N-benzylisatin sulfonamide analogues as potent caspase-3 inhibitors: Synthesis, in vitro activity, and molecular modeling studies

Article abstract of DOI:10.1021/jm0506625

A number of isatin sulfonamide analogues were prepared and their potencies for inhibiting caspase-1, -3, -6, -7, and -8 were evaluated in vitro. Several compounds displaying a nanomolar potency for inhibiting the executioner caspases, caspase-3 and caspas

Full text of DOI:10.1021/jm0506625

J. Med. Chem. 2005, 48, 7637-7647
7637
N-Benzylisatin Sulfonamide Analogues as Potent Caspase-3 Inhibitors:
Synthesis, in Vitro Activity, and Molecular Modeling Studies
Wenhua Chu,† Jun Zhang,† Chenbo Zeng, Justin Rothfuss, Zhude Tu, Yunxiang Chu, David E. Reichert,
Michael J. Welch, and Robert H. Mach*
Division of Radiological Sciences, Washington University School of Medicine, 510 South Kingshighway Boulevard,
St. Louis, Missouri 63110
Received July 13, 2005
A number of isatin sulfonamide analogues were prepared and their potencies for inhibiting
caspase-1, -3, -6, -7, and -8 were evaluated in vitro. Several compounds displaying a nanomolar
potency for inhibiting the executioner caspases, caspase-3 and caspase-7, were identified. These
compounds were also observed to have a low potency for inhibiting the initiator caspases,
caspase-1 and caspase-8, and caspase-6. Molecular modeling studies provided further insight
into the interaction of this class of compounds with activated caspase-3. The results of the
current study revealed a number of non-peptide-based caspase inhibitors that may be useful
in assessing the role of inhibiting the executioner caspases in minimizing tissue damage in
disease conditions characterized by unregulated apoptosis.
Introduction
Apoptosis, or programmed cell death, is a conserved
process that is mediated by the activation of a series of
cysteine aspartyl-specific proteases termed 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 ob-
served in large number of pathological conditions,
including ischemia-reperfusion injury (stroke and myo-
cardial infarction), cardiomyopathy, neurodegeneration
(Alzheimer’s Disease, Parkinson’s Disease, Huntington’s
Disease, ALS), sepsis, Type I diabetes, fulminant liver
disease, and allograft rejection.2,3 Therefore, the devel-
opment of drugs that can halt the process of apoptosis
has been an active area of research in the pharmaceuti-
Figure 1. Structure of isatin sulfonamide analogues reported
previously.
cal industry.2,4
There are two different classes of caspases involved
in apoptosis, the initiator caspases and the executioner
caspases.5 The initiator caspases, which include caspase-
6, -8, -9, and -10, are located at the top of the signaling
cascade; their primary function is to activate the
executioner caspases, caspase-2, -3, and -7. The execu-
tioner caspases are responsible for the physiological
(e.g., cleavage of the DNA repair enzyme PARP-1,
nuclear laminins, and cytoskeleton proteins) and mor-
phological changes (DNA strand breaks, nuclear mem-
brane damage, membrane blebbing) that occur in apo-
ptosis.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.
been shown to reduce the amount of cellular and tissue
damage in cell culture and animal models of disease.6,7
To date, most inhibitors of caspase-3 have been peptide-
based compounds that either reversibly or irreversibly
inhibit the catalytic activity of this enzyme.6-13 In vivo
studies have shown that the peptide-based inhibitors
of caspase-3, M-791 and M-826, reduce apoptosis and
tissue damage in animal models of sepsis and neonatal
hypoxic-ischemic brain injury.7,13
One of the potential problems of peptide-based caspase
inhibitors is their poor metabolic stability and poor cell
penetration,12 which has resulted in a search for non-
peptide-based inhibitors of caspase-3 and caspase-7.
Recently, a number of isatin-based inhibitors of caspase-3
and caspase-7 have been reported. One compound, (S)-
(+)-5-[1-(2-methoxymethyl-pyrrolidine)sulfonyl]isatin, 1
(Figure 1), has been shown to reduce tissue damage in
an isolated rabbit heart model of ischemic injury.14,15
Additional structure-activity relationship studies have
revealed that replacement of the 2-methoxymethyl
group with a phenoxymethyl moiety and the introduc-
tion of an alkyl group on the isatin nitrogen group
Although it was initially thought that apoptosis could
not be stopped once the process was initiated, recent
studies suggest that pharmacological intervention can
minimize cell death and tissue damage via these
pathways. For example, inhibitors of caspase-3 have
* To whom correspondence should be addressed. E-mail: rhmach@
mir.wustl.edu. Phone: (314) 362-8538. Fax: (314) 362-0039.
† These authors contributed equally to this work.
10.1021/jm0506625 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/05/2005

7638 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Chu et al.
Figure 2. Strategy used in the current structure-activity relationship study.
Scheme 1a
resulted in a dramatic improvement in potency for
inhibiting caspase-3 activity (2) (Figure 1).16 An ad-
ditional improvement in potency was also reported when
the pyrrolidine ring of 3 was replaced with an azetidine
ring to give compound 4.16 The goal of the current study
is to extend the structure-activity relationship study
of this class of compounds by incorporating the following
changes into lead compound 2 (Figure 2): (1) substitut-
ing the para position of the N-benzyl group in order to
determine if there are any substituent effects with
respect to caspase-3 inhibition potency; (2) replacing the
pyrrolidine ring with an azetidine ring; and, (3) replac-
ing the benzene ring of the phenoxymethyl moiety with
a pyridine ring. Docking studies were performed in
order to gain insight into structural features which
determine potency of this class of compounds for inhib-
iting caspase-3 activity.
Results
Chemistry. The synthesis of 5-(2-phenoxymethyl-
pyrrolidine-1-sulfonyl)isatin analogues is shown in
Scheme 1. The 5-chlorosulfonylisatin 6 was prepared by
reaction of 5-isatinsulfonic acid, sodium salt hydrate (5)
with phosphorus oxychloride in tetramethylene sulfone
at 60 °C for 3 h. The hydroxyl group of N-Boc-2-
pyrrolmethanol (7) was first tosylated with p-toluene-
sulfonyl chloride in pyridine to give compound 8,
followed by displacement of the tosylate group by
sodium phenoxide in DMF to afford N-Boc-2-(phe-
noxymethyl)pyrrolidine 9. The N-Boc group of 9 was
removed with TFA, and the secondary amine was
coupled with 6 in THF using triethylamine as an acid
scavenger to afford the 5-(2-phenoxymethyl-pyrrolidine-
sulfonyl)-1H-2,3-dione 10 in 84% yield. The isatin
nitrogen was alkylated by treatment of 10 with sodium
hydride in DMF at 0 °C followed by addition of various
alkyl halides to give compounds 2 and 11a-e,g-i.
Compound 11f was prepared by hydrolysis of 11e with
sodium hydroxide in aqueous methanol.
The synthesis of 5-(2-phenoxymethyl-azetidine-1-sul-
fonyl)isatin analogues is shown in Scheme 2. The
intermediate (S)-N-Boc-2-azetidinemethanol 14 was
prepared from (S)-2-azetidinecarboxylic acid 12 accord-
ing to the literature method.17 The hydroxy group of 14
was tosylated with p-toluenesulfonyl chloride in pyri-
dine to afford compound 15, which was converted to the
corresponding phenoxyl group as described above to give
16. Compound 16 was deprotected with TFA, and the
secondary amine was coupled with 6 using triethyl-
amine as the base to afford 17 in 63% yield. The
nitrogen of 17 was alkylated by the same procedure as
that of 10 to give compounds 18a-i. Similarly, the 5-(2-
a Reagents: (a) POCl3; (b) p-toluenesulfonyl chloride, pyridine;
(c) phenol, NaH, THF; (d) (1) TFA, CH2Cl2, (2) 6, triethylamine;
(e) NaH, DMF, R-CH2X (X ) Cl, Br, I).
pyridin-3-yl-oxymethyl)pyrrolidine-1-sulfonyl)isatin ana-
logues were prepared by using the same sequence of
reactions described in the synthesis of 11a-i to afford
compounds 21a-e (Scheme 3). The synthesis of the
4-pyridyl analogue 23 is also outlined in Scheme 3.

Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7639
Scheme 2a
Scheme 3a
a Reagents: (a) Di-tert-butyl dicarbonate; (b) BH3, THF; (c)
p-toluenesulfonyl chloride, pyridine; (d) phenol, NaH, THF; (e) (1)
TFA, (2) 6, triethylamine; (f) NaH, DMF, R-CH2X (X ) Cl, Br, I).
