Design, synthesis, and biological evaluation of isoquinoline-1,3,4-trione derivatives as potent caspase-3 inhibitors

Article abstract of DOI:10.1021/jm050896o

A series of isoquinoline-1,3,4-trione derivatives were identified as novel and potent inhibitors of caspase-3 through structural modification of the original compound from high-throughput screening. Various analogues (2, 6, 9, 13, and 14) were synthesized and identified as caspase inhibitors, and the introduction of a 6-N-acyl group (compound 13) greatly improved their activity. Some of them showed low nanomolar potency against caspase-3 in vitro (for example, for 6k, IG₅₀ = 40 nM) and significant protection against apoptosis in a model cell system. Additionally, compound 13f demonstrated a dose-dependent decrease in infarct volume in the transient MCA occlusion stroke model. The present small-molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.

Full text of DOI:10.1021/jm050896o

J. Med. Chem. 2006, 49, 1613-1623
1613
Design, Synthesis, and Biological Evaluation of Isoquinoline-1,3,4-trione Derivatives as Potent
Caspase-3 Inhibitors
Yi-Hua Chen,† Ya-Hui Zhang,† Hua-Jie Zhang,† Da-Zhi Liu, Min Gu, Jing-Ya Li, Fang Wu, Xing-Zu Zhu,* Jia Li,* and
Fa-Jun Nan*
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, 189 Guoshoujing Road,
Zhangjiang Hi-Tech Park, Shanghai, 201203, PRC
ReceiVed September 12, 2005
A series of isoquinoline-1,3,4-trione derivatives were identified as novel and potent inhibitors of caspase-3
through structural modification of the original compound from high-throughput screening. Various analogues
(2, 6, 9, 13, and 14) were synthesized and identified as caspase inhibitors, and the introduction of a 6-N-
acyl group (compound 13) greatly improved their activity. Some of them showed low nanomolar potency
against caspase-3 in vitro (for example, for 6k, IC50 ) 40 nM) and significant protection against apoptosis
in a model cell system. Additionally, compound 13f demonstrated a dose-dependent decrease in infarct
volume in the transient MCA occlusion stroke model. The present small-molecule caspase-3 inhibitor with
novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic
strategies directed against diseases involving abnormally up-regulated apoptosis.
Introduction
Apoptosis, or programmed cell death, has received increasing
attention over the past 20 years for its critical roles in several
neurodegenerative disorders. Excessive levels of apoptosis,
which can be triggered by a number of stimuli,1,2 are detrimental.
Among the more studied stimuli, cysteinyl-aspartate-specific
proteases (caspases) are characterized by their specific cleavage
Figure 1. Original hit isoquinoline-1,3,4-trione (1).
of an aspartic acid residue from their respective peptide
substrates in the presence of a histidine residue. This property
is known to play essential and distinct roles in apoptotic cell
death, and caspases are consequently potential targets for drug
therapies.3 So far, a total of 11 human caspases have been
reported since the identification and isolation of interleukin-
1â-converting enzyme (ICE, caspase-1), the first human caspase
described.4 They can be divided into three groups according to
the extent of their sequence identity, their biological function,
and substrate specificity. Group I caspases 1, 4, 5, and 13 are
involved primarily in inflammation. Group II caspases 6, 8, 9,
and 10 are initiators of apoptosis, and group III caspases 2, 3,
and 7 are the dominant effector caspases in apoptosis.5 Although
various apoptotic signaling pathways have been discovered,
caspase-3 is activated in nearly every model of apoptosis and
is believed to be the “central executioner” of the apoptotic
pathway.6 Until now, several inhibitors of caspase-3 have been
reported, but most were designed based on a tetrapeptide
substrate, and their utility was limited by intrinsic properties
such as poor selectivity, whole-cell activity, and low stability
in vivo.7
(Figure 1). In the present paper, we reported our discovery of
a series of isoquinoline-1,3,4-trione derivatives as novel and
potent inhibitors of caspases through structural modification
based on screening hit 1. A series of isoquinoline-1,3,4-trione
derivatives showed low nanomolar potency against caspase-3
in vitro, and some of them showed significant protection against
apoptosis in a model cell system and a dose-dependent protec-
tion efficiency in the transient MCA occlusion stroke model.
Chemistry
Isoquinoline-1,3,4-trione derivatives were prepared according
to Scheme 1. Compound 1 was alkylated by treatment with
potassium carbonate or sodium hydride in dimethylformamide
(DMF) for the appropriate time, and then the resulting salt
reacted with an alkyl halide at ambient temperature to give
N-alkyl-isoquinoline-1,3,4-trione (2).8
For preparing compounds 6, 5-nitro-isoquinoline (3) was used
as the starting material to become 5-nitro-isoquinolone (4)
according to the literature,9 and 4 was further reduced with tin-
(II) chloride dihydrate to produce 5-amino-isoquinolone (5).10
The corresponding acryl chlorides were reacted with 5-amino-
isoquinolone (5) in the presence of pyridine and were oxidized
with potassium dichromate to yield the desired target compounds
(6).11
Compounds 9, 12, and 13 were derived from 6-nitro-4H-
isoquinoline-1,3-dione (7). Compound 8 was obtained from 7
easily by the usual catalytic hydrogenation in DMF under a
hydrogen atmosphere, which was further converted to com-
pounds 9 in the presence of the corresponding acyl chloride
and pyridine as the base and subsequently oxidized with
To find small-molecular caspase-3 inhibitors with a novel
scaffold directed against diseases involving abnormally upregu-
lated apoptosis, our work was initially based on high-throughput
screening of a diverse small-molecule library of 8000 com-
pounds using recombinant caspase-3. Isoquinoline-1,3,4-trione
(1), with an IC50 of 0.15 µM, was identified as a leadlike hit
* To whom all correspondence should be addressed. F.-J.N.: tel/fax,
+86-21-50800954; e-mail, fjnan@mail.shcnc.ac.cn. J.L.: tel/fax, +86-21-
50801552; e-mail, jli@mail.shcnc.ac.cn. X.-Z.Z.: tel/fax, +86-21-50806096;
e-mail, xzzhu@mail.shcnc.ac.cn.
† Those authors contributed equally.
10.1021/jm050896o CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/14/2006

1614 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Scheme 1. Synthesis of Substituted Isoquinoline-1,3,4-trione
Chen et al.
selenium(IV) oxide.12 Treatment of 8 with succinic anhydride
or glutaric anhydride (10) afforded compound 11 with a good
yield.13 Compounds 13 were synthesized by coupling acid 11
with diverse commercially available amines, followed by
oxidation with selenium(IV) oxide. N-(1,3,4-Trioxo-1,2,3,4-
tetrahydro-isoquinolin-7-yl)-amides (14) were produced using
a procedure similar to that used to produce compounds 9.
Table 1. N-Substituted Isoquinoline-1,3,4-trione
compound
R
IC50 (µM)
Results and Discussion
Ac-DEVD-CHO
1
0.021 ( 0.001
0.149 ( 0.015
0.255 ( 0.008
0.261 ( 0.037
0.331 ( 0.039
0.544 ( 0.016
4.63 ( 0.56
Compound 1 was obtained to confirm the scaffold compound,
and a series of derivatives were synthesized to evaluate their
potential in terms of modification of the initial compound, to
clarify their structure-activity relationships. The replacement
of the hydrogen atom at the imide group of 1 with an alkyl
(2a, 2d), allyl (2b), benzyl (2c), or substituted benzyl (2e, 2f)
led to a decrease in inhibitory potency (Table 1). Related
experiments also suggested that the N-substitution contributed
to a loss of stability in aqueous solution over a long period11
compared with the original compound 1. Consequently, we
considered that N-substitution would reduce the potency and
stability of the derivatives and was therefore not suitable for
further modifications.
H
CH3-
2a
2b
2c
2d
2e
2f
CH2CHCH2-
C6H5CH2-
C6H5CH2CH2-
p-MeO-C6H4CH2-
p-F-C6H4CH2-
0.63 ( 0.26
small library with the general structure shown in Table 2 was
designed, in which four easily obtainable acyl groups were
attached directly to the 5, 6, or 7 position of the parent
compound by an amide backbone. A total of 12 compounds
were synthesized. The activity results for these compounds are
listed in Table 2. It seems that caspase-3 is sensitive to the
position of the substituted group on the benzene ring. The
We next turned our attention to modifying the benzene ring
of compound 1. To determine the best substitution position, a

Potent Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1615
Table 2. Importance of the Position of a Substitution
Table 4. Further Modification Based on the Scaffold of Compound 12a
IC50 (µM) to caspase-3
position 6
0.110 ( 0.014 0.176 ( 0.007 0.262 ( 0.043
(6a) (9a) (14a)
o-MeO-C6H4- 0.096 ( 0.011 0.103 ( 0.008 0.437 ( 0.022
(6b) (9b) (14b)
p-NO2-C6H4- 0.124 ( 0.021 0.054 ( 0.006 1.38 ( 0.26
(6c) (9c) (14c)
0.150 ( 0.007 0.078 ( 0.004 0.384 ( 0.032
(6d) (9d) (14d)
entry
1
R
position 5
position 7
Ph-
2
3
4
o-Cl-C6H4-
Table 3. Diverse Compounds at the 5 and 6 Positions
1, and several compounds showed potency lower than 100 nM
(compounds 6b, 6g, 6h). Insertion of CH2 between the aromatic
ring and the carbonyl group commonly led to an indefinite result
(compounds 6l, 6m, 6n), but an acetyloxy substitution in CH2
significant increased activity (compound 6k). These results
indicated some groups may be tolerated in this position and
provide us a new direction for further modification. To the 6
positions, most of the compounds showed potent inhibition
activity when the benzene ring was electron-deficient (com-
pounds 9c, 9d, 9g, 9i). Although the attachment of a fatty
acylamino directly to the parent scaffold decreased the inhibitory
activity, the replacement of the terminal hydrogen atom of the
chain by an electron-deficient group, such as Cl, COOH,
enhanced potency significantly (compounds 9k, 12a), especially
when these groups were isolated by two CH2.
