Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: Introduction of a hydrophilic group increases potency in a whole cell assay

Article abstract of DOI:10.1016/j.bmcl.2011.03.015

A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.

Full text of DOI:10.1016/j.bmcl.2011.03.015

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2192–2197
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of isatin analogs as caspase-3 inhibitors:
Introduction of a hydrophilic group increases potency in a whole cell assay
⇑
Wenhua Chu, Justin Rothfuss, Dong Zhou, Robert H. Mach
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group,
and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both
in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic
group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells
compared to the corresponding unsubstituted N-phenyl isatin analogs.
Received 30 January 2011
Revised 2 March 2011
Accepted 3 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Caspase-3
Apoptosis
Cell death
The caspase family of cysteine proteases consists of two differ-
ent classes of enzymes involved in apoptosis, the initiator caspases
and the executioner caspases.1 The initiator caspases, which in-
clude caspase-2, -8, -9, and -10, are located at the top of the signal-
ing cascade; their primary function is to activate the executioner
caspases, caspase-3, -6, and -7. The executioner caspases are
responsible for the physiological changes (e.g., cleavage of the
DNA repair enzyme poly(ADP-ribose) polymerase-1, nuclear lami-
nins, and cytoskeleton proteins) and morphological changes (DNA
strand breaks, nuclear membrane damage, and membrane bleb-
bing) that occur in apoptosis. Apoptosis plays an important role
in a wide variety of normal cellular processes including fetal devel-
opment, tissue homeostasis, and maintenance of the immune sys-
tem. However, abnormal apoptosis has been observed in a large
number of pathological conditions, including ischemia-reperfusion
injury (stroke and myocardial infarction), cardiomyopathy, neuro-
degeneration (Alzheimer’s disease, Parkinson’s disease, Hunting-
induce or halt apoptosis would be of tremendous value to the re-
search and clinical community.
Recently, a number of isatin-based inhibitors of caspase-3/7
have been reported.10–21 Structure–activity relationship studies
have revealed that the following structural changes to 1 (Fig. 1) re-
sulted in a dramatic improvement in potency for inhibiting cas-
pase-3 activity in enzymatic activity assays: (1) replacement of
the 2-methoxymethyl group with a phenoxymethyl or 2-pyridin-
3-yl-oxymethyl moiety; (2) replacement of the pyrrolidine ring
with an azetidine ring; and, (3) the introduction of an alkyl or aryl-
alkyl on the isatin nitrogen group.10,12 For example, isatin analog in
2 (Fig. 1) had a significantly enhanced potency for inhibiting cas-
pase-3 activity in enzymatic assays relative to 1; however, the po-
tency for inhibiting caspase-3 activity using a whole cell assay was
not as high as that observed in enzymatic assays using the recom-
binant enzyme. The introduction of a benzyl group on the isatin
nitrogen and replacement of the methoxymethyl group with a
phenoxymethyl group dramatically increased the lipophilicity of
the compound; this increased lipophilicity is expected to have a
ton’s disease, ALS), sepsis, Type
I diabetes, and allograft
rejection.2,3 The development of drugs that can halt the process
of apoptosis has been an active area of research in the pharmaceu-
tical industry.2 In addition, the benefits of many drugs, especially
antitumor drugs, can be attributed to their activation of the apop-
totic process.4–9 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
O
O
O
O
O
O
S
S
N
N
O
O
N
N
H
O
O
CH3
⇑
Corresponding author. Address: Division of Radiological Sciences, Washington
1
2
University School of Medicine, Campus Box 8225, 510 S. Kingshighway Blvd., St.
Louis, MO 63110, USA. Tel.: +1 3143628538; fax: +1 3143620039.
E-mail address: rhmach@mir.wustl.edu (R.H. Mach).
