 1).
The inhibition mechanism was further investigated by using
11d and its reaction with benzylmercaptan as a model. There
are two possible Michael addition products (13a or 13b)
produced by attack of the thiol nucleophile of benzylmercaptan
to 11d. The products depend on the position of attack of the
thiol group of benzylmercaptan on the carbon-carbon double
bond of 11d (Scheme 2). Initially, we hoped to purify the
Michael addition product in order to obtain a crystal structure
by X-ray diffraction. Therefore, benzylmercaptan was reacted
with 11d in CH2Cl2, and a white solid was obtained following
evaporation of the CH2Cl2 and excess benzylmercaptan in vacuo.
However, when the white solid was recrystallized from ethyl
acetate, a purple solid was produced and NMR structural
analysis revealed it was the starting material, 11d. This result
shows that the Michael addition product is easily reversible and
leads to the formation of the starting material. Hence, 11d is a
reversible Michael acceptor inhibitor. This result is consistent
with our inhibition studies of human caspase-3 with IMA
inhibitors. Human caspase-3 activity is inhibited when incubated
with caspase-3 and the IMA inhibitor, yet caspase-3 activity
can be recovered when the IMA inhibitor is removed by gel
filtration and washed with water (Supporting Information, Figure
2). In an effort to better understand the chemical structure of
the Michael addition product, a series of detailed NMR studies
were carried out. The proton and carbon chemical shifts for the
Michael addition product were assigned through two-dimen-
sional correlation spectroscopy (COSY, HMQC, and HMBC,
see Supporting Information, Figures 2-6). The results show
that the structure of the Michael addition product is 13b instead
of 13a, thereby demonstrating that the thiol group of benzyl-
mercaptan prefers to attack the indol ring carbon versus the
exocyclic methylene group of 11d.
Experimental Section
1-(4-Bromo-benzyl)-5-(2-Phenoxymethyl-pyrrolidine-1-sulfo-
nyl)-1H-indole-2,3-dione (9d). NaH (60%, 10 mg, 0.25 mmol) was
added to a solution of 619,21 (97 mg, 0.25 mmol) in DMF (3 mL)
at 0 °C. The mixture was stirred 15 min at 0 °C and then
4-bromobenzyl bromide (125 mg, 0.5 mmol) was added. The
mixture was stirred 1 h at room temperature, ethyl acetate (50 mL)
was added, washed with water (30 mL) and saturated NaCl (30
mL), and dried over Na2SO4. After evaporation of the solvent, the
crude product was purified with hexane-CH2Cl2-ether (1:1:1) to
afford 108 mg (78%) of 9d as a yellow solid, mp 112.1-113.4 °C.
1H NMR (300 MHz, CDCl3) δ 8.02 (s, 1H), 7.96 (d, J ) 8.1 Hz,
1H), 7.50 (d, J ) 8.4 Hz, 2H), 7.20 (m, 5H), 6.92 (t, J ) 7.8 Hz,
1H), 6.80 (d, J ) 8.1 Hz, 2H), 4.87 (s, 2H), 4.15 (m, 1H), 3.93 (m,
2H), 3.49 (m, 1H), 3.23 (m, 1H), 2.02 (m, 2H), 1.79 (m, 2H).
1-(4-Hydroxy-benzyl)-5-[2-(pyridin-3-yl-oxymethyl)-pyrroli-
dine-1-sulfonyl]-1H-indole-2,3-dione (10b). 1-[4-(tert-Butyl-
diphenyl-silanyloxy)-benzyl]-5-[2-(pyridin-3-yloxymethyl)-pyrrolidine-
1-sulfonyl]-1H-indole-2,3-dione (150 mg, 0.2 mmol) and n-Bu4NF
(53 mg, 0.2 mmol) in THF (6 mL) and water (2 mL) was stirred
for 2 h, ethyl acetate (50 mL) was added, and the mixture was
washed with water (30 mL) and saturated NaCl (30 mL) and dried
over Na2SO4. The crude product was purified with ether-ethyl
acetate (1:1) to afford 65 mg (66%) of 10b as a yellow solid, mp
1
126.7-128.8 °C. H NMR (300 MHz, CDCl3) δ 8.21 (m, 2H),
8.01 (s, 1H), 7.95 (d, J ) 8.1 Hz, 1H), 7.26 (m, 2H), 7.19 (d, J )
8.4 Hz, 2H), 6.90 (d, J ) 8.7 Hz, 1H), 6.84 (d, J ) 8.4 Hz, 2H),
4.87 (s, 2H), 4.19 (m, 1H), 3.95 (m, 2H), 3.48 (m, 2H), 3.19 (m,
1H), 2.00 (m, 2H), 1.79 (m, 2H).