Compounds 12a and 12b interested us because of their
conspicuous inhibitory potency against caspase-3 and the ease
for advanced modification, as well as their structural feature of
two carbonyl groups isolated by two or three CH2 groups. These
compounds indicated that a suitable distance between the
substituted group and the benzene ring may be required for
potent inhibition. With the goal being to increase inhibition
activity and the hydrophobicity of the compounds to permeate
the cell more easily, 12a was further explored by amidation of
the acid on the basis that the structure should be as concise as
possible. A set of diverse, commercially available amines were
used as substrates to couple with 12a to afford compounds 13
by the usual method. The effects of these compounds on
caspase-3 are summarized in Table 4. Most of the compounds
derived from fatty amines showed excellent inhibition of
caspase-3 (IC50 values in the range of 10-60 nM). Compound
13c had IC50 values of 25 nM, which means its potency of
tendency is obvious: The results for a group substituted at the
5 or 6 position were superior to those for the group substituted
at the 7 position (entries 1-4), but there was no apparent
difference between the 5 position and 6 position of the
derivatives examined. These results indicate that the benzene
ring of compound 1 is suitable for modification to improve the
activity of the inhibitor and that the 5 and 6 positions are much
more effective in increasing the inhibitory potency than is the
7 position.
To further improve the inhibitory potency and determine the
effect of the substituted group at the 5 and 6 positions, a series
of compounds substituted at the 5 or 6 positions with a diverse
series of groups, including fatty acylamino and aromatic
acylamino groups, was prepared, and their biological activity
was measured. The inhibitory activities of these compounds
toward caspase-3 are summarized in Table 3. To the 5 position,
the introduction of a simple alkyl or cycloalkyl substitution did
not increase the inhibitory potency (compounds 6e, 6f). Com-
pounds with the aryl acylamino group showed enhanced
inhibitory activity compared with that of the initial compound

1616 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Chen et al.
Table 5. Selectivity of Inhibitors among Various Protease
IC50 (µM)
some selectivity for caspases 3 and 7 compared with caspases
2, 6, and 8; however, these derivatives should be considered as
broad-spectrum caspases inhibitors (Table 6). Given that apo-
ptosis signal transduction involves the activation of multiple
caspases, the ability to inhibit most of the caspases is a desirable
feature in a cytoprotective drug when optimal inhibition of
apoptosis is being pursued.15 Although the exact mechanism of
action for this type of inhibitor is not clear, the characteristics
of the noncompetitive routine active site could be suggested on
the basis of our preliminary experiments, which will be reported
elsewhere.
compd
caspase-3
calpain-1 proteasome papain trypsin thrombin
1
6k
9k
0.149 ( 0.015
0.040 ( 0.003
0.083 ( 0.010
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
>5
12a 0.068 ( 0.006
13c
13f
14c
0.025 ( 0.003
0.113 ( 0.011
1.38 ( 0.26
inhibition was increased more than 5-fold relative to that of
the original compound 1. In general, amides derived from the
corresponding amines bearing a fatty amine resulted in an
increase in potency. The introduction of an aromatic ring with
an electron-donating and hydrophobic chain increased potency
compared to the corresponding nonsubstituted aniline, and the
result was more obvious when the substitution was at the meta
position of aniline. Also, its effectiveness was increased if the
size of the substituted group was suitable, like compound 13j.
Selectivity. The selectivity of seven inhibitory compounds
against five cysteine or serine proteases and five caspases was
determined (Table 5 and Table 6). Although keto-amide
compounds were thought to inhibit the activity of cysteine or
serine proteases, the results of selectivity experiments indicated
that the present compounds have excellent selectivity for
caspases over other cysteine or serine proteases, which suggested
that these compounds are not general protease inhibitors, as
shown in Table 5.14 With respect to selectivity among caspase
family members, the compounds derived from hit 1 showed
The activities of these compounds were initially evaluated
in human Jurkat T cells, as a cell-based model of apoptosis.
The cells were treated with camptothecin to induce apoptosis.
The ability of the compounds to inhibit apoptosis was assessed
by staining the cells with acridine orange and ethidium bromide
for morphological analysis under fluorescence microscopy (data
not shown) and by detecting caspase-3-like enzyme activity
through the hydrolysis of the caspase substrate Ac-DEVD-AMC.
Ac-DEVD-CHO and Z-VAD-fmk were used as positive controls
in parallel with compounds 1 and 6k. Compounds 1 and 6k
demonstrated some level of protection, as reflected in the level
of caspase-3 activity (Figure 2). Compound 6k showed much
better protective effects than compound 1, corresponding to their
different inhibition potency against caspase enzyme activity.
The results of the studies with Jurkat T cells revealed a
significant shift in the action of the inhibitors against enzyme
activities isolated in vitro compared with the corresponding cell-
based activities. Such shifts in activity are normally associated
Figure 2. Concentration-dependent inhibition of caspase activity (Ac-DEVD-AMC) in camptothecin-induced apoptotic Jurkat T cells by caspase-3
inhibitors 1 and 6k. After treatment with 2 µM camptothecin only, caspase-3 activity increased from 2.7 ( 0.1 RFU (relative fluoresence unit) µg-1
min-1(negative control) to 18.3 ( 0.7 RFU µg-1 min-1 in cell lysate. Treated with four caspase-3 inhibitors and camptothecin, caspase-3 presented
a low activity compared with camptothecin only. The caspase-3 activities were 3.4 ( 0.1 and 3.6 ( 0.3 RFU µg-1 min-1 when treated with two
positive inhibitors, Ac-DEVD-CHO (2 µM) and Z-VAD-fmk (2 µM), respectively. With 40 µM compounds 1 and 6k, the caspase-3 activities were
8.7 ( 0.3 and 4.8 ( 0.5 RFU µg-1 min-1, respectively: (//) denotes P < 0.005 compared with camptothecin treatment alone.
Table 6. Selectivity of Inhibitors among Various Caspases
IC50 (µM)
compd
caspase-2
caspase-3
caspase-6
caspase-7
caspase-8
1
6k
9k
12a
13c
13f
14c
1.53 ( 0.24
0.149 ( 0.015
0.040 ( 0.003
0.083 ( 0.010
0.068 ( 0.006
0.025 ( 0.003
0.113 ( 0.011
1.38 ( 0.26
0.474 ( 0.083
0.216 ( 0.014
0.283 ( 0.061
0.201 ( 0.006
0.124 ( 0.013
0.137 ( 0.006
2.85 ( 0.36
0.386 ( 0.034
0.063 ( 0.007
0.139 ( 0.015
0.136 ( 0.014
0.064 ( 0.006
0.218 ( 0.024
2.71 ( 0.26
1.91 ( 0.15
0.233 ( 0.027
0.53 ( 0.18
0.425 ( 0.055
0.575 ( 0.003
1.12 ( 0.043
0.288 ( 0.003
0.835 ( 0.016
24.7 ( 0.63
0.859 ( 0.073
0.231 ( 0.034
0.537 ( 0.035
5.67 ( 0.36

Potent Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1617
Figure 3. Detection of DNA fragments in Jurkat T cell apoptosis induced with camptothecin. (A) Compound 1: lane 1, control cells without
camptothecin; lanes 2-6, cells treated with 2 µM camptothecin and varying concentrations of inhibitors; lane 7, compound 1 only, without
camptothecin; lane 8, positive control 2 µM Ac-DEVD-CHO with 2 µM camptothecin; lane 9, 100-bp DNA ladder used as standard. (B) Compound
6k: lane 1, control cells without camptothecin; lanes 2-6, cells treated with 2 µM camptothecin and varying concentrations of inhibitors; lane 7,
compound 6k only, without camptothecin; lane 8, positive control 2 µM Ac-DEVD-CHO with 2 µM camptothecin; lane 9, positive control 2 µM
Z-VAD-fmk with 2 µM camptothecin; lane 10, 100-bp DNA ladder used as standard.
with factors that limit the ability of a compound to reach its
intracellular target (e.g., cell permeability, protein binding, etc.).