Figure 1. Structures of the lead compounds for the current study.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.015

W. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2192–2197
2193
O
O
O
Boc
N
S
N
N
Boc
b
N
a
O
N
H
O
O
O
Ts
N
5
3
4
O
O
O
O
O
O
S
S
c
N
d
N
O
O
n
N
n
N
H
O
O
R
X
X
O
6 n=1, X=CH
9: n=1, X=CH, R=CH2CH2F
10: n=1 X=CH, R=CH2CH2OCH2CH2F
11: n=1, X=CH, R=CH2CH2OCH2CH2OCH2CH2F
5
7
8
n=1, X=N
n=2, X=CH
n=2, X=N
12:
n=1, X=N, R=CH2CH2F
13:n=1, X=N, R=CH3
14: n=2, X=CH, R=CH2CH2OCH2CH2F
15: n=2, X=CH, R=CH2CH2OCH2CH2OCH2CH2F
16:
n=2, X=N, R=CH2CH2F
NC
O
O
CN
O
17: n=1, X=CH, R=CH2CH2F
S
18:
19:
n=1, X=CH, R=CH2CH2OCH2CH2F
n=1, X=N, R=CH2CH2F
N
n
N
20: n=1, X=N, R=CH3
21: n=2, X=CH, R=CH2CH2F
O
22:
n=2, X=CH, R=CH2CH2OCH2CH2F
R
23: n=2, X=N, R=CH2CH2F
X
O
Scheme 1. Synthesis of the pegylated analogs. Reagents and conditions: (a) 3-hydroxypyridine, NaH, THF/DMF; (b) (1) TFA, CH2C12, (2) 2,3-dioxoindoline-5-sulfonyl chloride,
Et3N; (c) (1) NaH, DMF, (2) BrCH2C6H4(OCH2CH2)nF (n = l,2,3); (d) CNCH2CN, MeOH.
negative effect on the ability of the inhibitor to cross the cell mem-
brane and inhibit caspase-3 in the whole cell assay versus in an
in vitro enzymatic assay using recombinant protein.10 The goal of
the current study is to extend the structure–activity relationship
study of this class of compounds by introducing a hydrophilic
group in the isatin nitrogen, including an oligo-ethylene glycol
b
a
Br
O
OMs
H3C
H3C
O
O
H3C
c
O
25
O
24
F
O
F
Br
26
27
a
d
O
H3C
H3C
OH
H3C
O
Br
28
29
b
O
O
F
O
OMs
H3C
O
O
31
30
c
O
F
O
O
Br
32
Scheme 2. Synthesis of intermediates 27 and 32. Reagents and conditions: (a) AgOMs, acetonitrile; (b) 2-fluoroethanol, NaH, DMF; (c) NBS, CC14; (d) (BrCH2CH2)2O, K2CO3,
acetone.

2194
W. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2192–2197
O
O
O
O
O
O
O
O
O
S
S
c
S
N
d
N
N
O
O
O
n
N
n
N
n
N
H
O
F
O
O
N
N
N
X
X
X
6 n=1, X=CH
5 n=1, X=N
7 n=2, X=CH
8 n=2, X=N
33 n=1, X=CH
34 n=1, X=N
35 n=2, X=CH
36 n=2, X=N
37 n=1, X=CH
38 n=1, X=N
39 n=2, X=CH
40 n=2, X=N
O
O
O
a
Boc
N
Boc
N
b
S
N
O
n
n
n
F
OH
N
H
O
F
F
O
O
F
F
Ts
2,4-difluorophenol
3 n=1
41 n=2
44 n=1
45 n=2
42 n=1
43 n=2
F
O
O
O
O
O
O
S
c
S
d
N
N
O
O
n
N
n
N
F
O
F
F
O
F
R
N
N
N
F
46 n=1, R=
47 n=2, R=
50 n=1
51 n=2
F
O
48 n=1, R=
F
O
49 n=2, R=
Scheme 3. Synthesis of the triazole analogs. Reagents and conditions: (a) 2,4-difluorophenol, 60% NaH, DMF/THF; (b) (1) TFA, (2) 2,3-dioxoindoline-5-sulfonyl chloride, Et3N,
CH2C12; (c) (1) NaH, DMF, (2) 3-bromoprop-l-yne (46, 47), BrCH2C6H4OCH2CH2F (33–36, 46, 47); (d) FCH2CH2N3, CuSO4, sodium ascorbate, DMF.
group or a triazole ring, in order to reduce the lipophilicity of the
isatin analogs and improve the potency for inhibiting caspase-3
activity in the whole cell assay. Here we report a new series of isat-
in analogs as caspase-3 inhibitors with improved potency for
inhibiting caspase-3 activity in both the in vitro enzyme assay
and in cyclosporine-induced caspase activation in HeLa cells.