1-(4-Bromo-benzyl)-5-[2-(pyridin-3-yl-oxymethyl)-pyrrolidine-
1-sulfonyl]-1H-indole-2,3-dione (10c). Compound 10c was pre-
pared according to the same procedure for compound 9d, except
using 7 and 4-bromobenzyl bromide, purified with CH2Cl2-ethyl
acetate (1:1) to afford 53 mg (38%) of 10c as a yellow solid, mp
The current study is focused on the development of caspase-3
inhibitors as radiotracers that are specific for imaging apoptosis
using PET, as well as drugs for therapy of the numerous diseases
associated with unregulated apoptosis. We have reported that
biodistribution studies using [18F]3, a reversible isatin-based
caspase-3 inhibitor, revealed higher uptake in liver and spleen
of cycloheximide-treated rats, an animal model of apoptosis,
relative to control animals.22 MicroPET imaging studies also
showed a high uptake of the radiotracer in the liver of
1
92.1-93.3 °C. H NMR (300 MHz, CDCl3) δ 8.24 (m, 2H), 8.04
(s, 1H), 7.98 (d, J ) 8.1 Hz, 1H), 7.51 (d, J ) 8.1 Hz, 2H), 7.23
(m, 4H), 6.86 (d, J ) 8.4 Hz, 1H), 4.90 (s, 2H), 4.23 (m, 1H), 3.97
(m, 2H), 3.50 (m, 1H), 3.17 (m, 1H), 2.02 (m, 2H), 1.78 (m, 2H).
2-[2-Oxo-5-(2-phenoxymethyl-pyrrolidine-1-sulfonyl)-1,2-di-
hydro-indol-3-yl-idene]-malononitrile (8). A solution of 6 (97 mg,
0.25 mmol) and malononitrile (18 mg, 0.27 mmol) in methanol (4

3754 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Brief Articles
mL) was heated to reflux for 1 h, then cooled to room temperature.
The solid was filtered out and dried in vacuum to afford 93 mg
(86%) of 8 as a red solid, mp 245.7-248.4 °C. 1H NMR (300 MHz,
DMSO) δ 11.66 (s, 1H), 8.23 (s, 1H), 8.02 (d, J ) 8.7 Hz, 1H),
7.25 (t, J ) 8.7 Hz, 2H), 7.09 (d, J ) 8.7 Hz, 1H), 6.90 (m, 3H),
4.05 (m, 1H), 3.92 (m, 2H), 3.39 (m, 1H), 3.15 (m, 1H), 1.90 (m,
2H), 1.72 (m, 2H).
2-{1-(4-Methoxy-benzyl)-2-oxo-5-[2-(pyridin-3-yloxymethyl)-
pyrrolidine-1-sulfonyl]-1,2-dihydro-indol-3-yl-idene}-malononi-
trile (12d). Compound 12d was prepared according to the same
procedure for compound 8, except using 10d,21 to afford 91 mg
(82%) of 12d as a purple solid, mp 132.4 °C (dec). 1H NMR (300
MHz, CDCl3) δ 8.47 (s, 1H), 8.20 (m, 2H), 7.95 (dd, J ) 8.4 Hz,
J ) 1.8 Hz, 1H), 7.26 (d, J ) 8.7 Hz, 2H), 7.19 (m, 2H), 6.92 (d,
J ) 8.4 Hz, 1H), 6.89 (d, J ) 8.7 Hz, 2H), 4.88 (s, 2H), 4.21 (m,
1H), 4.02 (m, 2H), 3.80 (s, 3H), 3.50 (m, 1H), 3.29 (m, 1H), 2.05
(m, 2H), 1.86 (m, 2H).
2-[1-Methyl-2-oxo-5-(2-phenoxymethyl-pyrrolidine-1-sulfonyl)-
1,2-dihydro-indol-3-yl-idene]-malononitrile (11a). Compound 11a
was prepared according to the same procedure for compound 8,
except using 9a,19,21 to afford 39 mg (87%) of 11a as a red solid,
Acknowledgment. The authors would like to thank Ms.
Yunxuang Chu for her editorial assistance. This work was
supported by CA121952 and HL13851.
1
mp 217.5 °C (dec). H NMR (300 MHz, CDCl3) δ 8.47 (s, 1H),
8.06 (dd, J ) 8.6 Hz, J ) 1.8 Hz, 1H), 7.21 (t, J ) 7.8 Hz, 2H),
6.93 (t, J ) 7.5 Hz, 1H), 6.89 (d, J ) 8.7 Hz, 1H), 6.73 (d, J ) 7.8
Hz, 2H), 4.11 (m, 1H), 4.08 (m, 1H), 4.00 (m, 1H), 3.49 (m, 2H),
3.26 (s, 3H), 2.11-1.88 (m, 4H).