However, it is possible that high inhibitor concentrations are
required for activity in cells because the inhibition of a caspase-
(s) other than caspase 3 or 7 (which would inhibit much less
potently than caspase 3 or 7) is also required for antiapoptotic
activity.
The detection of DNA fragments is a diagnostic symbol of
cell apoptosis. Jurkat T cells were treated with camptothecin
for 4 h with or without caspase inhibitors, and the soluble DNA
was extracted from the cells and separated by electrophoresis
(Figure 3). Significant DNA laddering was observed in the
absence of any caspase inhibitor or with the addition of a weakly
active compound (1). Compounds 1 and 6k demonstrated dose-
dependent inhibition of DNA laddering. The inhibitory activities
of compounds 1 and 6k were similar to those observed in the
Jurkat T-cell-based apoptosis assays.
Figure 4. Effects of 1 (1 mg/kg, sc) and 13f (0.5 and 1 mg/kg, sc) on
the infarct volume of ischemic damage in the transient MCA occlusion
model. The bifurcation of the left common carotid artery was exposed,
and the right MCA was occluded by insertion of a silicon-coated nylon
suture (USS-DG, Dermalon) through the common carotid artery. After
closure of the operative sites, the animals were temporarily transferred
to a cage with a heating lamp and the suture was gently removed at
1.5 h after MCA occlusion. Drugs were dissolved in DMSO and
subcutaneously injected 5 min after the start of MCA occlusion at a
volume of 0.5 mL/kg. Transient occlusion of MCA for 1.5 h resulted
in tissue damage 24 h after occlusion. Each column and vertical bar
represents the mean ( SE of results from eight rats. The operated
animals showed statistical significance at P < 0.01 compared with
saline-treated animals (one-way ANOVA followed by Dunnett’s post
hoc comparison).
Compound 1, 6h, 6k, 13c, 13j, and 13f with structural
diversity and various in vitro potencies were selected for primary
animal brain ischemia studies in the widely accepted transient
middle cerebral artery (MCA) occlusion stroke model.16 These
studies were performed using a method described previously,
with minor modification.17 Five of them, compounds 6h, 6k,
13c, 13j, and 13f showed obvious protection efficiency, and
compound 1 was inactive at 1 mg/kg sc dose (data not shown).
Compound 13f was selected for further evaluation due to its
advantage in preparation and solubility. Administration of 13f
produced a dose-dependent decrease in infarct volume (Figure
4) of up to 77% at 1 mg/kg sc even though its parent compound
1 (1 mg/kg, sc) did not show a significant efficiency at the same
dose. These results show that the caspases inhibitor compound,
designed with structure modification from the original HTS hit,
also inhibits apoptosis in vivo.
Conclusion
In summary, isoquinoline-1,3,4-trione (1) was identified as
a caspase-3 inhibitor by high-throughput screening. The activity
results indicate that the potency of this set of caspase inhibitors
could be optimized. Various analogues (2, 6, 9, 13, and 14)
were synthesized and identified as caspase inhibitors, and the
introduction of a 6-N-acyl group (compound 13) greatly
improved their activity. Additionally, compound 13f demon-
strated a dose-dependent decrease in infarct volume in the
transient MCA occlusion stroke model. These results reveal a
new direction for therapeutic strategies directed against diseases

1618 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Chen et al.
2-(4-Methoxy-benzyl)isoquinoline-1,3,4-trione (2e) was pre-
pared according to the procedure for compound 2c except using
4-methoxybenzyl chloride in 10% yield. H NMR (CDCl3, 300
MHz) δ: 8.34 (dd, J ) 1.2, 7.8 Hz, 1H), 8.19 (dd, J ) 1.5, 7.8
Hz, 1H), 7.89 (ddd, J ) 1.2, 7.5, 7.8 Hz, 1H), 7.81 (ddd, J ) 1.5,
7.5, 7.8 Hz, 1H), 7.47 (d, J ) 8.7 Hz, 2H), 6.82 (d, J ) 8.7 Hz,
2H), 5.17 (s, 2H), 3.76 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ:
174.8, 162.3, 159.5, 157.1, 136.2, 134.6, 131.3, 130.9, 130.0 (2),
128.3, 127.9, 114.1, 55.4, 43.9. EIMS (m/z): 295 (M+). HPLC
purity: system A, 99.5%; system B, 99.3%.
involving abnormally up-regulated apoptosis. Further work is
in progress to optimize the structures of compounds 13 with
the aim of increasing their potency, as well as efficiency, in
vivo.
1
Experimental Section
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded
on a Varian Mercury-VX300 Fourier transform spectrometer. The
chemical shifts were reported in δ (ppm) using the δ 7.26 signal
of CDCl3 (1H NMR) and the δ 77.23 signal of CDCl3 (13C NMR)
as internal standards and using the δ 2.50 signal of DMSO (1H
NMR) and the δ 39.51 signal of DMSO (13C NMR) as internal
standards. Low-resolution mass spectra of 2a-f were obtained on
a SHIMADZU GCMS-QP5050A spectrometer, high-resolution
mass spectra of 6a-n, 9a-l, 12a,b, and 13a-k were obtained on
a Finnigan MAT95 spectrometer, and HPLC analyses data were
performed on an HP 1100 series LC system (HP ChemStation
A.06.03). System A: column, Zorbax SB-C18, 4.6 mm × 150 mm,
5 µm; mobile phase, CH3CN/H2O; flow rate, 1.0 mL/min; UV
wavelength, 254 nm; temperature, ambient temperature; injection
volume, 10 µL. System B: column, Phenomenex Gemini C18, 4.6
mm × 150 mm, 5 µm; mobile phase, CH3OH/H2O (with 0.1%
TFA); flow rate, 1.0 mL/min; UV wavelength, 254 nm; temperature,
ambient temperature; injection volume, 10 µL.
2-(4-Fluoro-benzyl)isoquinoline-1,3,4-trione (2f) was prepared
according to the procedure for compound 2c except using 4-fluo-
1
robenzyl chloride in 38% yield. H NMR (CDCl3, 300 MHz) δ:
8.34 (dd, J ) 1.2, 7.8 Hz, 1H), 8.20 (dd, J ) 1.2, 7.2 Hz, 1H),
7.90 (ddd, J ) 1.2, 7.2, 7.8 Hz, 1H), 7.82 (ddd, J ) 1.2, 7.2, 7.8
Hz, 1H), 7.53-7.48 (m, 2H), 7.01-6.95 (m, 2H), 5.19 (s, 2H).
13C NMR (CDCl3, 75 MHz) δ: 174.7, 164.3, 162.3, 161.0, 157.1,
136.3, 134.8, 131.9, 131.8, 131.7, 130.9, 130.1, 129.9, 128.1, 115.8,
115.5, 43.8. EIMS (m/z): 283 (M+). HPLC purity: system A,
95.8%; system B, 98.6%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-5-yl)-benza-
mide (6a). To a solution of 5-amino-2H-isoquinolin-1-one 5 (53
mg, 0.33 mmol) in 4 mL of acetone was added pyridine (66 µL,
0.83 mmol) and benzoyl chloride (96 µL, 0.83 mmol). The mixture
was stirred for 4-8 h at room temperature, removed of acetone
under vacuum, and then diluted with 10 mL of ethyl acetate and
10 mL of H2O. The aqueous phase was extracted with 10 mL of
ethyl acetate. The combined organic layer was dried over anhydrous
Na2SO4 and evaporated under reduced pressure to afford the crude
intermediate product without further purification. To the mixture
in 2 mL of glacial acetic acid a solution of potassium dichromate
(250 mg, 0.85 mmol) in 2 N HCl (2 mL) was added dropwise at
such a rate that the reaction temperature did not exceed 30 °C.
The mixture was then further stirred at room temperature for 3 h
and extracted with 30 mL of ethyl acetate, and then the combined
ethyl acetate/extract solution was washed with saturated sodium
bicarbonate solution and brine, dried over anhydrous Na2SO4, and
evaporated to dryness under reduced pressure. The product was
purified by chromatography on silica gel using CH2Cl2/acetone in
24% yield. 1H NMR (DMSO, 300 MHz) δ: 12.40 (br, 1H), 12.16
(br, 1H), 9.08 (dd, J ) 1.5, 7.8 Hz, 1H), 8.04 (dd, J ) 1.5, 8.1 Hz,
1H), 7.99-7.92 (m, 3H), 7.69-7.65 (m, 2H), 7.51 (dd, J ) 7.8,
7.8 Hz, 1H). HRMS: calcd for C16H10N2O4, 294.0640; found,
294.0641. HPLC purity: system A, 95.3%; system B, 95.4%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-5-yl)-2-meth-
oxybenzamide (6b) was prepared according to the procedure for
compound 6a except using 2-methoxybenzoyl chloride in 30%
2-Methylisoquinoline-1,3,4-trione (2a). To a solution of iso-
quinoline-1,3,4-trione 1 (40 mg, 0.228 mmol) in 1.5 mL of
anhydrous DMF at 25 °C was added K2CO3 (80 mg, 0.570 mmol),
and then iodomethane was added (28 µL, 0.456 mmol). The reaction
mixture was stirred at room temperature for 2 h. It was diluted
with 10 mL of ethyl acetate and 10 mL of H2O, and the aqueous
phase was extracted with 10 mL of ethyl acetate. The combined
organic phases were processed in the usual way and chromato-
graphed to yield 2a (23 mg, 53%). 1H NMR (CDCl3, 300 MHz) δ:
8.36 (dd, J ) 1.2, 7.8 Hz, 1H), 8.23 (dd, J ) 1.8, 7.5 Hz, 1H),
7.94-7.81 (m, 2H), 3.50 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ:
174.7, 162.6, 157.5, 136.2, 134.7, 130.9, 130.1, 129.9, 128.0, 27.7.