Enzyme Assays. Inhibition of recombinant human
caspase-3 and other caspases by the isatin analogues
was assessed using a fluorometric assay by measuring
the accumulation of a fluorogenic product, 7-amino-4-
methylcoumarin (7-AMC). All of the tested compounds
inhibited caspase-3 and caspase-7 in a concentration-
dependent manner with similar potency. The IC50
values from the enzyme assays are summarized in
Tables 1-3. Alkylation of the isatin nitrogen of 10 with
a benzyl group (i.e., 2) or substituted benzyl group (i.e.,
11c-e) resulted in a 10 to 20-fold increase in potency
for inhibiting caspase-3, and a 9 to 37-fold increase in
potency for inhibiting caspase-7. The isatin analogues
were also evaluated for their inhibitory activity against
a panel of three other caspases (caspases-1, -6, and -8).
As shown in Table 1, they demonstrated high selectivity
against caspase-3 and -7, with IC50 values at least 100-
fold higher versus caspases-1, -6, and -8.
The azetidine analogue 17 had a similar potency for
inhibiting caspase-3 as that of the corresponding pyr-
rolidine analogue 10. However, compound 17 was >2-
fold less potent for inhibiting caspase-7 relative to the
corresponding pyrrolidine analogue, 10. Substitution of
17 with either a benzyl (i.e., 18b), a substituted benzyl
(18c-f), or a pyridylmethyl group (18g-i) resulted in
a 10 to 50-fold increase in potency against caspase-3 and
a 10 to 80-fold increase in potency for inhibiting
caspase-7 relative to 17 (Table 2). Again, these com-
pounds exhibited at least 100-fold greater selectivity for
caspase-3 and -7 versus caspases-1, -6, and -8.
a Reagents: (a) 3-Hydroxypyridine, NaH, THF/DMF; (b) (1)
TFA, CH2Cl2, (2) 6, triethylamine; (c) NaH, DMF, R-CH2X (X )
Cl, Br, I); (d) 4-hydroxypyridine, NaH, THF/DMF.
methyl)pyrrolidine moiety with a pyridine ring (Table
3). All pyridine-containing analogues had a lower IC50
value for inhibiting caspase-3 than the corresponding
benzene-containing congeners (eg., 11a vs 21a, 11d vs
21d). Compound 21c was found to be the most potent
inhibitor of caspase-3, with IC50 of 3.9 nM. These
compounds demonstrated similar potency against cas-
pase-3 and 7, but at least 100 fold less potent versus
caspases-1, -6, and -8.
Kinetic studies were also conducted in order to
determine the mechanism of inhibition of caspase-3
activity by compound 21c. The kinetic pattern indicated
that 21c displays competitive inhibition versus Ac-
DEVD-AMC with a calculated Ki value of 4.4 nM (Figure
3). These data are consistent with previous studies
demonstrating that the isatin analogues bind to the
catalytic site of activated caspase-3.16
Modeling Studies. Becker et al.12 analyzed a series
of seven caspase-3 inhibitor complexes and found that
the tertiary and quaternary structures were remarkably
similar regardless of the inhibitors involved. The key
interactions found in these crystal structures, particu-
larly the covalent attachment to Cys285, hydrogen
bonds to Cys285 and Gly238, and polar interactions
with Arg179 and Gln283, were remarkably similar to
those found in the isatin-based inhibitor developed by
Lee et al.14 (PDB ID: 1GFW), shown in Figure 4. Lee
Interestingly, a higher caspase-3 potency was achieved
upon replacing the benzene ring of the 2-(phenoxy-

7640 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Chu et al.
Table 1. Inhibitor Selectivity of Pyrrolidine Isatin Analogues for Caspases-1, -3, -6, -7, and -8
IC50 (nM)
caspase-1
caspase-3
caspase-6
caspase-7
caspase-8
Log P
10
11a
2
>10000
>20000
>10000
>10000
>50000
>50000
>50000
>5000
240.0 ( 10.0
119.2 ( 17.0
12.2 ( 0.3
14.5 ( 1.6
12.1 ( 2.1
12.4 ( 2.1
12.0 ( 1.5
13.5 ( 2.4
10.3 ( 1.5
21.3 ( 3.2
9.1 ( 1.8
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
540.0 ( 56.6
310.0 ( 14.1
28.0 ( 0.7
21.8 ( 3.5
23.0 ( 1.4
41.0 ( 1.4
34.8 ( 0.4
44.0 ( 0.1
14.5 ( 0.9
58.0 ( 2.8
22.2 ( 4.0
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
2.23
2.27
4.05
3.96
4.1
4.54
3.39
3.31
2.67
2.67
2.67
11b
11c
11d
11e
11f
11g
11h
11i
>50000
>50000
>50000
standard
Ac-YVAD-CHO
Ac-DEVD-CHO
Ac-VEID-CHO
Ac-IETD-CHO
8.1 ( 2.1
-
-
-
-
-
-
-
3.8 ( 0.8
-
8.5 ( 1.0
-
-
-
60.5 ( 7.6
-
-
-
-
4.7 ( 0.9
Table 2. Inhibitor Selectivity of the Azetidine Isatin Analogues 17, 18a-i for Caspases-1, -3, -6, -7, and -8
IC50 (nM)
caspase-1
caspase-3
caspase-6
caspase-7
caspase-8
Log P
17
>10000
>10000
>10000
>50000
>50000
>10000
>10000
>50000
>50000
>10000
286.7 ( 24.7
91.7 ( 7.6
9.7 ( 1.6
8.4 ( 1.2
11.3 ( 1.2
8.8 ( 1.4
9.4 ( 0.3
10.9 ( 1.4
29.2 ( 5.2
5.8 ( 1.0
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
1350.0 ( 141.4
362.5 ( 3.5
29.5 ( 4.9
23.2 ( 3.0
26.7 ( 7.2
21.0 ( 5.6
26.0 ( 5.2
17.0 ( 3.0
135.0 ( 7.1
22.7 ( 3.1
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
>50000
1.66
1.71
3.49
3.4
3.97
3.54
3.54
2.11
2.11
2.11
18a
18b
18c
18d
18e
18f
18g
18h
18i
Table 3. Selectivity Profile of the Pyridine Analogues within the Caspase Family
IC50 (nM)
caspase-1
caspase-3
caspase-6
caspase-7
caspase-8
Log P
20
>5000
>10000
>10000
>10000
>50000
>10000
>5000
58.3 ( 7.6
23.3 ( 3.1
5.2 ( 1.6
3.9 ( 0.9
4.4 ( 1.4
8.4 ( 2.0
20.4 ( 1.7
>5000
>5000
>5000
>5000
>5000
>5000
>5000
214.9 ( 49.5
94.9 ( 21.6
14.1 ( 3.4
15.1 ( 1.2
23.3 ( 0.7
15.1 ( 0.1
142.3 ( 22.6
>50000
>50000
>50000
>50000
>50000
>50000
>50000
1.17
1.21
2.99
2.91
3.48
3.04
1.04
21a
21b
21c
21d
21e
23
performed using the program Gold. The binding site of
caspase-3 for these inhibitors is quite wide and shallow
with extensive solvent accessibility which tends to lead
to a large number of accessible poses in docking studies.
By utilizing covalent constraints, Gold was able to
successfully dock all twenty-eight compounds. The
compounds all bound in a fashion similar to that of the
isatin MSI found in the crystal structure 1GFW. Figure
5 shows the superimposition of the highest scoring poses
of the pyrrolidines 2, 11a-i, and the azetidines 18a-i.
Discussion
It was previously reported that isatin sulfonamides
are potent and selective non-peptide-based inhibitors of
the executioner caspases, caspase-3 and -7.16 The key
structural properties that resulted in high potency for
inhibiting caspase-3 and -7 identified in this initial
structure-activity relationship study were the presence
of an N-benzyl moiety off the isatin nitrogen atom and
an (S)-2-phenoxymethyl moiety in the 2-position of the
pyrrolidine ring (Figure 1). The goal of the current study
was to extend the structure-activity relationships of
Figure 3. Competitive inhibition of caspase-3 by 21c. The
concentration of 21c was 0 (O), 5 (b), 10 (0), and 20 nM (9).
et al.14 found that the isatin-based inhibitors developed
their selectivity through hydrophobic contacts between
the pyrrolidine ring and the residues of the S2 pocket.