Supporting Information Available: Experimental details cor-
responding to the synthesis of the compounds described in this
paper, elemental analysis (C, H, N) results, enzyme inhibition
assays, reversibility assay, and two-dimensional NMR study. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2-[1-Benzyl-2-oxo-5-(2-phenoxymethyl-pyrrolidine-1-sulfonyl)-
1,2-dihydroindol-3-yl-idene]-malononitrile (11b). Compound 11b
was prepared according to the same procedure for compound 8,
except using 9b,21 to afford 92 mg (88%) of 11b as a purple solid,
1
mp 196.6 °C (dec). H NMR (300 MHz, CDCl3) δ 8.43 (s, 1H),
7.93 (dd, J ) 8.6 Hz, J ) 1.8 Hz, 1H), 7.39-7.29 (m, 5H), 7.14
(t, J ) 7.2 Hz, 2H), 6.89 (t, J ) 7.2 Hz, 1H), 6.83 (d, J ) 8.7 Hz,
1H), 6.68 (d, J ) 7.8 Hz, 2H), 4.90 (s, 2H), 4.05 (m, 2H), 3.97 (m,
1H), 3.45 (m, 2H), 2.07-1.85 (m, 4H).
2-[1-(Hydroxy-benzyl-2-oxo-5-(2-phenoxymethyl-pyrrolidine-
1-sulfonyl)-1,2-dihydro-indol-3-yl-idene]-malononitrile (11c). Com-
pound 11c was prepared according to the same procedure for
compound 8, except using 9c,21 to afford 68 mg (84%) of 11c as
a purple solid, mp 174.9 °C (dec). 1H NMR (300 MHz, DMSO) δ
9.51 (s, 1H), 8.30 (s, 1H), 8.10 (dd, J ) 8.6 Hz, J ) 1.8 Hz, 1H),
7.32-7.20 (m, 5H), 6.95-6.84 (m, 3H), 6.76 (d, J ) 8.7 Hz, 2H),
4.87 (s, 2H), 4.09 (m, 1H), 3.97 (m, 2H), 3.40 (m, 1H), 3.20 (m,
1H), 1.90 (m, 2H), 1.71 (m, 2H).
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1-sulfonyl)-1,2-dihydro-indol-3-yl-idene]-malononitrile (11d). Com-
pound 11d was prepared according to the same procedure for
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1
purple solid, mp 237.0 °C (dec). H NMR (300 MHz, CDCl3) δ
8.46 (s, 1H), 7.95 (d, J ) 8.1 Hz, 1H), 7.51 (d, J ) 8.4 Hz, 2H),
7.20-7.13 (m, 4H), 6.90 (t, J ) 7.5 Hz, 1H), 6.79 (d, J ) 8.4 Hz,
1H), 6.68 (d, J ) 7.8 Hz, 2H), 4.84 (m, 2H), 4.06 (m, 2H), 3.97
(m, 1H), 3.46 (m, 2H), 2.08-1.86 (m, 4H).
(7) O’Brien, T.; Lee, D. Prospects for Caspase inhibitors. Mini-ReV. Med.
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2-{1-Benzyl-2-oxo-5-[2-(pyridine-3-yloxymethyl)-pyrrolidine-
1-sulfonyl]-1,2-dihydro-indol-3-yl-idene}-malononitrile (12a).
Compound 12a was prepared according to the same procedure for
compound 8, except using 10a,21 to afford 59 mg (75%) of 12a as
a purple solid, mp 216.5 °C (dec). 1H NMR (300 MHz, CDCl3) δ
8.53 (s, 1H), 8.23 (m, 2H), 7.98 (dd, J ) 8.4 Hz, J ) 1.8 Hz, 1H),
7.42-7.35 (m, 5H), 7.20 (m, 2H), 6.91 (d, J ) 8.7 Hz, 1H), 4.98
(s, 2H), 4.23 (m, 1H), 4.05 (m, 2H), 3.55 (m, 1H), 3.33 (m, 1H),
2.08 (m, 2H), 1.90 (m, 2H).
2-{1-(4-Hydroxy-benzyl)-2-oxo-5-[2-(pyridine-3-yloxymethyl)-
pyrrolidine-1-sulfonyl]-1,2-dihydro-indol-3-yl-idene}-malononi-
trile (12b). Compound 12b was prepared according to the same
procedure for compound 8, except using 10b, to afford 46 mg (85%)
of 12b as a purple solid, mp 203.3 °C (dec). 1H NMR (300 MHz,
DMSO) δ 9.50 (s, 1H), 8.31 (s, 1H), 8.25 (d, J ) 2.7 Hz, 1H),
8.17 (dd, J ) 4.5 Hz, J ) 1.5 Hz, 1H), 8.10 (d, J ) 8.1 Hz, 1H),
7.34 (m, 3H), 7.24 (d, J ) 8.4 Hz, 2H), 6.76 (d, J ) 8.7 Hz, 2H),
4.88 (s, 2H), 4.16 (m, 1H), 4.08 (m, 1H), 3.96 (m, 1H), 3.41 (m,
1H), 3.18 (m, 1H), 1.91 (m, 2H), 1.71 (m, 2H).
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1
of 12c as a purple solid, mp 232.3 °C (dec). H NMR (300 MHz,
CDCl3) δ 8.50 (s, 1H), 8.21 (m, 1H), 8.16 (s, 1H), 7.96 (d, J ) 8.4
Hz, 1H), 7.51 (d, J ) 8.1 Hz, 2H), 7.20 (m, 4H), 6.84 (d, J ) 8.7
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