EIMS (m/z): 189 (M+). HPLC purity: system A, 99.8%; system
B, 99.5%.
2-Allylisoquinoline-1,3,4-trione (2b) was prepared according
to the procedure for compound 2a except using allyl chloride in
1
27% yield. H NMR (CDCl3, 300 MHz) δ: 8.35 (ddd, J ) 0.6,
1.2, 7.5 Hz, 1H), 8.22 (ddd, J ) 0.6, 1.5, 7.5 Hz, 1H), 7.94-7.81
(m, 2H), 5.96-5.86 (m, 1H), 5.37-5.22 (m, 2H), 4.66 (dd, J )
1.5, 6.0 Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ: 174.7, 162.1,
156.9, 136.3, 134.7, 134.5, 131.0, 130.1, 130.0, 128.1, 119.4, 43.4.
EIMS (m/z): 215 (M+). HPLC purity: system A, 99.5%; system
B, 99.4%.
1
yield. H NMR (DMSO, 300 MHz) δ: 12.72 (br, 1H), 12.10 (br,
2-Benzylisoquinoline-1,3,4-trione (2c). To a solution of iso-
quinoline-1,3,4-trione 1 (40 mg, 0.228 mmol) in 1.5 mL of
anhydrous DMF at -15 °C was added 60% sodium hydride (14
mg, 0.350 mmol). After the mixture was stirred for 20 min, benzyl
chloride (200 µL, 1.710 mmol) was added. The reaction mixture
was then stirred at room temperature for 2 h and was diluted with
10 mL of ethyl acetate and 20 mL of H2O, and the aqueous phase
was extracted with 10 mL of ethyl acetate. The combined organic
phases were then processed in the usual way and chromatographed
to yield 2c in 23% yield. 1H NMR (CDCl3, 300 MHz) δ: 8.34 (d,
J ) 7.5 Hz, 1H), 8.19 (d, J ) 7.5 Hz, 1H), 7.91-7.79 (m, 2H),
7.51-7.48 (m, 2H), 7.32-7.26 (m, 3H), 5.23 (s, 2H). 13C NMR
(CDCl3, 75 MHz) δ: 174.8, 162.3, 157.1, 136.3, 136.0, 134.7,
131.0, 130.1, 130.0, 129.6, 128.8, 128.2, 128.0, 44.5. EIMS (m/z):
266 (M + 1)+. HPLC purity: system A, 99.6%; system B, 96.9%.
2-Phenethylisoquinoline-1,3,4-trione (2d) was prepared ac-
cording to the procedure for compound 2c except using phenylethyl
1H), 9.18 (dd, J ) 3.0, 6.3 Hz, 1H), 8.04 (d, J ) 7.5 Hz, 1H),
8.06-7.92 (m, 2H), 7.66-7.62 (m, 1H), 7.31 (d, J ) 7.8 Hz, 1H),
7.16 (t, J ) 7.2 Hz, 1H), 4.10 (s, 3H). HRMS: calcd for
C17H12N2O5, 324.0746; found, 324.0749. HPLC purity: system A,
98.5%; system B, 98.4%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-5-yl)-4-nitro-
benzamide (6c) was prepared according to the procedure for
compound 6a except using 4-nitrobenzoyl chloride in 10% yield.
1H NMR (DMSO, 300 MHz) δ: 12.39 (br, 1H), 12.17 (br, 1H),
9.04 (d, J ) 1.8 Hz, 1H), 8.18 (d, J ) 8.7 Hz, 2H), 8.24 (d, J )
8.7 Hz, 2H), 8.00-7.96 (m, 2H). HRMS: calcd for C16H9N3O6,
339.0492; found, 339.0498. HPLC purity: system A, 98.9%; system
B, 97.8%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-5-yl)-2-
chlorobenzamide (6d) was prepared according to the procedure
for compound 6a except using 2-chlorobenzoyl chloride in 15%
yield. 1H NMR (CDCl3, 300 MHz) δ: 12.03 (br, 1H), 9.37 (d, J )
8.1 Hz, 1H), 9.16 (br, 1H), 8.13 (d, J ) 7.5 Hz, 1H), 8.01 (dd, J
) 7.5, 8.1 Hz, 1H), 7.70 (d, J ) 7.8 Hz, 1H), 7.50-7.40 (m, 3H).
HRMS: calcd for C16H9ClN2O4, 328.0251; found, 328.0252. HPLC
purity: system A, 98.3%; system B, 97.0%.
1
bromide in 47% yield. H NMR (CDCl3, 300 MHz) δ: 8.33 (dd,
J ) 1.2, 8.1 Hz, 1H), 8.20 (dd, J ) 1.5, 7.5 Hz, 1H), 7.90 (ddd, J
) 1.5, 7.2, 8.1 Hz, 1H), 7.83 (ddd, J ) 1.2, 7.2, 7.5 Hz, 1H), 7.29-
7.18 (m, 5H), 4.29-4.23 (m, 2H), 2.99-2.93 (m, 2H). 13C NMR
(CDCl3, 75 MHz) δ: 174.7, 162.2, 157.0, 138.0, 136.2, 134.6,
130.9, 129.9 (2), 129.1, 128.8, 127.9, 126.9, 42.5, 34.1. EIMS (m/
z): 279 (M+). HPLC purity: system A, 97.0%; system B, 98.5%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-5-yl)-acet-
amide (6e) was prepared according to the procedure for compound

Potent Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1619
1
6a except using acetyl chloride in 23% yield. H NMR (DMSO,
300 MHz) δ: 12.10 (br, 1H), 11.34 (br, 1H), 8.84 (d, J ) 7.5 Hz,
1H), 7.89-7.87 (m, 2H), 2.25 (s, 3H). HRMS: calcd for C11H8N2O4,
232.0484; found, 232.0484. HPLC purity: system A, 96.4%; system
B, 96.2%.
HRMS: calcd for C17H11FN2O4, 326.0703; found, 326.0699. HPLC
purity: system A, 99.1%; system B, 97.7%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-5-yl)-2-phen-
ylacetamide (6n) was prepared according to the procedure for
compound 6a except using phenylacetyl chloride in 10% yield. 1H
NMR (DMSO, 300 MHz) δ: 12.05 (br, 1H), 11.39 (br, 1H), 8.87
(d, J ) 7.8 Hz, 1H), 7.87-7.85 (m, 2H), 7.41-7.32 (m, 5H), 3.88
(s, 2H). HRMS: calcd for C17H12N2O4, 308.0798; found, 308.0807.
HPLC purity: system A, 95.7%; system B, 96.6%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-benz-
amide (9a). To a solution of compound 8 (53 mg, 0.300 mmol) in
20 mL of dioxane was added benzoyl chloride (70 µL, 0.602 mmol)
and pyridine (60 µL, 0.750 mmol). The reaction mixture was stirred
at room temperature for 5 h and diluted with 30 mL of ethyl acetate
and 30 mL of H2O. The aqueous phase was extracted with 30 mL
of ethyl acetate. The combined organic phases were dried over Na2-
SO4, filtered, and concentrated under reduced pressure to give a
solid, which was transferred to 3 mL of dioxane. SeO2 (33 mg,
0.300 mmol) was added, the mixture was heated to reflux for 1-3
h, and then the solvent was evaporated under reduced pressure and
chromatographed to yield 9a (33 mg, 37%). 1H NMR (DMSO, 300
MHz) δ: 11.87 (br, 1H), 10.81 (br, 1H), 8.55 (d, J ) 2.1 Hz, 1H),
8.31 (dd, J ) 2.1, 8.4 Hz, 1H), 8.13 (d, J ) 8.4 Hz, 1H), 8.03-
7.93 (m, 2H), 7.65-7.48 (m, 3H). HRMS: calcd for C16H10N2O4,
294.0640; found, 294.0638. HPLC purity: system A, 95.8%; system
B, 97.8%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-5-yl)cyclopro-
panecarboxamide (6f) was prepared according to the procedure
for compound 6a except using cyclopropanecarbonyl chloride in
11% yield. 1H NMR (DMSO, 300 MHz) δ: 12.08 (br, 1H), 11.61
(br, 1H), 8.71 (d, J ) 7.2 Hz, 1H), 7.89-7.84 (m, 2H), 1.90-1.88
(m, 1H), 0.94-0.91 (m, 4H). HRMS: calcd for C13H10N2O4,
258.0641; found, 258.0637. HPLC purity: system A, 98.8%; system
B, 98.9%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-5-yl)-4-
fluorobenzamide (6g) was prepared according to the procedure
for compound 6a except using 4-fluorobenzoyl chloride in 10%
1
yield. H NMR (DMSO, 300 MHz) δ: 12.35 (br, 1H), 12.17 (br,
1H), 9.04 (d, J ) 8.1 Hz, 1H), 8.11-8.08 (m, 2H), 7.98-7.94 (m,
2H), 7.52 (dd, J ) 8.7, 8.7 Hz, 2H). HRMS: calcd for C16H9-
FN2O4, 312.0546; found, 312.0540. HPLC purity: system A,
98.6%; system B, 95.8%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-5-yl)-3-nitro-
benzamide (6h) was prepared according to the procedure for
compound 6a except using 3-nitrobenzoyl chloride in 28% yield.