To understand how the compounds described in this
work interact with caspase-3, docking studies were

Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7641
from the current study was the absence of a substituent
effect in the aromatic ring of the N-benzyl moiety of
compound 2. The results outlined in Table 1 indicate
that either substitution of the para position of 2 or
replacement of the benzene ring with a pyridine ring
results in little change in potency for inhibiting caspase-3
and caspase-7. These results are consistent with the
earlier observations regarding the substitution of the
isatin nitrogen with hydrophobic substituents.16
A second, and somewhat unexpected, observation was
the similar potency between the pyrrolidine analogues
11b-i and the azetidine analogues 18b-i, given the
difference in potency for inhibiting caspase-3 by com-
pound 3 and compound 4 (Figure 1). However, the
results were explained by molecular modeling studies
which revealed a high degree of overlap in binding of
the azetidine and pyrrolidine analogues to activated
caspase-3 (Figure 5). An analysis of the docked poses
for 11a-i and 18a-i found little difference between the
two classes in terms of the relative orientation of the
phenoxymethyl group and the isatin core. For each
molecule, two planes were defined: one containing the
phenyl carbons of the phenoxymethyl group, and the
second containing the isatin heavy atoms. For the
pyrrolidines, these two planes intersected with a mean
angle of 43.4 ( 1.7°, while the azetidines had a mean
intersection angle of 55.6 ( 3.7°. These differences are
too small to have a major effect on the binding mode of
the molecules.
Figure 4. Binding interactions of the isatin-based inhibitor
MSI found in the crystal structure 1GFW. Hydrogen bonds
are shown as green lines; hydrophobic contacts are shown as
radial hemispheres. Binding interactions were calculated with
the program LigPlot.20
this class of compounds by using the strategy outlined
in Figure 2. The first conclusion that can be reached
The predominant noncovalent interactions appear to
be a π-π interaction between the phenyl of the isatin
Figure 5. Superimposition of the top poses of pyrrolidines 2, 11a-i and azetidines 18a-i in the active site of caspase-3.

7642 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Chu et al.
Figure 6. Compound 21c (space filling) superimposed on the contact statistics surface of the binding site of caspase-3 (1RHJ).
Hydrophobic preferences are shown in green, and the hydrophilic preferences are shown in red.
core and Tyr338, and a stabilization of the oxyanion
through interactions with the amide hydrogen of Ser339
or the amide hydrogen of Cys285 (also found in the
crystal structure 1GFW). A final key interaction appears
to be a π-π interaction between the aromatic group in
Region III (Figure 2) and Phe381.
analogues.18,19 For example, compound 2 has a calcu-
lated log P value of 4.05 whereas the corresponding
pyridine analogue, 21b, has a calculated log P value of
2.99. Therefore, the pyridine analogues may have a
higher potency for inhibiting activated caspase-3 in
which there is an intact cell membrane. Studies evalu-
ating the potency of these compounds in cell culture and
animal models of apoptosis are currently ongoing in our
laboratory.
In conclusion, we have completed the synthesis and
in vitro evaluation of a series of isatin analogues having
a high potency for inhibiting the executioner caspases,
caspase-3, and caspase-7. The results of this study have
extended the structure-activity relationships of this
class of compounds and provide further insight into the
development of non-peptide-based inhibitors of caspase-3
and caspase-7. The compounds described above may be
useful probes for determining the effectiveness of in-
hibiting caspase-3 and caspase-7 for minimizing tissue
damage in pathological conditions characterized by
unregulated apoptosis.
Another unexpected observation was the high potency
of the pyridine analogues 21b-e relative to their phenyl
congeners, 2 and 11b-d. These data suggest a possible
hydrophilic interaction between the phenoxymethyl
moiety and the S3 binding domain of caspase-3 (Figure
6). As already mentioned, all compounds were found to
involve a π-π interaction with Phe381. The pyridine
analogues 21b-e also appear perfectly oriented to
involve a hydrogen bond between the pyridine nitrogen
and the hydroxyl group of Ser381. In compound 21c this
distance is 2.21 Å, although this distance is slightly long
it falls within the observed range of this interaction
found in the Cambridge database. Comparison of the
distance between the same ring position for the phenyl
congener 11b to the Ser381 hydroxyl was 2.92 Å.
Compound 23, with the pyridine attached at the four
position instead of at the 3 position, showed a distance
of 2.60 Å. While a single hydrogen bond is unlikely to
be the sole cause of the differences in inhibitory poten-
cies, the ability to hydrogen bond with Ser381 is
consistent with the assay data.
Experimental Section
All reactions were carried out under an inert nitrogen
atmosphere with dry solvents using anhydrous conditions
unless otherwise stated. Reagents and grade solvents were
used without further purification. Flash column chromatog-
raphy was conducted using Scientific Adsorbents, Inc. silica
gel, 60a, “40 Micron Flash” (32-63 µm). Melting points were
determined using MEL-TEMP 3.0 apparatus and uncorrected.
1H NMR spectra were recorded at 300 MHz on a Varian
Substitution of the pyridine ring for benzene ring in
the phenoxymethyl moiety also resulted in a dramatic
reduction in the overall lipophilicity of the isatin

Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7643
Mercury-VX spectrometer. All chemical shift values are re-
ported in ppm (δ). Elemental analyses (C, H, N) were deter-
mined by Atlantic Microlab, Inc.
1H), 8.01 (s, 1H), 7.25 (t, J ) 8.4 Hz, 2H), 6.92 (m, 3H), 6.81
(d, J ) 7.8 Hz, 2H), 4.15 (dd, J ) 9.0 Hz, J ) 2.7 Hz, 1H), 4.00
(m, 1H), 3.92 (m, 1H), 3.51 (m, 1H), 3.30 (m, 1H), 3.26 (s, 3H),
2.04 (m, 2H), 1.81 (m, 2H). Anal. Calcd for C20H20N2O5S: C,
59.99, H, 5.03, N, 7.00. Found: C, 59.80, H, 5.03, N, 6.91.
(S)-1-Benzyl-5-(2-phenoxymethyl-pyrrolidine-1-sulfo-
nyl)-1H-indole-2,3-dione (2) was prepared according to the
same procedure for compound 11a, except using benzyl
bromide, and purified with hexanes-ether (1:2) to afford 152
mg (64%) of 2 as a yellow solid, mp 97.2-99.1 °C. 1H NMR
(300 MHz, CDCl3) δ 8.01 (d, J ) 1.5 Hz, 1H), 7.94 (dd, J ) 8.4
Hz, J ) 1.8 Hz, 1H), 7.36 (m, 5H), 7.22 (m, 2H), 6.95-6.79
(m, 4H), 4.92 (s, 2H), 4.15 (dd, J ) 8.85 Hz, J ) 2.4 Hz, 1H),
3.97-3.87 (m, 2H), 3.49 (m, 1H), 3.23 (m, 1H), 2.01 (m, 2H),
1.78 (m, 2H). Anal. Calcd for C26H24N2O5S: C, 65.53, H, 5.08,
N, 5.88. Found: C, 65.27, H, 5.32, N, 5.58.
2,3-Dioxo-2,3-dihydro-1H-indole-5-sulfonyl Chloride
(6).16 Phosphorus oxychloride (13.2 mL, 141.6 mmol) was
added to a solution of 5-isatinsulfonic acid (5), sodium salt
hydrate (8.0 g, 30.0 mmol) in tetramethylene sulfone (40 mL).
The mixture was heated to 60 °C for 3 h, then cooled to 0 °C.
The reaction mixture was poured into 150 g of ice. The solid
was filtered out and washed with cold water, then the solid
was dissolved in ethyl acetate (100 mL), washed with water
(50 mL × 2) and saturated NaCl (50 mL), and dried over Na2-
SO4. The ethyl acetate was evaporated in reduced pressure to
afford 6.12 g (83%) of 6 as a pale yellow solid, mp 188.2-190.1
1
°C. H NMR (300 MHz, DMSO) δ 11.1 (s, 1H), 7.82 (dd, J )
8.4 Hz, J ) 1.8 Hz, 1H), 7.60 (s, 1H), 6.89 (d, J ) 8.1 Hz, 1H).
(S)-1-(4-Methoxybenzyl)-5-(2-phenoxymethyl-pyrroli-
dine-1-sulfonyl)-1H-indole-2,3-dione (11b) was prepared
according to the same procedure for compound 11a, except
using 4-methoxybenzyl chloride, and purified with hexanes-
ether (1:3) to afford 175 mg (69%) of 11b as a yellow solid, mp
126.7-128.8 °C. 1H NMR (300 MHz, CDCl3) δ 8.00 (s, 1H),
7.95 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.28-7.21 (m, 4H),
6.96-6.80 (m, 6H), 4.86 (s, 2H), 4.18-4.11 (m, 1H), 3.97-3.88
(m, 2H), 3.80 (s, 3H), 3.50 (m, 1H), 3.23 (m, 1H), 2.02 (m, 2H),
1.78 (m, 2H). Anal. Calcd for C27H26N2O6S: C, 64.02, H, 5.17,
N, 5.53. Found: C, 64.76, H, 5.24, N, 5.06.