The synthesis of 5-(2-phenoxymethyl-pyrrolidine-1-sulfo-
nyl)isatin and 5-(2-phenoxymethyl-azetidine-1-sulfonyl)isatin
analogs is shown in Scheme 1. The pyridin-3-yloxy group was
introduced via displacement of the tosylate group of 3 with sodium
pyridin-3-olate in DMF to afford N-Boc-2-(phenoxymethyl)azeti-
dine 4. The N-Boc group of 4 was removed with TFA and the sec-
ondary amine was coupled with 2,3-dioxoindoline-5-sulfonyl
chloride in THF using triethylamine as an acid scavenger to afford
the (S)-5-(2-((pyridin-3-yloxy)methyl)azetidin-1-ylsulfonyl)indo-
line-2,3-dione 5. The isatin nitrogen was alkylated by treatment
of 5 with sodium hydride in DMF at 0 °C followed by addition of
1-(bromomethyl)-4-(2-fluoroethoxy)benzene or 1-(chloromethyl)-
4-methoxybenzene to give compounds 12 and 13. The isatin com-
pound 12 or 13 were treated with malononitrile in methanol to
give the corresponding isatin Michael addition acceptor (IMA) ana-
logs 19 and 20 as a red solid. Similarly, the isatin nitrogen of 6–8,
was alkylated with alkyl halide intermediates to give 9–11, and
14–16, which were converted to their IMA analogs 17, 18, 21–23,
respectively. The preparation of alkyl halide intermediates is
shown in Scheme 2.
The synthesis of the triazole-containing isatin analogs is shown
in Scheme 3. The isatin nitrogen of 5–8 was first alkylated by treat-
ment of 5–8 with sodium hydride in DMF at 0 °C followed by addi-
tion of propargyl bromide to give alkyne analogs 33–36,
respectively. The triazole analogs 37–40 were prepared by cop-
per(I) catalyzed 1,3-dipolar cycloaddition (i.e., ‘Click reaction’) of
2-fluoroethylazide with 33–36, respectively. While this research
was ongoing, Aboagye and colleagues reported
a series of

W. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2192–2197
2195
Table 1
Inhibition activities for caspase-1, -3, -6, -7, and -8
b
Compoundsa
IC50 (nM)
Log Pc
Caspase-1
Caspase-3
Caspase-6
Caspase-7
Caspase-8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
37
38
39
40
48
49
50
51
>20,000
>50,000
>25,000
>10,000
>15,000
>25,000
>50,000
>20,000
4775 178
5733 1102
2900 90
>36,000
>10,000
>10,000
3500 960
>20,000
>20,000
>20,000
>20,000
>50,000
>50,000
>20,000
>20,000
8.6 1.0
27.9 3.6
26.9 4.4
3.6 0.5
3.3 0.1
10.9 1.2
13.7 1.0
3.1 0.4
22.4 2.1
24.4 2.0
7.1 0.6
6.0 0.8
18.3 0.4
23.3 0.4
7.5 0.2
11.0 0.2
5.6 0.5
12.2 1.5
4.5 0.7
12.0 2.1
15.2 1.5
13.4 2.0
10.8 0.4
4930 1240
>10,000
2987 118
8700 140
9200 600
>10,000
7666 1361
6900 850
747 91
1012 55
509 55
26.1 1.5
28.9 4.1
19.2 1.2
17.6 0.4
10.2 1.1
11.9 2.0
17.6 0.4
11.3 0.6
292 60
68.3 6.9
45.0 4.3
50.0 11.6
96.3 20.7
70.0 4.5
26.5 1.3
18.0 2.8
6.1 1.8
>25,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>25,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
>50,000
4.2 0.2
3.63
3.23
2.87
2.57
2.34
3.79
3.44
3.13
3.08
2.68
2.02
1.80
3.65
3.25
2.59
1.22
0.16
1.78
0.73
3.79
4.36
1.39
1.95
450 43
927 35
1165 256
770 119
6950 915
7750 676
9967 1079
13,450 636
3475 727
9125 2647
4700 712
4425 532
12.4 1.4
3.8 0.2
15.4 1.3
12.3 2.2
15.3 2.5
11.6 1.1
Ac-IETD-CHO
Ac-YVAD-CHO
Ac-VEID-CHO
Ac-DEVD-CHO
8.4 0.6
8.0 2.0
2.2 0.4
2.9 0.4
a
b
c
All tested compounds in the manuscript possess a purity of at least 95% as determined by elemental analysis.