1H NMR (DMSO, 300 MHz) δ: 12.42 (br, 1H), 12.19 (br, 1H),
9.01 (dd, J ) 1.8, 8.1 Hz, 1H), 8.87 (d, J ) 1.8 Hz, 1H), 8.56 (dd,
J ) 0.9, 7.2 Hz, 1H), 8.45 (dd, J ) 0.9, 7.2 Hz, 1H), 8.02-7.96
(m, 3H). 13C NMR (DMSO, 75 MHz) δ: 178.4, 163.4, 162.7, 157.0,
148.2, 140.7, 136.7, 135.2, 133.3, 131.2, 130.9, 127.2, 124.8, 123.8,
122.1, 118.8. HRMS: calcd for C16H9N3O6, 339.0492; found,
339.0490. HPLC purity: system A, 99.3%; system B, 97.8%.
5-Nitro-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-5-yl)-
isophthalamic acid methyl ester (6i) was prepared according to
the procedure for compound 6a except using 3-chlorocarbonyl-5-
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-6-yl)-2-meth-
oxybenzamide (9b) was prepared according to the procedure for
compound 9a except using 2-methoxybenzoyl chloride in 31%
1
yield. H NMR (CDCl3, 300 MHz) δ: 11.87 (br, 1H), 10.69 (br,
1H), 8.50 (d, J ) 1.8 Hz, 1H), 8.17 (dd, J ) 1.8, 8.4 Hz, 1H), 8.10
(d, J ) 8.4 Hz, 1H), 7.63 (dd, J ) 1.5, 7.5 Hz, 1H), 7.57-7.51
(m, 1H), 7.21 (d, J ) 8.1 Hz, 1H), 7.13-7.06 (m, 1H), 3.90 (s,
3H). HRMS: calcd for C17H12N2O5, 324.0746; found, 324.0745.
HPLC purity: system A, 95.3%; system B, 96.0%.
1
nitro-benzoic acid methyl ester in 12% yield. H NMR (DMSO,
300 MHz) δ: 12.41 (br, 1H), 12.20 (br, 1H), 8.98-8.96 (m, 2H),
8.90 (s, 1H), 8.52 (s, 1H), 8.01-7.98 (m, 2H), 3.99 (s, 3H).
HRMS: calcd for C18H11N3O8, 397.0547; found, 397.0547. HPLC
purity: system A, 97.3%; system B, 95.9%.
(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-5-yl)-carbamic acid
benzyl ester (6j) was prepared according to the procedure for
compound 6a except using benzyl chloroformate in 10% yield. 1H
NMR (DMSO, 300 MHz) δ: 12.10 (br, 1H), 11.07 (br, 1H), 8.64
(d, J ) 8.1 Hz, 1H), 7.92 (dd, J ) 7.2, 8.1 Hz, 1H), 7.83 (d, J )
7.2 Hz, 1H), 7.44-7.40 (m, 5H), 5.25 (s, 2H). HRMS: calcd for
C17H12N2O5, 324.0746; found, 327.0749. HPLC purity: system A,
98.5%; system B, 97.6%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-6-yl)-4-nitro-
benzamide (9c) was prepared according to the procedure for
compound 9a except using 4-nitrobenzoyl chloride in 12% yield.
1H NMR (DMSO, 300 MHz) δ: 11.89 (br, 1H), 11.09 (br, 1H),
8.53 (d, J ) 2.1 Hz, 1H), 8.41 (d, J ) 9.0 Hz, 2H), 8.30 (dd, J )
2.1, 8.4 Hz, 1H), 8.25 (d, J ) 9.0 Hz, 2H), 8.16 (d, J ) 8.4 Hz,
1H). HRMS: calcd for C16H9N3O6, 339.0492; found, 339.0494.
HPLC purity: system A, 95.6%; system B, 95.5%.
2-Chloro-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
benzamide (9d) was prepared according to the procedure for
compound 9a except using 2-chlorobenzoyl chloride in 62% yield.
1H NMR (DMSO, 300 MHz) δ: 11.89 (br, 1H), 11.12 (br, 1H),
8.48 (s, 1H), 8.13 (br, 2H), 7.68-7.39 (m, 4H). HRMS: calcd for
C16H9ClN2O4, 328.0251; found, 328.0257. HPLC purity: system
A, 97.0%; system B, 98.5%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-6-yl)propion-
amide (9e) was prepared according to the procedure for compound
9a except using propionyl chloride in 14% yield. 1H NMR (DMSO,
300 MHz) δ: 11.84 (br, 1H), 10.50 (br, 1H), 8.34 (d, J ) 2.1 Hz,
1H), 8.07 (d, J ) 8.7 Hz, 1H), 8.02 (dd, J ) 2.1, 8.7 Hz, 1H), 2.42
(q, J ) 7.5 Hz, 2H), 1.10 (t, J ) 7.5 Hz, 3H). HRMS: calcd for
C12H10N2O4, 246.0641; found, 246.0642. HPLC purity: system A,
98.1%; system B, 97.8%.
(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-5-ylcarbamoyl)-
(phenyl)methyl acetate (6k) was prepared according to the
procedure for compound 6a except using 2-acetoxy-2-phenylacetyl
chloride in 10% yield. 1H NMR (DMSO, 300 MHz) δ: 12.13 (br,
1H), 12.07 (br, 1H), 8.82-8.81(m, 1H), 7.89-7.87 (m, 2H), 7.55-
7.52 (m, 2H), 7.43-7.40 (m, 3H), 6.16 (s, 1H), 2.29 (s, 3H). 13C
NMR (DMSO, 75 MHz) δ: 178.3, 169.4, 168.1, 162.7, 157.0,
140.1, 136.5, 134.9, 130.8, 129.2, 128.9, 127.4, 123.9, 123.5, 118.4,
75.7, 20.7. HRMS: calcd for C19H14N2O6, 366.0852; found,
366.0858. HPLC purity: system A, 95.7%; system B, 97.3%.
2-(4-Nitro-phenyl)-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquin-
olin-5-yl)-acetamide (6l) was prepared according to the procedure
for compound 6a except using (4-nitro-phenyl)-acetyl chloride in
Cyclohexanecarboxylic acid (1,3,4-trioxo-1,2,3,4-tetrahydro-
isoquinolin-6-yl)-amide (9f) was prepared according to the pro-
cedure for compound 9a except using cyclohexanecarbonyl chloride
1
6% yield. H NMR (DMSO, 300 MHz) δ: 12.07 (br, 1H), 11.37
1
(br, 1H), 8.79 (dd, J ) 1.5, 7.5 Hz, 1H), 8.23 (d, J ) 8.4 Hz, 2H),
7.87-7.82 (m, 2H), 7.68 (d, J ) 8.4 Hz, 2H), 4.09 (s, 2H).
HRMS: calcd for C17H11N3O6, 353.0648; found, 353.0656. HPLC
purity: system A, 95.4%; system B, 96.3%.
in 23% yield. H NMR (DMSO, 300 MHz) δ: 11.84 (br, 1H),
10.45 (br, 1H), 8.35 (d, J ) 2.1 Hz, 1H), 8.04 (br, 2H), 2.42-2.38
(m, 1H), 1.85-1.14 (m, 10H). HRMS: calcd for C16H16N2O4,
300.1110; found, 300.1105. HPLC purity: system A, 98.4%; system
B, 97.0%.
2-(4-Fluoro-phenyl)-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-5-yl)-acetamide (6m) was prepared according to the
procedure for compound 6a except using (4-fluoro-phenyl)-acetyl
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-6-yl)-2-nitro-
benzamide (9g) was prepared according to the procedure for
compound 9a except using 2-nitrobenzoyl chloride in 29% yield.
1H NMR (DMSO, 300 MHz) δ: 11.89 (br, 1H), 11.25 (br, 1H),
8.42 (d, J ) 1.8 Hz, 1H), 8.21 (d, J ) 8.1 Hz, 1H), 8.14 (d, J )
1
chloride in 8% yield. H NMR (DMSO, 300 MHz) δ: 12.04 (br,
1H), 11.32 (br, 1H), 8.85 (dd, J ) 1.8, 7.8 Hz, 1H), 7.89-7.82
(m, 2H), 7.46-7.42 (m, 2H), 7.24-7.18 (m, 2H), 3.88 (s, 2H).