(S)-1-(4-Fluorobenzyl)-5-(2-phenoxymethyl-pyrrolidine-
1-sulfonyl)-1H-indole-2,3-dione (11c) was prepared accord-
ing to the same procedure for compound 11a, except using
4-fluorobenzyl bromide, and purified with hexanes-ether (1:
2) to afford 196 mg (79%) of 11c as an orange solid, mp 74.5-
75.4 °C. 1H NMR (300 MHz, CDCl3) δ 7.99 (s, 1H), 7.95 (dd, J
) 8.25 Hz, J ) 2.1 Hz, 1H), 7.34-7.19 (m, 4H), 7.06 (t, J )
8.7 Hz, 2H), 6.92 (t, J ) 7.2 Hz, 1H), 6.87-6.79 (m, 3H), 4.89
(s, 2H), 4.13 (m, 1H), 3.93 (m, 2H), 3.47 (m, 1H), 3.23 (m, 1H),
2.01 (m, 2H), 1.78 (m, 2H). Anal. Calcd for C26H23FN2O5S: C,
63.15, H, 4.69, N, 5.66. Found: C, 63.05, H, 4.69, N, 5.60.
(S)-1-(4-Methylthiobenzyl)-5-(2-phenoxymethyl-pyrro-
lidine-1-sulfonyl)-1H-indole-2,3-dione (11d) was prepared
according to the same procedure for compound 11a, except
using 4-methylthiobenzyl bromide, and purified with hexanes-
ether (1:2) to afford 152 mg (64%) of 11d as a yellow solid, mp
175.4-176.8 °C. 1H NMR (300 MHz, CDCl3) δ 8.05 (s, 1H),
7.99 (d, J ) 10.5 Hz, 1H), 7.27 (m, 6H), 6.96 (t, J ) 7.2 Hz,
1H), 6.86 (t, J ) 8.1 Hz, 3H), 4.90 (s, 2H), 4.19 (d, J ) 8.7 Hz,
1H), 3.96 (m, 2H), 3.53 (m, 1H), 3.25 (m, 1H), 2.50 (s, 3H),
2.05 (m, 2H), 1.82 (m, 2H). Anal. Calcd for C27H26N2O5S2: C,
62.05, H, 5.01, N, 5.36. Found: C, 61.81, H, 4.95, N, 5.34.
((S)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)methyl 4-
Methylbenzesulfonate (8). A solution of 7 (5.03 g, 25.0
mmol) and pyridine (15 mL) in CH2Cl2 (50 mL) was reacted
with p-toluenesulfonyl chloride (5.96 g, 31.2 mmol) at 0 °C.
The mixture was stirred overnight at room temperature, then
CH2Cl2 (50 mL) was added. The solution was washed with
water (50 mL × 2), 10% citric acid (50 mL × 2), and saturated
NaCl (50 mL) and dried over Na2SO4. After evaporation of the
CH2Cl2, the crude product was purified with hexanes-ethyl
ether (1:1) to afford 8.9 g (100%) of 8 as a colorless oil. 1H NMR
(300 MHz, DMSO) δ 7.78 (d, J ) 8.4 Hz, 2H), 7.49 (d, J ) 8.1
Hz, 2H), 4.02 (m, 2H), 3.83 (m, 1H), 3.18 (m, 2H), 2.43 (s, 3H),
1.92 (m, 1H), 1.72 (m, 3H), 1.35 and 1.29 (s, 9H).
(S)-tert-Butyl 2-(Phenoxymethyl)pyrrolidine-1-car-
boxylate (9). A solution of phenol (7.37 g, 78.4 mmol) in THF
(100 mL) was reacted with 60% NaH (3.14 g, 78.4 mmol) at 0
°C in 20 min. The mixture was warmed to room temperature
and stirred 20 min, then a solution of 8 (5.57 g, 15.7 mmol) in
THF (25 mL) was added. The mixture was heated to reflux
for 24 h. After evaporation of the THF, ether (200 mL) was
added, washed with water (40 mL), 1 N NaOH (40 mL × 3),
and saturated NaCl (40 mL), and dried over Na2SO4. After
evaporation of the ether, the crude product was purified with
hexanes-ether (2:1) to afford 2.37 g (54%) of 9 as a colorless
oil. 1H NMR (300 MHz, DMSO) δ 7.28 (t, J ) 8.4 Hz, 2H),
6.95 (m, 3H), 4.04 (m, 2H), 3.87 (m, 1H), 3.27 (m, 2H), 1.93-
1.80 (m, 4H), 1.41 (s, 9H).
(S)-5-(2-Phenoxymethyl-pyrrolidine-1-sulfonyl)-1H-in-
dole-2,3-dione (10). To a solution of 9 (1.46 g, 5.2 mmol) in
CH2Cl2 (5 mL) was added trifloroacetic acid (5 mL) at 0 °C.
The mixture was stirred at 0 °C for 15 min. After evaporation
of the solvent in vacuo, CH2Cl2 (15 mL) and triethylamine (2
mL) were added, then a solution of 6 (1.44 g, 5.9 mmol) in
THF (25 mL) was added at 0 °C. The reaction mixture was
stirred overnight at room temperature. The solvent was
evaporated in vacuo, then ethyl acetate (150 mL) was added,
washed with water (50 mL × 2) and saturated NaCl (50 mL),
and dried over Na2SO4. After evaporation of the ethyl acetate,
the crude product was purified with ether to afford 1.7 g (84%)
of 10 as a yellow solid, mp 204.5-205.9 °C. 1H NMR (300 MHz,
CDCl3) δ 8.94 (s, 1H), 7.77 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H),
7.67 (s, 1H), 7.02 (t, J ) 8.7 Hz, 2H), 6.84 (d, J ) 8.1 Hz, 1H),
6.69 (t, J ) 7.2 Hz, 1H), 6.63 (d, J ) 7.8 Hz, 2H), 3.89 (m,
1H), 3.75-3.66 (m, 2H), 3.23 (m, 1H), 2.96 (m, 1H), 1.72 (m,
2H), 1.54-1.42 (m, 2H). LRMS (FAB) m/e: 387.1 (M + H, 100).
Anal. Calcd for C19H18N2O5S: C, 59.06, H, 4.70, N, 7.25.
Found: C, 58.99, H, 4.74, N, 7.11.
Acetic Acid (S)-4-[2,3-Dioxo-5-(2-phenoxymethyl-pyr-
rolidine-1-sulfonyl)-2,3-dihydroindol-1-yl-methyl]phen-
yl Ester (11e). This compound was prepared according to the
same procedure for compound 11a, except using 4-(chloro-
methyl)phenyl acetate, and purified with hexanes-ether (1:
2) to afford 112 mg (42%) of 11e as a yellow solid, mp 191.5-
1
193.2 °C. H NMR (300 MHz, CDCl3) δ 8.03 (d, J ) 2.1 Hz,
1H), 7.98 (d, J ) 8.4 Hz, 1H), 7.35 (d, J ) 8.4 Hz, 2H), 7.23 (t,
J ) 7.2 Hz, 2H), 7.10 (d, J ) 8.7 Hz, 2H), 6.95-6.81 (m, 4H),
4.90 (s, 2H), 4.17 (m, 1H), 3.93 (m, 2H), 3.51 (m, 1H), 3.23 (m,
1H), 2.30 (s, 3H), 2.02 (m 2H), 1.79 (m, 2H). Anal. Calcd for
C28H26N2O7S: C, 62.91, H, 4.90, N, 5.24. Found: C, 62.99, H,
5.02, N, 5.13.