IC50 values are the mean SD of at least three independent assays.
Calculated value using the C log P.
triazole-containing isatin analogs containing a fluorine-substituted
benzene ring in the phenoxymethyl moiety.19,20 These analogs
showed high caspase-3 inhibition potency and improved in vivo
stability relative to the corresponding unsubstituted phenoxy-
methyl analogs.19,20 In fact, [18F]51 has shown potential for imag-
ing chemotherapy-induced caspase-3 activation in microPET
imaging studies of an animal model of lymphoma.20 Therefore
2,4-difluorophenyloxy isatin analogs 48–50 were also prepared
using the same procedure as that of 12 and 37, respectively
(Scheme 3) and compared with 51 in the enzymatic and whole cell
assays for inhibiting caspase-3 activity.
described previously;12 the IC50 values for each compound are
summarized in Table 1. All of the tested compounds have no inhi-
bition of caspase-8, and little or very weak inhibition of caspase-1.
The isatin analogs 9–16 and their IMA analogs 17–23 show high
potency and selectivity for the inhibition of caspase-3. Compounds
9–16 show comparable inhibition potency for caspase-7, whereas
the IMA analogs 17–23 show weaker inhibition for caspase-7 than
for caspase-3. The pyridine-containing analogs, 5-(2-pyridin-3-yl-
oxymethylpyrrolidine-1-sulfonyl)isatin 16 (IC50: 3.1 nM) and its
IMA analog 23 (IC50: 7.5 nM) have higher potency for inhibition
of caspase-3 in the enzyme assays than the corresponding phen-
oxymethyl-containing analog, WC-II-89 (IC50: 9.7 nM) and its
IMA analog 21 (IC50: 18.3 nM). The same trend holds for 5-(2-pyri-
din-3-yl-oxymethylazetidine-1-sulfonyl)isatin and its IMA analog
The inhibition potency of the isatin and representative IMA ana-
logs for recombinant human caspase-3 and other caspases in the
enzyme assays was assessed using a standard fluorometric assay
Table 2
Inhibition activities for caspase-3 in HeLa cell
O
O
O
S
N
O
N
n
R
O
X
a
a
Compound
n
X
R
EC50
(
lM)
Compd
n
X
R
EC50
(l
M)
6
7
8
1
2
2
1
2
2
CH
CH
N
CH
CH
N
H
H
H
CH3
CH3
CH3
9.57 2.27
11.50 1.50
1.83 0.29
3.25 0.25
7.67 2.02
1.80 0.26
55b
2
1
2
2
1
1
2
CH
CH
N
CH
N
Phenyl
Phenyl
Phenyl
4-MeO–phenyl
4-MeO–phenyl
4-MeO–Phenyl
2.80 0.72
3.67 0.76
0.65 0.18
3.07 1.01
0.45 0.12
0.45 0.15
56b
5b
52b
53b
54b
13
58b
N
a
EC50 values are the mean SD of at least three independent assays.
Ref. 12.