1620 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Chen et al.
8.4 Hz, 1H), 8.07 (dd, J ) 1.8, 8.4 Hz, 1H), 7.95-7.90 (m, 1H),
7.86-7.81 (m, 1H), 7.80-7.77 (m, 1H). HRMS: calcd for
C16H9N3O6, 339.0492; found, 339.0492. HPLC purity: system A,
96.5%; system B, 96.7%.
the reaction mixture was diluted with 10 mL of ethyl acetate and
10 mL of H2O, and the aqueous phase was extracted with 10 mL
of ethyl acetate. The combined organic phases were dried and then
concentrated under reduced pressure. The solid was transferred to
3 mL of dioxane and SeO2 (18 mg, 0.162 mmol) was added, the
mixture was heated to reflux for 3-5 h, and then the solvent was
evaporated at reduced pressure and chromatographed to yield 13a
(8 mg, 15%). 1H NMR (DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.57
(br, 1H), 8.35 (d, J ) 1.8 Hz, 1H), 8.06 (d, J ) 8.4 Hz, 1H), 7.98
(dd, J ) 1.8, 8.4 Hz, 1H), 7.88 (t, J ) 5.1 Hz, 1H), 2.99 (q, J )
6.6 Hz, 2H), 2.62 (t, J ) 6.6 Hz, 2H), 2.43 (t, J ) 6.6 Hz, 2H),
1.42-1.35 (m, 2H), 0.83 (t, J ) 7.5 Hz, 3H). 13C NMR (DMSO,
75 MHz) δ: 176.4, 171.6, 171.0, 162.7, 157.5, 144.0, 133.0, 129.5,
124.1, 123.9, 115.5, 40.3, 31.6, 29.8, 22.2, 11.3. HRMS: calcd for
C16H17N3O5, 331.1169; found, 331.1160. HPLC purity: system A,
98.4%; system B, 97.8%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-6-yl)-3-nitro-
benzamide (9h) was prepared according to the procedure for
compound 9a except using 3-nitrobenzoyl chloride in 29% yield.
1H NMR (DMSO, 300 MHz) δ: 11.90 (br, 1H), 11.11 (br, 1H),
8.89-8.83 (m, 1H), 8.53 (d, J ) 2.4 Hz, 1H), 8.50-8.45 (m, 2H),
8.33 (dd, J ) 2.4, 8.4 Hz, 1H), 8.16 (d, J ) 8.4 Hz, 1H), 7.91-
7.86 (m, 1H). HRMS: calcd for C16H9N3O6, 339.0492; found,
339.0493. HPLC purity: system A, 95.2%; system B, 95.8%.
Methyl 3-(1, 2, 3, 4-tetrahydro-1, 3, 4-trioxoisoquinolin-6-
ylcarbamoyl)-5-nitro-benzoate (9i) was prepared according to the
procedure for compound 9a except using methyl 3-(chlorocarbonyl)-
1
5-nitrobenzoate in 40% yield. H NMR (DMSO, 300 MHz) δ:
11.91 (br, 1H), 11.25 (br, 1H), 9.09 (dd, J ) 1.8, 1.8 Hz, 1H),
8.95 (dd, J ) 1.8, 1.8 Hz, 1H), 8.80 (dd, J ) 1.8, 1.8 Hz, 1H),
8.51 (d, J ) 2.1 Hz, 1H), 8.33 (dd, J ) 2.1, 8.4 Hz, 1H), 8.16 (d,
J ) 8.4 Hz, 1H), 3.99 (s, 3H). HRMS: calcd for C18H11N3O8,
397.0547; found, 397.0546. HPLC purity: system A, 99.1%; system
B, 98.8%.
4-Chloro-N-(1,2,3,4-tetrahydro-1,3,4-trioxoisoquinolin-6-yl)-
butanamide (9j) was prepared according to the procedure for
compound 9a except using 4-chlorobutanoyl chloride in 40% yield.
1H NMR (DMSO, 300 MHz) δ: 11.85 (br, 1H), 10.61 (br, 1H),
8.35 (d, J ) 1.8 Hz, 1H), 8.07 (d, J ) 8.4 Hz, 1H), 8.00 (dd, J )
1.8, 8.4 Hz, 1H), 3.72 (t, J ) 6.3 Hz, 2H), 2.73 (t, J ) 6.3 Hz,
2H), 2.08-2.01 (m, 2H). HRMS: calcd for C13H11ClN2O4,
294.0407; found, 294.0402. HPLC purity: system A, 97.1%; system
B, 97.8%.
3-Chloro-N-(1,2,3,4-tetrahydro-1,3,4-trioxoisoquinolin-6-yl)-
propanamide (9k) was prepared according to the procedure for
compound 9a except using 3-chloropropanoyl chloride in 12% yield.
1H NMR (DMSO, 300 MHz) δ: 11.86 (br, 1H), 10.70 (br, 1H),
8.35 (d, J ) 2.4 Hz, 1H), 8.09 (d, J ) 8.4 Hz, 2H), 8.01 (dd, J )
2.4, 8.4 Hz, 1H), 3.91 (t, J ) 6.3 Hz, 2H), 2.91 (t, J ) 6.3 Hz,
2H). HRMS: calcd for C12H9ClN2O4, 280.0251; found, 280.0252.
HPLC purity: system A, 96.3%; system B, 96.2%.
2-Chloro-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
acetamide (9l) was prepared according to the procedure for
compound 9a except using 2-chloroacetyl chloride in 40% yield.
1H NMR (DMSO, 300 MHz) δ: 11.88 (br, 1H), 10.91 (br, 1H),
8.33 (d, J ) 2.1 Hz, 1H), 8.10 (d, J ) 8.4 Hz, 1H), 8.01 (dd, J )
2.1, 8.4 Hz, 1H), 4.35 (s, 2H). 13C NMR (DMSO, 75 MHz) δ:
175.4, 165.7, 162.8, 157.6, 143.4, 133.4, 129.7, 124.8, 124.5, 115.9,
43.6. HRMS: calcd for C11H7ClN2O4, 266.0095; found, 266.0092.
HPLC purity: system A, 97.7%; system B, 96.8%.
N-Allyl-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
succinamide (13b) was prepared according to the procedure for
compound 13a except using allylamine in 20% yield. H NMR
1
(DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.57 (br, 1H), 8.35 (d, J
) 1.8 Hz, 1H), 8.06 (d, J ) 8.7 Hz, including NH, 2H), 7.98 (dd,
J ) 2.1, 8.7 Hz, 1H), 5.83-5.73 (m, 1H), 5.17-5.02 (m, 2H), 3.69
(t, J ) 5.4 Hz, 2H), 2.64 (d, J ) 7.2 Hz, 2H), 2.08 (br, 2H).
HRMS: calcd for C16H15N3O5, 329.1011; found, 329.1013. HPLC
purity: system A, 96.6%; system B, 97.8%.
4-Oxo-4-piperidin-1-yl-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-butyramide (13c) was prepared according to the
procedure for compound 13a except using piperidine in 27% yield.
1H NMR (DMSO, 300 MHz) δ: 11.82 (br, 1H), 10.57 (br, 1H),
8.35 (d, J ) 2.1 Hz, 1H), 8.06 (d, J ) 8.4 Hz, 1H), 7.99 (dd, J )
2.1, 8.4 Hz, 1H), 3.42-3.39 (m, 4H), 2.63 (br, 4H), 1.57-1.51
(m, 4H), 1.40 (br, 2H). 13C NMR (DMSO, 75 MHz) δ: 175.6,
172.0, 169.3, 162.9, 157.6, 144.3, 133.2, 129.6, 124.1, 124.0, 115.5,
45.7, 42.1, 31.7, 27.4, 26.0, 25.4, 24.1. HRMS: calcd for
C18H19N3O5, 357.1325; found, 357.1334. HPLC purity: system A,
96.6%; system B, 96.4%.