(S)-1-(4-Hydroxybenzyl)-5-(2-phenoxymethyl-pyrroli-
dine-1-sulfonyl)-1H-indole-2,3-dione (11f). To a solution of
11e (53 mg, 0.1 mmol) in methanol (3 mL) and water (1 mL)
was added NaOH (4.4 mg, 0.11 mmol) at ambient temperature.
The mixture was stirred overnight, then acidified with 1 M
HCl to pH of 4 and extracted with ethyl acetate (50 mL). The
ethyl acetate was washed with NaCl (30 mL) and dried over
Na2SO4. After evaporation of the solvent, the crude product
was purified with ether to afford 36 mg (73%) of 11f as a yellow
(S)-1-Methyl-5-(2-phenoxymethyl-pyrrolidine-1-sulfo-
nyl)-1H-indole-2,3-dione (11a). To a solution of 10 (193 mg,
0.5 mmol) in DMF (3 mL) was added 60% NaH (30 mg, 0.75
mmol) at room temperature. The mixture was stirred 15 min,
then iodomethane (0.5 mL) was added. The mixture was
stirred overnight at ambient temperature, then ether (75 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 ether to afford
1
solid, mp 170.5-172.4 °C. H NMR (300 MHz, CDCl3) δ 8.01
1
85 mg (43%) of 11a as a yellow solid, mp 160.1-160.9 °C. H
(s, 1H), 7.97 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.24-7.19 (m,
4H), 6.96-6.80 (m, 6H), 4.85 (s, 2H), 4.16 (m, 1H), 3.98-3.88
NMR (300 MHz, CDCl3) δ 8.07 (dd, J ) 8.25 Hz, J ) 2.1 Hz,

7644 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Chu et al.
(m, 2H), 3.49 (m, 1H), 3.21 (m, 1H), 2.03 (m, 2H), 1.80 (m,
2H). Anal. Calcd for C26H24N2O6S‚0.25H2O: C, 62.83, H, 4.97,
N, 5.64. Found: C, 62.87, H, 4.74, N, 5.69.
hexanes-ether (2:1) to afford 0.81 g (79%) of 16 as a colorless
oil. 1H NMR (300 MHz, CDCl3) δ CDCl3 7.30 (m, 2H), 6.94
(m, 3H), 4.53 (m, 1H), 4.26 (m, 1H), 4.12 (m, 1H), 3.93 (m,
2H), 2.33 (m, 2H), 1.43 (s, 9H).
(S)-1-(6-Fluoropyridin-3-yl-methyl)-5-(2-phenoxymethyl-
pyrrolidine-1-sulfonyl)-1H-indole-2,3-dione (11g) was pre-
pared according to the same procedure for compound 11a,
except using 5-(bromomethyl)-2-fluoropyridine,21 and purified
with ether to afford 94 mg (76%) of 11g as yellow solid, mp
113.3-114.7 °C. 1H NMR (300 MHz, CDCl3) δ 8.28 (s, 1H),
8.03 (m, 2H), 7.82 (m 1H), 7.27-7.20 (m, 2H), 7.00-6.79 (m,
5H), 4.92 (s, 2H), 4.13 (m, 1H), 3.95 (m, 2H), 3.50 (m, 1H),
3.26 (m, 1H), 2.05 (m, 2H), 1.80 (m, 2H). Anal. Calcd for C25H22-
FN3O5S: C, 60.60, H, 4.47, N, 8.48. Found: C, 60.60, H, 4.59,
N, 8.33.
(S)-5-(2-Phenoxymethyl-azetidine-1-sulfonyl)-1H-indole-
2,3-dione (17) was prepared according to the same procedure
for compound 10, except using compound 16, and purified with
ether to afford 715 mg (63%) of 17 as a yellow solid, mp 173.2-
174.5 °C. 1H NMR (300 MHz, DMSO) δ 11.48 (s, 1H), 7.98 (dd,
J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.77 (s, 1H), 7.27 (m, 2H), 7.10
(d, J ) 8.1 Hz, 1H), 6.91 (d, J ) 7.8 Hz, 3H), 4.20-4.02 (m,
3H), 3.70 (m, 1H), 3.55 (m, 1H), 2.22 (m, 1H), 2.02 (m, 1H).
LRMS (FAB) m/e: 373.0 (M + H, 100). Anal. Calcd for
C18H16N2O5S‚0.5H2O: C, 56.68, H, 4.49, N, 7.34. Found: C,
56.96, H, 4.39, N, 7.30.
(S)-1-Methyl-5-(2-phenoxymethyl-azetidine-1-sulfonyl)-
1H-indole-2,3-dione (18a) was prepared according to the
same procedure for compound 11a, except using compound 17,
and purified with ether to afford 46 mg (48%) of 18a as an
orange solid, mp 173.5-174.9 °C. 1H NMR (300 MHz, CDCl3)
δ 8.04 (m, 2H), 7.24 (m, 2H), 6.94 (m, 2H), 6.79 (m, 2H), 4.46
(m, 1H), 4.10 (m, 2H), 3.86 (m, 2H), 3.25 (m, 3H), 2.30 (m,
2H). Anal. Calcd for C19H18N2O5S: C, 59.06, H, 4.70, N, 7.25.
Found: C, 58.98, H, 4.75, N, 7.19.
(S)-1-(2-Fluoro-pyridin-4-yl-methyl)-5-(2-phenoxy-
methyl-pyrrolidine-1-sulfonyl)-1H-indole-2,3-dione (11h)
was prepared according to the same procedure for compound
11a, except using 4-(bromomethyl)-2-fluoropyridine,21 and
purified with ether to afford 41 mg (33%) of 11h as a yellow
1
solid, mp 180.1-181.9 °C. H NMR (300 MHz, CDCl3) δ 8.25
(d, J ) 5.4 Hz, 1H), 8.07 (s, 1H), 8.00 (d, J ) 8.4 Hz, 1H), 7.23
(m, 2H), 7.12 (d, J ) 4.2 Hz, 1H), 6.96-6.73 (m, 5H), 4.94 (s,
2H), 4.13 (m, 1H), 4.00-3.89 (m, 2H), 3.49 (m, 1H), 3.28 (m,
1H), 2.04 (m, 2H), 1.82 (m, 2H). Anal. Calcd for C25H22-
FN3O5S: C, 60.60, H, 4.47, N, 8.48. Found: C, 60.32, H, 4.34,
N, 8.35.
(S)-1-Benzyl-5-(2-phenoxymethyl-azetidine-1-sulfonyl)-
1H-indole-2,3-dione (18b) was prepared according to the
same procedure for compound 11a, except using compound 17
and benzyl bromide, and purified with hexanes-ether (1:2)
to afford 92 mg (80%) of 18b as an orange solid, mp 157.1-
(S)-1-(6-Fluoro-pyridin-2-yl-methyl)-5-(2-phenoxy-
methyl-pyrrolidine-1-sulfonyl)-1H-indole-2,3-dione (11i)
was prepared according to the same procedure for compound
11a, except using 6-(bromomethyl)-2-fluoropyridine,21 and
purified with ether to afford 57 mg (46%) of 11i as a yellow
1
158.9 °C. H NMR (300 MHz, CDCl3) δ 8.05 (d, J ) 2.1 Hz,
1H), 7.95 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.34 (m, 5H), 7.22
(m, 2H), 6.94 (m, 1H), 6.87-6.78 (m, 3H), 4.93 (s, 2H), 4.46
(m, 1H), 4.10 (m, 2H), 2.82 (m, 2H), 2.32 (m 2H). Anal. Calcd
for C25H22N2O5S: C, 64.92, H, 4.79, N, 6.06. Found: C, 64.82,
H, 4.79, N, 7.97.
1
solid, mp 128.6-129.4 °C. H NMR (300 MHz, CDCl3) δ 8.02
(m, 2H), 7.82 (m, 1H), 7.28-7.10 (m, 4H), 6.92 (m, 2H), 6.85
(m, 2H), 4.96 (s, 2H), 4.14 (m, 1H), 3.94 (m, 2H), 3.51 (m, 1H),
3.23 (m, 1H), 2.03 (m, 2H), 1.79 (m, 2H). Anal. Calcd for C25H22-
FN3O5S‚0.25H2O: C, 60.05, H, 4.54, N, 8.40. Found: C, 60.06,
H, 4.49, N, 8.24.