b

2196
W. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2192–2197
Table 3
(12 IC50: 3.6 nM, 19 IC50: 7.1 nM) versus the corresponding
phenoxymethyl-containing analogs (9 IC50: 8.6 nM, and 17 IC50
Inhibition activities for caspase-3 in HeLa cells
:
22.4 nM). The IMA analogs have higher inhibition potencies for
caspase-6 than their corresponding isatin analogs, which is consis-
tent with our previous observations.15 The 2,4-difluorophenoxym-
ethyl-containing
analogs,
5-(2-(2,4-difluorophenoxymethyl)-
pyrrolidine-1-sulfonyl)isatin 49 and 5-(2-(2,4-difluorophenoxym-
ethyl)azetidine-1-sulfonyl)isatin 48, have high selectivity and
enzymatic activity for inhibition of caspase-3 and caspase-7. The
triazole analogs, compounds 37–40, 50, and 51, also showed high
selectivity and activity for inhibition of caspase-3 and caspase-7
in the enzyme assays. It is interesting to note that the triazole-con-
taining isatin analogs have a high potency for inhibiting caspase-7
activity, with IC50 values for inhibiting caspase-7 similar to that for
caspase-3 (e.g., 38 IC50: 5.6 nM and 40 IC50: 4.5 nM for caspase-3 vs
38 IC50: 6.1 nM and 40 IC50: 3.8 nM for caspase-7). Otherwise, for
most of the isatin compounds reported, their inhibition potency
for caspase-3 is usually 3–4 times higher than that for caspase-7.
To our knowledge, this is the first report of caspase inhibitors dis-
playing inhibition potencies for caspase-7 similar to or better than
for caspase-3.
a
Compound
n
X
R
EC50 (lM)
9
1
CH
2.00 0.44
2.15 0.45
4.50 1.10
1.69 0.18
1.30 0.25
0.38 0.07
0.39 0.09
OCH2CH2F
OCH2CH2 2
OCH2CH2F
OCH2CH2 2
OCH2CH2 3
OCH2CH2F
OCH2CH2F
10
1
2
2
2
1
2
CH
CH
CH
CH
N
F
WC-II-89b
The Apo-OneÒ Homogeneous Caspase-3/7 Assay (Promega Cor-
poration, Madison, WI) was used to screen potential apoptosis
inhibitors against caspase-3 activity in cell culture following the
manufacturer’s protocol with slight modifications. HeLa cells,
grown under appropriate cell culture conditions, were plated at a
density of 1–2 Â 104 cells per well in 96-well format and allowed
to adhere overnight at 37 °C in a 5% CO2 atmosphere. Cells were
14
15
12
16
F
F
N
then induced into apoptosis following treatment with 0.5 lg/ml
staurosporine in the presence of potential caspase-3 inhibitors.
After induction, a cell permeable caspase-3/7 substrate Z-DEVD-
R110 was added and the hydrolysis of the substrate was measured
N
N
N
N
N
37
38
39
1
1
2
CH
N
0.80 0.06
0.42 0.10
0.93 0.21
N
F
F
F
F
using
a
Victor3V microplate fluorometer (Perkin Elmer Inc.,
N
Waltham, MA) at excitation wavelength 485 nm and emission
wavelength 535 nm. Dose–response curves were plotted and EC50
values were determined for each compound. EC50 values were
calculated from no less than three independent experiments. Data
from the whole cell assays are shown in Tables 2 and 3. The phen-
oxymethyl analog 5-(2-phenoxymethylazetidine-1-sulfonyl)isatin
N
N
CH
N
N
N
40
2
n
1
0.35 0.04
N
6 (EC50: 9.57
pase-3 in whole cell assays than the 5-(2-phenoxymethylpyrroli-
dine-1-sulfonyl)isatin (EC50 11.50 M), but the more
hydrophilic pyridine analog, 5-(2-pyridin-3-yl-oxymethylpyrroli-
dine-1-sulfonyl)isatin 8 (EC50: 1.83 M) has the highest potency
lM) has a slightly higher inhibition potency for cas-
a
Compd
48
R
EC50
(l
M)
7
:
l
1.39 0.40
2.50 0.80
OCH2CH2F
OCH2CH2F
l
for inhibiting caspase-3 in the whole cell assay. A good correlation
exists between the relative whole cell assays with their in vitro en-
zyme assays for caspase-3 or -7 inhibition activities.11,12,15 As re-
ported previously, alkylation of the isatin nitrogen with methyl,
benzyl or a substituted benzyl group also resulted in an increase
in potency for inhibition of caspase-3 in the whole cell assay. The
azetidine analogs, 5-(2-phenoxymethylazetidine-1-sulfonyl)isatin
52 and 55, have higher potency for inhibition of caspase-3 than
their corresponding pyrrolidine analogs, 5-(2-phenoxymethylpyrr-
olidine-1-sulfonyl)isatin 53 and 2, and the pyridine-containing
5-(2-pyridin-3-yl-oxymethylpyrrolidine-1-sulfonyl)isatin analogs
have increased potency for inhibiting caspase-3 in whole cell as-
says compared with their corresponding phenoxymethyl isatin
analogs. As shown in Table 3, the results from the whole cell assay
are mirrored in the enzyme assay results shown in Table 1. For the
5-(2-phenoxymethylpyrrolidine-1-sulfonyl)isatin analogs, starting
from the fluoroethoxy benzyl analog (WC-II-89), increasing the
chain with an ethylene glycol unit resulted in higher potency for
inhibiting caspase-3 in the whole cell assay as well as in the
49
50
2
1
N
N
N
1.06 0.20
0.68 0.39
N
F
F
N
51
2
N
a
IC50 values are the mean SD of at least three independent assays.