N-Benzyl-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
succinamide (13d) was prepared according to the procedure for
1
compound 13a except using benzylamine in 20% yield. H NMR
(DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.59 (br, 1H), 8.43 (t, J )
6.0 Hz, 1H), 8.37 (d, J ) 2.1 Hz, 1H), 8.07 (d, J ) 8.7 Hz, 1H),
7.99 (dd, J ) 2.1, 8.7 Hz, 1H), 7.33-7.22 (m, 5H), 4.27 (d, J )
6.0 Hz, 2H), 2.67 (t, J ) 6.6 Hz, 2H), 2.52 (t, J ) 6.6 Hz, 2H). 13
C
NMR (DMSO, 75 MHz) δ: 175.6, 171.8, 171.3, 162.9, 157.7,
144.3, 139.6, 133.3, 129.7, 128.4, 127.2, 126.8, 124.2, 124.1, 115.7,
42.2, 31.9, 30.0. HRMS: calcd for C20H17N3O5, 379.1169; found,
379.1188. HPLC purity: system A, 97.0%; system B, 97.5%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
succinamic acid (12a). SeO2 (35 mg, 0.315 mmol) was added to
the acid 11a (80 mg, 0.290 mmol) in 5 mL of dry dioxane. The
mixture was heated to reflux for 3-5 h, and then the solvent was
evaporated at reduced pressure and chromatographed to yield 12a
N-Phenyl-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
succinamide (13e) was prepared according to the procedure for
compound 13a except using aniline in 20% yield. 1H NMR (DMSO,
300 MHz) δ: 11.83 (br, 1H), 10.64 (br, 1H), 10.02 (br, 1H), 8.35
(d, J ) 1.8 Hz, 1H), 8.07 (d, J ) 8.7 Hz, 1H), 8.01 (dd, J ) 1.8,
8.7 Hz, 1H), 7.58 (d, J ) 7.5 Hz, 2H), 7.28 (dd, J ) 7.2, 7.5 Hz,
2H), 7.02 (dd, J ) 7.2, 7.2 Hz, 1H), 2.71 (br, 4H). HRMS: calcd
for C19H15N3O5, 365.1012; found, 365.0995. HPLC purity: system
A, 96.8%; system B, 95.2%.
1
(45 mg, 54%). H NMR (DMSO, 300 MHz) δ: 12.11 (br, 1H),
11.84 (br, 1H), 10.60 (br, 1H), 8.34 (d, J ) 1.8 Hz, 1H), 8.07 (d,
J ) 8.7 Hz, 1H), 7.99 (dd, J ) 1.8, 8.7 Hz, 1H), 2.64-2.62 (m,
2H), 2.57-2.55 (m, 2H). HRMS: calcd for C13H10N2O6, 290.0538;
found, 290.0541. HPLC purity: system A, 97.8%; system B, 98.2%.
4-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-6-ylcarbamoyl)-
butyric acid (12b) was prepared according to the procedure for
compound 12a except using 11b in 30% yield. 1H NMR (DMSO,
300 MHz) δ: 12.11 (br, 1H), 11.84 (br, 1H), 10.53 (br, 1H), 8.34
(d, J ) 2.1 Hz, 1H), 8.07 (d, J ) 8.4 Hz, 1H), 8.01 (dd, J ) 2.1,
8.4 Hz, 1H), 2.44 (t, J ) 7.2 Hz, 2H), 2.30 (t, J ) 7.2 Hz, 2H),
1.85-1.80 (m, 2H). HRMS: calcd for C14H12N2O6, 304.0695;
found, 304.0691. HPLC purity: system A, 96.2%; system B, 97.3%.
N-Propyl-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-
succinamide (13a). EDCa (39 mg, 0.203 mmol) was added to a
mixture of acid 11a (43 mg, 0.156 mmol), propylamine (26 µL,
0.311 mmol), and HOBt (23 mg, 0.171 mmol) in 1.5 mL of
anhydrous DMF. After being stirred at room temperature for 10 h,
N-(2-Methoxy-phenyl)-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-succinamide (13f) was prepared according to the
procedure for compound 13a except using 2-methoxyaniline in 16%
1
yield. H NMR (DMSO, 300 MHz) δ: 11.82 (br, 1H), 10.60 (br,
1H), 9.18 (br, 1H), 8.36 (d, J ) 1.8 Hz, 1H), 8.07 (d, J ) 8.4 Hz,
1H), 8.01 (dd, J ) 1.8, 8.4 Hz, 1H), 7.94 (d, J ) 8.1 Hz, 1H),
7.05-7.01 (m, 2H), 6.90-6.85 (m, 1H), 3.83 (s, 3H), 2.75-2.71
(m, 4H). 13C NMR (DMSO, 75 MHz) δ: 175.5, 171.6, 170.3, 162.8,
157.5, 149.4, 144.1, 133.2, 129.5, 127.4, 124.1, 124.0 (2), 121.7,
a Abbreviations: pNA, p-nitroaniline; AMC, 7-amino-4-methylcoumarin;
EDC, 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide; HOBt, 1-hydroxy-
benzotriazole hydrate.

Potent Caspase-3 Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1621
1
120.1, 115.5, 111.1, 55.6, 31.5, 30.8. HRMS: calcd for C20H17N3O6,
395.1117; found, 395.1109. HPLC purity: system A, 98.3%; system
B, 98.3%.
22%). H NMR (DMSO, 300 MHz) δ: 10.89 (br, 1H), 10.19 (br,
1H), 8.76 (d, J ) 2.1 Hz, 1H), 8.43 (dd, J ) 2.1, 8.7 Hz, 1H), 8.18
(d, J ) 8.7 Hz, 1H), 8.10-8.07 (m, 2H), 7.67-7.62 (m, 1H), 7.59-
7.54 (m, 2H). HRMS: calcd for C16H10N2O4, 294.0640; found,
294.0650. HPLC purity: system A, 95.1%; system B, 96.7%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-7-yl)-2-meth-
oxybenzamide (14b) was prepared according to the procedure for
compound 14a except using 2-methoxybenzoyl chloride in 15%
N-(4-Methoxy-phenyl)-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-succinamide (13g) was prepared according to the
procedure for compound 13a except using 4-methoxyaniline in 19%
1
yield. H NMR (DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.63 (br,
1H), 9.88 (br, 1H), 8.35 (d, J ) 2.1 Hz, 1H), 8.07 (d, J ) 8.7 Hz,
1H), 8.00 (dd, J ) 2.1, 8.7 Hz, 1H), 7.49 (d, J ) 9.0 Hz, 2H), 6.85
(d, J ) 9.0 Hz, 2H), 3.70 (s, 3H), 2.72-2.66 (m, 4H). 13C NMR
(DMSO, 75 MHz) δ: 175.5, 171.6, 169.6, 162.9, 157.6, 155.0,
144.2, 133.2, 132.5, 129.6, 124.0 (2), 120.5, 115.5, 113.8, 55.1,
31.4, 30.7. HRMS: calcd for C20H17N3O6, 395.1117; found,
395.1121. HPLC purity: system A, 95.3%; system B, 96.0%.
N-(3-Methoxy-phenyl)-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-succinamide (13h) was prepared according to the
procedure for compound 13a except using 3-methoxyaniline in 31%
1
yield. H NMR (DMSO, 300 MHz) δ: 11.97 (br, 1H), 10.76 (br,
1H), 8.59 (d, J ) 2.1 Hz, 1H), 8.14 (dd, J ) 2.1, 8.4 Hz, 1H), 8.06
(d, J ) 8.4 Hz, 1H), 7.63 (dd, J ) 2.1, 7.5 Hz, 1H), 7.58-7.52
(m, 1H), 7.21 (d, J ) 8.4 Hz, 1H), 7.09 (dd, J ) 7.5, 7.5 Hz, 1H),
3.90 (s, 3H). HRMS: calcd for C17H12N2O5, 324.0746; found,
324.0745. HPLC purity: system A, 98.5%; system B, 96.9%.
N-(1,2,3,4-Tetrahydro-1,3,4-trioxoisoquinolin-7-yl)-4-nitro-
benzamide (14c) was prepared according to the procedure for
compound 14a except using 2-methoxybenzoyl chloride in 38%
1
1
yield. H NMR (DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.63 (br,
yield. H NMR (DMSO, 300 MHz) δ: 12.07 (br, 1H), 11.02 (br,
1H), 10.00 (br, 1H), 8.35 (d, J ) 1.8 Hz, 1H), 8.07 (d, J ) 8.7 Hz,
1H), 8.00 (dd, J ) 1.8, 8.7 Hz, 1H), 7.30 (br, 1H), 7.18 (dd, J )
8.1, 8.1 Hz, 1H), 7.11 (d, J ) 8.1 Hz, 1H), 6.60 (dd, J ) 1.5, 8.1
Hz, 1H), 3.71 (s, 3H), 2.72-2.68 (m, 4H). 13C NMR (DMSO, 75
MHz) δ: 175.5, 171.6, 170.3, 162.8, 159.5, 157.6, 144.1, 140.5,
133.2, 129.6, 129.5, 124.0 (2), 115.5, 111.2, 108.4, 104.7, 54.9,
31.3, 30.9. HRMS: calcd for C20H17N3O6, 395.1117; found,
395.1117. HPLC purity: system A, 98.3%; system B, 97.5%.
N-(4-Fluoro-phenyl)-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-succinamide (13i) was prepared according to the
procedure for compound 13a except using 4-fluoroaniline in 18%
1H), 8.61 (d, J ) 1.5 Hz, 1H), 8.37 (dd, J ) 1.5, 8.4 Hz, 1H), 8.29
(d, J ) 9.0 Hz, 2H), 8.22 (d, J ) 9.0 Hz, 2H), 8.08 (d, J ) 8.7 Hz,
1H). HRMS: calcd for C16H9N3O6, 339.0492; found, 339.0495.
HPLC purity: system A, 96.1%; system B, 95.6%.