(S)-1-(4-Methoxybenzyl)-5-(2-phenoxymethyl-azetidine-
1-sulfonyl)-1H-indole-2,3-dione (18c) was prepared accord-
ing to the same procedure for compound 11a, except using
compound 17 and 4-methoxybenzyl chloride, and purified with
hexanes-ether (1:2) to afford 62 mg (50%) of 18c as an orange
(S)-1-(tert-Butoxycarbonyl)azetidine-2-carboxylic Acid
(13). To a solution of (S)-2-azetidinecarboxylic acid 12 (1.0 g,
10.0 mmol) and di-tert-butyl dicarbonate (2.83 g, 12.5 mmol)
in ethanol (20 mL) and water (10 mL) was added NaOH (420
mg, 10.5 mmol) at 0 °C. The mixture was stirred overnight at
ambient temperature. After evaporation of the ethanol, water
(20 mL) was added, then acidified with diluted HCl to a pH of
3 and extracted with ethyl acetate (50 mL × 3). The combined
ethyl acetate was washed with water (30 mL) and saturated
NaCl (30 mL), and dried over Na2SO4. After evaporation of
the ethyl acetate to afford 1.98 g (100%) of 13 as a white solid.
1H NMR (300 MHz, CDCl3) δ 4.79 (m, 1H), 3.93 (m, 2H), 2.46
(m, 2H), 1.48 (s, 9H).
1
solid, mp 159.8-161.5 °C. H NMR (300 MHz, CDCl3) δ 8.04
(s, 1H), 7.96 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.25 (m, 4H),
6.97-6.87 (m, 4H), 6.80 (d, J ) 7.8 Hz, 2H), 4.87 (s, 2H), 4.45
(m, 2H), 4.11 (m, 2H), 3.84 (m, 2H), 3.81 (s, 3H), 2.32 (m, 2H).
Anal. Calcd for C26H24N2O6S: C, 63.40, H, 4.91, N, 5.69.
Found: C, 63.65, H, 4.93, N, 5.59.
(S)-1-(4-Methylthiobenzyl)-5-(2-phenoxymethyl-azeti-
dine-1-sulfonyl)-1H-indole-2,3-dione (18d) was prepared
according to the same procedure for compound 11a, except
using compound 17 and 4-methylthiobenzyl bromide, and
purified with hexanes-ether (1:2) to afford 57 mg (45%) of 18d
(S)-tert-Butyl 2-(Hydroxymethyl)azetidine-1-carboxy-
late (14).17 To a solution of 13 (0.94 g, 4.7 mmol) in THF (10
mL) was added slowly a 1 M BH3 in THF (21.0 mL) at 0 °C.
The mixture was stirred 2 days at ambient temperature, then
cold water (20 mL) was added at 0 °C. After evaporation of
the THF in vacuo, an 10% aqueous solution of citric acid (15
mL) was added and extracted with ethyl acetate (50 mL × 2).
The combined ethyl acetate was washed with saturated
NaHCO3 (30 mL) and NaCl (30 mL), and dried over Na2SO4.
Evaporation of the ethyl acetate in vacuo afforded 0.86 g
1
as an orange solid, mp 167.6-169.2 °C. H NMR (300 MHz,
CDCl3) δ 8.05 (d, J ) 1.5 Hz, 1H), 7.96 (dd, J ) 8.4 Hz, J )
1.8 Hz, 1H), 7.25 (m, 6H), 6.95 (t, J ) 7.2 Hz, 1H), 6.86-6.78
(m, 3H), 4.89 (s, 2H), 4.46 (m, 1H), 4.11 (m, 2H), 3.82 (m, 2H),
2.49 (s, 3H), 2.39-2.25 (m, 2H). Anal. Calcd for C26H24-
N2O5S2: C, 61.40, H, 4.76, N, 5.51. Found: C, 60.99, H, 4.71,
N, 5.36.
(S)-1-(4-Fluorobenzyl)-5-(2-phenoxymethyl-azetidine-
1-sulfonyl)-1H-indole-2,3-dione (18e) was prepared accord-
ing to the same procedure for compound 11a, except using
compound 17 and 4-fluorobenzyl bromide, and purified with
hexanes-ether (1:2) to afford 85 mg (71%) of 18e as an orange
1
(100%) of 14 as a colorless oil. H NMR (300 MHz, CDCl3) δ
4.40 (m, 1), 3.85-3.70 (m, 3H), 2.13 (m, 1H), 1.90 (m, 1H),
1.42 (s, 9H).
1
((S)-1-(tert-Butoxycarbonyl)azetidine-2-yl)methyl 4-
methylbenzenesulfonate (15) was prepared according to the
same procedure for compound 8, except using compound 14,
and purified with hexanes-ether (1:1) to afford 1.34 g (86%)
solid, mp 164.6-165.7 °C. H NMR (300 MHz, CDCl3) δ 8.05
(d, J ) 1.8 Hz, 1H), 7.97 (dd, J ) 8.4 Hz, J ) 2.1 Hz, 1H),
7.34-7.20 (m, 4H), 7.07 (t, J ) 8.7 Hz, 2H), 6.94 (t, J ) 7.2
Hz, 1H), 6.86-6.77 (m, 3H), 4.90 (s, 2H), 4.47 (m, 1H), 4.10
(m, 2H), 3.85 (m, 2H), 2.36-2.22 (m, 2H). Anal. Calcd for
C25H21FN2O5S: C, 62.49, H, 4.41, N, 5.83. Found: C, 62.27,
H, 4.48, N, 5.69.
1
of 15 as a colorless oil. H NMR (300 MHz, CDCl3) δ 7.79 (d,
J ) 8.7 Hz, 2H), 7.34 (d, J ) 8.1 Hz, 2H), 4.33-4.24 (m, 2H),
4.10 (m, 1H), 3.78 (m 2H), 2.44 (s, 3H), 2.21 (m, 2H), 1.36 (s,
9H).
(S)-1-(2-Fluorobenzyl)-5-(2-phenoxymethyl-azetidine-
1-sulfonyl)-1H-indole-2,3-dione (18f) was prepared accord-
ing to the same procedure for compound 11a, except using
compound 17 and 2-fluorobenzyl bromide, and purified with
(S)-tert-Butyl 2-(phenoxymethyl)azetidine-1-carboxy-
late (16) was prepared according to the same procedure for
compound 9, except using compound 15, and purified with

Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7645
hexanes-ether (1:2) to afford 81 mg (68%) of 18f as an orange
to the same procedure for compound 11a, except using
compound 20 and benzyl bromide, and the crude product was
purified with ether to afford 61 mg (51%) of 21b as a yellow
solid, mp 79.6-80.7 °C. 1H NMR (300 MHz, CDCl3) δ 8.25 (s,
1H), 8.22 (t, J ) 2.7 Hz, 1H), 8.04 (d, J ) 2.1 Hz, 1H), 7.98
(dd, J ) 8.4 Hz, J ) 2.1 Hz, 1H), 7.36 (m, 5H), 7.22 (m, 2H),
6.91 (d, J ) 8.8 Hz, 1H), 4.97 (s, 2H), 4.25 (m, 1H), 3.99-3.94
(m, 2H), 3.55-3.48 (m, 1H), 3.21-3.15 (m, 1H), 2.10-1.97 (m,
2H), 1.84-1.75 (m, 2H). LRMS (FAB) m/e: 484.1 (M + Li, 100);
HRMS (FAB) m/e calcd for C25H23N3O5SLi (M + Li) 484.1518,
found 484.1539.
1-(4-Methoxybenzyl)-5-(2-(pyridin-3-yl-oxymethyl)-pyr-
rolidine-1-sulfonyl)-1H-indole-2,3-dione (21c) was pre-
pared according to the same procedure for compound 11a,
except using 20 and 4-methoxybenzyl chloride. The crude
product was purified with ether to afford 45 mg (36%) of 21c
as a yellow solid, mp 156.7-158.4 °C. 1H NMR (300 MHz,
CDCl3) δ 8.26 (s, 1H), 8.23 (t, J ) 2.7 Hz, 1H), 8.03 (d, J ) 1.5
Hz, 1H), 7.98 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.28 (d, J )
8.7 Hz, 2H), 7.22 (t, J ) 2.1 Hz, 2H), 6.94 (d, J ) 8.1 Hz, 1H),
6.90 (d, J ) 8.7 Hz, 2H), 4.90 (s, 2H), 4.24 (m, 1H), 4.01-3.90
(m, 2H), 3.81 (s, 3H), 3.55-3.49 (m, 1H), 3.20-3.15 (m, 1H),
2.05 (m, 2H), 1.78 (m, 2H). LCMS m/e:507.9 (M + H). Anal.