Ref. 14.
b
inhibition potency for caspase-3 in the whole cell assay compared
with their corresponding fluoroethoxybenzyl analogs (e.g., 37 vs 9;
39 vs WC-II-89). Similarly, the pyridine-containing isatin analogs
always give rise to the most potent inhibition for caspase-3 in
the whole cell assay, and even the introduction of triazole group
did not increase the potency further as shown. The 2,4-difluoro-
phenoxymethyl-containing isatin analogs afforded comparable re-
sults with the phenoxymethyl isatin analogs in the whole cell
assay. It is worthwhile to mention that compound 51, which was
reported by the Aboagye and colleagues19 as a potential PET imag-
ing tracer, also has very good inhibition potency for caspase-3 in
the whole cell assay.
enzyme assays (WC-II-89 EC50: 4.50
15 EC50: 1.30 M, respectively). For the phenoxymethyl isatin ana-
logs, the introduction of a triazole group also resulted in improved
lM, 14 EC50: 1.69 lM, and
l

W. Chu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2192–2197
2197
The current study was focused on the development of new cas-
Supplementary data
pase-3 inhibitors as radiotracers that are specific for imaging apop-
tosis using PET, as well as drugs for the treatment of diseases
associated with unregulated apoptosis. As an efficient drug or radi-
otracer for PET imaging, the inhibitor has to penetrate the cell eas-
ily in vivo to arrive at the target. For the neurodegenerative
diseases, the inhibitors need to cross the blood–brain barrier by a
passive diffusion process. The 5-(2-phenoxymethylpyrrolidine-1-
sulfonyl)isatin analogs having a pegylated moiety in the N-benzyl
region displayed an increase in potency for inhibiting caspase-3
in the whole cell assay and a lower log P value relative to WC-II-
89. Molecular modeling studies have also provided evidence of a
possible hydrophilic interaction between pyridyloxymethyl moiety
and the S3 binding domain of caspase-3.12,22 In addition to reduc-
ing the log P of the compound, this pyridine-for-benzene substitu-
Supplementary data (synthetic procedures, NMR and elemental
analysis data) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2011.03.015.
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tion results in a favorable
p–p interaction as well as a hydrogen
bond formation between the pyridine nitrogen and Phe381 in the
active site of caspase-3. Consequently, the pyridyloxymethyl isatin
analogs have a higher potency for inhibiting caspase-3 in both the
in vitro enzyme assay and the whole cell assay than the corre-
sponding benzoxymethyl isatin analogs. Furthermore, alkylation
of the isatin nitrogen with a substituted triazole ring resulted in
a further decrease in log P value and an increase in potency for
inhibiting caspase-3 in whole cell assay.
In conclusion, we synthesized a series of isatin analog com-
pounds as caspase-3 inhibitors and evaluated their activities for
inhibiting caspase-3 in enzyme and whole cell assays. The isatin
analogs containing a hydrophilic group, such as a pyridine ring,
polyethyleneglycol group, and a triazole ring, resulted in a dra-
matic increase in inhibiting caspase-3 and -7 in both the in vitro
enzyme assays using recombinant enzyme and in HeLa cells trea-
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Acknowledgment
This research was funded by CA121952 and HL13851 awarded
by the National Institutes of Health.
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