Calcd for C26H25N3O6S: C, 61.53, H, 4.96, N, 8.28. Found: C,
61.27, H, 4.95, N, 8.17.
1-(4-Methylthiobenzyl)-5-(2-(pyridin-3-yl-oxymethyl)-
pyrrolidine-1-sulfonyl)-1H-indole-2,3-dione (21d) was pre-
pared according to the same procedure for compound 11a,
except using 20 and 4-methylsulfanylbenzyl bromide. The
crude product was purified with ether to afford 57 mg (44%)
of 21d as a yellow solid, mp 81.5-83.1 °C. 1H NMR (300 MHz,
CDCl3) δ 8.25-8.21 (m, 2H), 8.03 (d, J ) 1.8 Hz, 1H), 7.24 (s,
3H), 7.21 (m, 1H), 6.89 (d, J ) 8.4 Hz, 1H), 4.91 (s, 2H), 4.23
(m, 1H), 4.00-3.89 (m, 2H), 3.51 (m, 1H), 3.14 (m, 1H), 2.47
(s, 3H), 2.02 (m, 2H), 1.78 (m, 2H). LRMS (FAB) m/e: 530.1
(M + Li, 100); HRMS (FAB) m/e calcd for C26H25N3O5S2Li (M
+ Li) 530.1396, found 530.1397.
1
solid, mp 147.1-148.0 °C. H NMR (300 MHz, CDCl3) δ 8.05
(d, J ) 1.8 Hz, 1H), 8.00 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H),
7.35 (m, 2H), 7.24-7.11 (m, 3H), 7.02-6.78 (m, 5H), 4.98 (s,
2H), 4.47 (m, 1H), 4.11 (m, 2H), 3.85 (m, 2H), 2.35-2.25 (m,
2H). Anal. Calcd for C25H21FN2O5S: C, 62.49, H, 4.41, N, 5.83.
Found: C, 62.25, H, 4.47, N, 5.68.
(S)-1-(6-Fluoropyridin-3-ylmethyl)-5-(2-phenoxymethyl-
azetidine-1-sulfonyl)-1H-indole-2,3-dione (18g) was pre-
pared according to the same procedure for compound 11a,
except using compound 17 and 5-(bromomethyl)-2-fluoropyri-
dine, and purified with ether to afford 74 mg (62%) of 18g as
an orange solid, mp 176.8-178.3 °C. 1H NMR (300 MHz,
CDCl3) δ 8.27 (d, J ) 2.4 Hz, 1H), 8.07 (d, J ) 2.1 Hz, 1H),
8.01 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H), 7.79 (td, J ) 8.1 Hz, J
) 2.4 Hz, 1H), 7.22 (m, 2H), 7.00-6.77 (m, 5H), 4.92 (s, 2H),
4.49 (m, 1H), 4.09 (m, 2H), 3.85 (m, 2H), 2.35-2.23 (m, 2H).
Anal. Calcd for C24H20FN3O5S: C, 59.87, H, 4.19, N, 8.73.
Found: C, 59.81, H, 4.16, N, 8.62.
(S)-1-(2-Fluoropyridin-4-yl-methyl)-5-(2-phenoxymethyl-
azetidine-1-sulfonyl)-1H-indole-2,3-dione (18h) was pre-
pared according to the same procedure for compound 11a,
except using compound 17 and 4-(bromomethyl)-2-fluoropyri-
dine, and purified with ether to afford 36 mg (30%) of 18h as
an orange solid, mp 159.0-159.9 °C. 1H NMR (300 MHz,
CDCl3) δ 8.23 (d, J ) 5.1 Hz, 1H), 8.08 (s, 1H), 7.98 (dd, J )
8.7 Hz, J ) 2.1 Hz, 1H), 7.22 (m, 2H), 7.10 (d, J ) 5.4 Hz,
1H), 6.93 (t, J ) 7.5 Hz, 1H), 6.84-6.72 (m, 4H), 4.93 (s, 2H),
4.49 (m, 1H), 4.07 (m, 2H), 3.91-3.81 (m, 2H), 2.35-2.22 (m,
2H). Anal. Calcd for C24H20FN3O5S‚0.5H2O: C, 58.77, H, 4.32,
N, 8.57. Found: C, 58.69, H, 4.45, N, 8.26.
(S)-1-(6-Fluoropyridin-2-yl-methyl)-5-(2-phenoxymethyl-
azetidine-1-sulfonyl)-1H-indole-2,3-dione (18i) was pre-
pared according to the same procedure for compound 11a,
except using compound 17 and 6-(bromomethyl)-2-fluoropyri-
dine, and purified with ether to afford 62 mg (52%) of 18i as
an orange solid, mp 144.7-146.1 °C. 1H NMR (300 MHz,
CDCl3) δ 8.03 (s, 1H), 8.00 (dd, J ) 8.25 Hz, J ) 2.1 Hz, 1H),
7.82 (m, 1H), 7.27-7.11 (m, 4H), 6.92 (m, 2H), 6.79 (m, 2H).
Anal. Calcd for C24H20FN3O5S: C, 59.87, H, 4.19, N, 8.73.
Found: C, 59.59, H, 4.27, N, 8.48.
1-(4-Fluorobenzyl)-5-(2-(pyridin-3-yl-oxymethyl)-pyr-
rolidine-1-sulfonyl)-1H-indole-2,3-dione (21e) was pre-
pared according to the same procedure for compound 11a,
except using 20 and 4-fluorobenzyl bromide. The crude product
was purified with ether to afford 35 mg (28%) of 21e as a
2-(Pyridin-3-yl-oxymethyl)-pyrrolidine-1-carboxylic
acid tert-butyl ester (19) was prepared according to the same
procedure for compound 9, except using 3-hydroxypyridine.
The crude product was purified with ether to afford 1.70 g
(61%) of 19 as a colorless oil. 1H NMR (300 MHz, CDCl3) δ
8.32 (s, 1H), 8.21 (s, 1H), 7.21 (m, 2H), 4.16 (m, 2H), 3.99-
3.86 (m, 1H), 3.38 (m, 2H), 2.05-1.84 (m, 4H), 1.47 (s, 9H).
5-(2-(Pyridin-3-yl-oxymethyl)-pyrrolidine-1-sulfonyl)-
1H-indole-2,3-dione (20) was prepared according to the same
procedure for compound 10, except using compound 19, and
the crude product was recrystallized from ethyl acetate to
afford 1.75 g (82%) of 20 as a yellow solid, mp 215.9-217.8
1
yellow solid, mp 77.1-78.3 °C. H NMR (300 MHz, CDCl3) δ
8.25 (m, 2H), 8.05 (s, 1H), 8.03-7.99 (m, 1H), 7.36-7.32 (m,
2H), 7.23 (m, 2H), 7.09 (t, J ) 8.7 Hz, 2H), 6.90 (d, J ) 8.7
Hz, 1H), 4.94 (s, 2H), 4.25 (d, J ) 6.0 Hz, 1H), 3.98 (m, 2H),
3.52 (m, 1H), 3.19 (m, 1H), 2.05 (m, 2H), 1.80 (m, 2H). LRMS
(FAB) m/e: 502.1 (M + Li, 100); HRMS (FAB) m/e calcd for
C25H22FN3O5SLi (M + Li) 502.1424, found 502.1420.
2-(Pyridin-4-yl-oxymethyl)-pyrrolidine-1-carboxylic
acid tert-butyl ester (22) was prepared according to the same
procedure for compound 9, except using 4-hydroxypyridine.
The crude product was purified with ethyl acetate to afford
1.31 g (47%) of 22 as a colorless oil. 1H NMR (300 MHz, CDCl3)
δ 8.42 (m, 2H), 6.87 (m, 2H), 4.15 (m, 3H), 3.43 (m, 2H), 1.98
(m, 4H), 1.50 (s, 9H).
1
°C. H NMR (300 MHz, DMSO) δ 11.42(s, 1H), 8.26 (d, J )
3.0 Hz, 1H), 8.16 (d, J ) 4.5 Hz, 1H), 8.02 (d, J ) 8.4 Hz, 1H),
7.78 (s, 1H), 7.38 (m, 1H), 7.33 (m, 1H), 7.05 (d, J ) 8.4 Hz,
1H), 4.15-4.02 (m, 2H), 3.90 (m, 1H), 3.34 (m, 1H), 3.12 (m,
1H), 1.87 (m, 2H), 1.67-1.54 (m, 2H). LCMS m/e: 387.8 (M +
H). Anal. Calcd for C18H17N3O5S‚0.5H2O: C, 54.54, H, 4.58,
N, 10.60. Found: C, 54.56, H, 4.70, N, 10.04.
5-(2-(Pyridin-4-yl-oxymethyl)-pyrrolidine-1-sulfonyl)-
1H-indole-2,3-dione (23) was prepared according to the same
procedure for compound 10, except using compound 22,
purified with ethyl acetate to afford 1.17 g (55%) of 23 as a
1
yellow solid, mp 204.2-205.3 °C. H NMR (300 MHz, CDCl3)
1-Methyl-5-(2-(pyridin-3-yl-oxymethyl)-pyrrolidine-1-
sulfonyl)-1H-indole-2,3-dione (21a) was prepared according
to the same procedure for compound 11a, except using
compound 20, and the crude product was purified with ethyl
acetate to afford 55 mg (55%) of 21a as a yellow solid, mp
142.1-143.4 °C. 1H NMR (300 MHz, CDCl3) δ 8.24 (d, J )
2.7, 1H), 8.22 (dd, J ) 3.9 Hz, J ) 2.1 Hz, 1H), 8.08 (dd, J )
8.4, J ) 2.1 Hz, 1H), 7.26 (s, 1H), 7.21 (m, 2H), 7.00 (d, J )
8.4 Hz, 1H), 4.22 (m, 1H), 3.98 (m, 2H), 3.53 (m, 1H), 3.30 (s,
3H), 3.22 (m, 2H), 2.03 (m, 2H), 1.80 (m, 2H). LCMS m/e:
401.84 (M + H). Anal. Calcd for C19H19N3O5S: C, 56.85, H,
4.77, N, 10.47. Found: C, 56.48, H, 4.87, N, 10.19.
δ 11.44 (s, 1H), 8.37 (d, J ) 5.7 Hz, 2H), 8.03 (dd, J ) 8.4 Hz,
J ) 2.1 Hz, 1H), 7.79 (s, 1H), 7.06 (d, J ) 8.4 Hz, 1H), 6.96 (d,
J ) 6.0 Hz, 2H), 4.17-4.05 (m, 2H), 3.90 (m, 1H), 3.32 (m,
1H), 3.10 (m, 1H), 1.85 (m, 2H), 1.60 (m, 2H). LCMS m/e: 387.9
(M + H). Anal. Calcd for C18H17N3O5S‚0.75H2O: C, 53.92, H,
4.65, N, 10.48. Found: C, 54.14, H, 4.39, N, 10.35.
Enzyme Inhibition Assays. Recombinant human caspases
(3, 6, 7, and 8) and their peptide-specific substrates (Ac-DEVD-
AMC, Ac-VEID-AMC, Ac-DEVD-AMC, and Ac-IETD-AMC,
respectively) were purchased from Sigma-Aldrich (St. Louis,
MO) with the, exception of caspase 1 and its substrate (Ac-
YVAD-AMC) which were obtained from BIOMOL Research
Laboratories (Plymouth Meeting, PA). The enzymatic activity
1-Benzyl-5-(2-(pyridin-3-yl-oxymethyl)-pyrrolidine-1-
sulfonyl)-1H-indole-2,3-dione (21b) was prepared according

7646 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24
Chu et al.
of caspases was determined by measuring the accumulation
of the fluorogenic product 7-amino-4-methylcoumarin (AMC).
All assays were prepared in 96-well format at a volume of 210
µL per well and consisted of 100 mM Na+ HEPES (pH 7.4),
10% sucrose, 100 mM NaCl, 0.1% CHAPS, 5 mM 2-mercap-
toethanol, 2 mM EDTA, 10 µM Ac-YVAD-AMC (caspase 1);
20 mM Na+ HEPES (pH 7.4), 10% sucrose, 100 mM NaCl, 0.1%
CHAPS, 2 mM EDTA, 10 µM Ac-DEVD-AMC (caspase 3); 20
mM Na+ HEPES (pH 7.4), 10% sucrose, 100 mM NaCl, 0.1%
CHAPS, 2 mM EDTA, 10 µM Ac-VEID-AMC (caspase 6); 20
mM Na+ HEPES (pH 7.4), 100 mM NaCl, 10% sucrose, 0.1%
CHAPS, 5 mM 2-mercaptoethanol, 2 mM EDTA, 10 µM Ac-
DEVD-AMC (caspase 7); 20 mM Na+ HEPES (pH 7.4), 10%
sucrose, 100 mM NaCl, 0.1% CHAPS, 2 mM EDTA, 10 µM
Ac-IETD-AMC (caspase 8).
Recombinant caspases were first assayed to determine the
optimal concentration for each experiment. Optimal concentra-
tions were based in the linear range of the enzyme activation
curves. Peptide inhibitors with known IC50 values were tested
together with the compounds as a control for each caspase
assay. Peptide inhibitors, Ac-DEVD-CHO (caspase-3 and -7),
Ac-VEID-CHO (caspase-6), and Ac-IETD-CHO (caspase-8)
were purchased from Sigma-Aldrich (St. Louis, MO) with
exception of caspase-1 specific inhibitor (Ac-YVAD-CHO) which
was acquired from BIOMOL Research Laboratories (Plymouth
Meeting, PA). Peptide and nonpeptide inhibitors were dis-
solved in DMSO, and a 2× serial dilution was performed prior
to screening in order to obtain desired concentrations. 10 µL
was added to each well containing 100 µL caspase solution
and allowed to incubate on ice for 30 min. A 100 µL substrate
solution was added to each well, and plates were incubated
for 1-2 h at 37 °C. The final concentration of DMSO in all
wells was 5% of the total volume. In caspase-1 and caspase-7
assays, 10 mM 2-mercaptoethanol was added to the substrate
solution for full activation of the enzymes.
search (LMCS) with a 25 kJ/mol energy window using the
MMFF94 force field. The lowest energy structures found were
then exported back into MOE and modified for covalent
docking into the protein. The inhibitors were modified to allow
for covalent docking by adding a sulfur to position 3 of the
isatin moieties, forming a tetrahedral carbon with sulfur and
oxyanion substituents. As this generates a chiral center, both
enantiomers were prepared and used in docking studies. The
sulfur serves as the link between the inhibitor and Cys285 of
the enzyme.
Docking. The ligands were then docked into the enzyme
using covalent constraints as implemented in Gold 2.2. A
substructure-based template mapping was used in order to
setup the covalent constraints so that multiple compounds
could be docked within one study. The template utilized
consisted of the isatin core with a sulfur covalently attached
at C3. Gold utilizes an evolutionary genetic algorthithm to
optimize the docked pose of the inhibitor within the enzyme.25
Each inhibitor was docked into the binding site, through a
covalent attachment between the isatin moiety and Cys285.
Each molecule was docked 25 times with early termination if
the top three poses are within 1.5 Å rmsd. Each pose was
ranked according to its GoldScore which consists of protein-
ligand hydrogen bond energy, protein-ligand van der Waals
(vdw) energy, ligand internal vdw energy, and ligand torsional
strain energy. A total of four docking runs were done with each
ligand, both enantiomers of the covalently modified ligands
as well as both enantiomers of the template utilized for the
substructure mapping. The fitness value from all runs were
combined and the highest scoring poses used for further
analysis.
Acknowledgment. This work was supported by
grants HL1385 awarded by the National Heart, Lung
and Blood Institute and EB1729 and EB00340 awarded
by the National Institute of Biomedical Imaging and
Bioengineering.
The amount of AMC released was determined by using a
Victor3 microplate fluorometer (Perkin-Elmer Life Sciences,
Boston, MA) at excitation and emission wavelengths 355 nm
and 460 nm, respectively. Compounds were tested in duplicate,
and IC50 curves were calculated for all inhibitors assayed. Final
IC50s were the average of three independent experiments.
Enzyme Kinetic Studies. The inhibition profile for com-
pound 21c was determined for caspase-3 in the assay buffer.
The concentration of Ac-DEVD-AMC was varied from 6.25 to
100 µM, and the concentration of 21c was varied from 0 to 20
nM. The kinetic parameters of 21c were obtained by fitting
initial-rate data to
Supporting Information Available: Elemental analysis
data. The structures of the molecules in their best poses are
also available as a mol2 file. This material is available free of
charge via the Internet at http://pubs.acs.org.
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VmS
v )
(1)
1
Km 1 +
+ S
(
)
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Caspase-3 Inhibitors
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