2-Chloro-N-(1,3,4-trioxo-1,2,3,4-tetrahydro-isoquinolin-7-yl)-
benzamide (14d) was prepared according to the procedure for
compound 14a except using 2-methoxybenzoyl chloride in 51%
1
yield. H NMR (DMSO, 300 MHz) δ: 11.98 (br, 1H), 10.88 (br,
1H), 8.62 (s, 1H), 8.28 (d, J ) 8.4 Hz, 1H), 8.13-8.07 (m, 2H),
8.03-7.98 (m, 1H), 7.45-7.39 (m, 1H), 7.35-7.29 (m, 1H). 13C
NMR (DMSO, 75 MHz) δ: 174.5, 165.8, 163.1, 157.8, 144.7,
136.1, 131.7, 131.3, 130.0, 129.8, 129.1, 128.7, 127.6, 127.5, 123.8,
117.9. HRMS: calcd for C16H9ClN2O4, 328.0251; found, 328.0245.
HPLC purity: system A, 97.2%; system B, 95.3%.
1
yield. H NMR (DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.63 (br,
1H), 10.09 (br, 1H), 8.35 (d, J ) 2.1 Hz, 1H), 8.07 (d, J ) 8.7 Hz,
1H), 8.00 (dd, J ) 2.1, 8.7 Hz, 1H), 7.62-7.57 (m, 2H), 7.15-
7.09 (m, 2H), 2.73-2.68 (m, 4H). HRMS: calcd for C19H14FN3O5,
383.0917; found, 383.0909. HPLC purity: system A, 98.6%; system
B, 95.9%.
Inhibition Assay of Caspase-3. Recombinant human caspase-3
catalytic domain was prepared according to previous literature with
minor modification.18 The typical assay of caspase-3 was carried
out in a 100 µL system including 50 mM HEPES pH 7.5, 150 mM
NaCl, 2 mM dithiothreitol, 1 mM EDTA, 100 µM Ac-DEVD-pNA,
and 20 nM caspase-3 in the presence or absence of 2 µL of inhibitor
in DMSO. In screening, the enzyme was incubated with inhibitors
for 30 min. The rate of hydrolysis product pNA was monitored
continuously by change of absorbance at 405 nm for 3 min, and
the initial rate of hydrolysis was determined using the early linear
region of the enzymatic reaction curve. Compounds were tested in
duplicate, and IC50 curves were calculated for all inhibitors assayed.
Final IC50 values were the average of three independent experi-
ments.
Selectivity. To study the inhibition selectivity of caspase-3
inhibitor on other caspases family members and cysteine or serine
protease, caspases 2 and 6-8 were overexpressed in Escherichia
coli and purified by affinity chromatography according to previous
reports with minor modification in our laboratory.19 Human
proteasome, human trypsin, thrombin, papain, and calpain-I were
purchased from Sigma. Caspases tetrapeptide substrates Ac-
VDVAD-pNA, Ac-VEAD-pNA, and Ac-LEHD-pNA were syn-
thesized in this laboratory. Peptide inhibitors Ac-DEVD-CHO and
Z-VAD-fmk and peptide substrates Suc-LY-AMC, Ac-LLVY-pNA,
and Nb-FVR-pNA were purchased from Bachem Bioscience (King
of Prussia, PA). The enzymatic activity of caspases 2, 6, 7, and 8
(0.38, 0.81, 0.51, and 1.42 µg, respectively) and human proteasome,
human trypsin, thrombin, papain, and calpain-I (0.23, 0.5, 5.0, 1.0,
and 0.38 µg, respectively) was determined from the intial rate of
hydrolysis of their respective substrates (10-100 µM). The enzymes
and caspase-3 inhibitors were diluted in respective buffer and
incubated for 30 min at their optimal pH value. These assays were
performed at 35 °C on 96-well clear polystyrene microplate. The
rate of hydrolysis product pNA was monitored continuously by
change of absorbance at 405 nm for 1-3 min using a SPECTRA
max 340 PC (Molecular Devices).
N-(3-Ethoxy-phenyl)-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-succinamide (13j) was prepared according to the
procedure for compound 13a except using 3-ethoxyaniline in 24%
1
yield. H NMR (DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.63 (br,
1H), 9.98 (br, 1H), 8.35 (d, J ) 1.8 Hz, 1H), 8.07 (d, J ) 8.7 Hz,
1H), 8.01 (dd, J ) 1.8, 8.7 Hz, 1H), 7.29 (br, 1H), 7.16 (dd, J )
7.8, 8.1 Hz, 1H), 7.08 (d, J ) 8.1 Hz, 1H), 6.58 (dd, J ) 1.5, 8.1
Hz, 1H), 3.97 (q, J ) 6.9 Hz, 2H), 2.72-2.68 (m, 4H), 1.30 (t, J
) 6.9 Hz, 3H). 13C NMR (DMSO, 75 MHz) δ: 175.5, 171.5, 170.2,
162.8, 158.8, 157.6, 144.1, 140.4, 133.2, 129.6, 129.4, 124.0 (2),
115.5, 111.1, 108.9, 105.2, 62.8, 31.3, 30.9, 14.7. HRMS: calcd
for C21H19N3O6, 409.1274; found, 409.1266. HPLC purity: system
A, 99.0%; system B, 98.5%.
N-(3-Propoxy-phenyl)-N′-(1,3,4-trioxo-1,2,3,4-tetrahydro-iso-
quinolin-6-yl)-succinamide (13k) was prepared according to the
procedure for compound 13a except using 3-propoxyaniline in 12%
1
yield. H NMR (DMSO, 300 MHz) δ: 11.83 (br, 1H), 10.63 (br,
1H), 9.98 (br, 1H), 8.35 (d, J ) 1.8 Hz, 1H), 8.07 (d, J ) 8.7 Hz,
1H), 8.01 (dd, J ) 1.8, 8.7 Hz, 1H), 7.31 (br, 1H), 7.16 (dd, J )
8.1, 8.1 Hz, 1H), 7.07 (d, J ) 8.1 Hz, 1H), 6.58 (d, J ) 8.1 Hz,
1H), 3.87 (t, J ) 6.3 Hz, 1H), 2.72-2.68 (m, 4H), 1.74-1.67 (m,
2H), 0.96 (t, J ) 7.5 Hz, 3H). HRMS: calcd for C22H21N3O6,
423.1431; found, 423.1444. HPLC purity: system A, 97.4%; system
B, 97.5%.
N-(1,3,4-Trioxo-1,2,3,4-tetrahydro-isoquinolin-7-yl)-benz-
amide (14a). To a solution of 7-amino-4H-isoquinoline-1,3-dione
(50 mg, 0.284 mmol) in 5 mL of ethyl acetate was added benzoyl
chloride (90 µL, 0.774 mmol) and pyridine (70 µL, 0.864 mmol).
The reaction mixture was stirred at room temperature for 5 h, and
diluted with 20 mL of ethyl acetate and 20 mL of H2O. The aqueous
phase was extracted with 20 mL of ethyl acetate. The combined
organic phases were dried over Na2SO4, filtered, and concentrated
under reduced pressure to give a solid, which was transferred to 3
mL of dioxane. SeO2 (34 mg, 0.306 mmol) was added, and the
mixture was heated to reflux for 1-3 h. The solvent was evaporated
at reduced pressure and chromatographed to yield 14a (18 mg,
Cell Culture and Treatment with Camptothecin. Jurkat cells
were incubated with 3 mL of 1 × 105 cells/mL in a 35 mm plate
for 30 min at 37 °C, and then caspase-3 inhibitors (Z-VAD-fmk,

1622 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Chen et al.
Ac-DEVD-CHO, 1, 6k) with a different concentration were added
to the cell media for 2 h followed by the addition of 2 µM
camptothecin. After 4 h, the cells were stained with acridine orange
and ethidium bromide for morphological analysis under a fluores-
cent microscope according to the literature.20,21
post hoc comparison. For all cases, significance of differences were
accepted at p < 0.05.
Acknowledgment. The Major State Hi-tech Research and
Development Program (Grant 2001AA234011), the State Key
Program of Basic Research of China (Grant 2004CB720300),
the Chinese Academy of Sciences, and the Shanghai Commis-
sion of Science and Technology are appreciated for their
financial support.
Detection of Caspase-3 Activity with Treated Cell Lysate.
Camptothecin treated cells were washed by phosphate-buffered
saline (PBS) one time, and resuspended in 200 µL of the lysis buffer
composed of 50 mM HEPES (pH 7.5), 10 mM dithiothreitol, 5
mM EDTA, 10 µg/mL proteinase K, 100 µg/mL phenylmethy-
sulfonyl fluoride (PMSF), 10 µg/mL pepstatin, and 10 µg/mL
leupeptin. Cells in lysis buffer were put on -80 °C and 4 °C by
turns for four times until cells were completely lysed. The samples
were centrifuged at 12000g for 20 min at 4 °C. The protein
concentration of each supernatant was measured using the Bradford
method. The caspase-3 activity was mearsured in a 100 µL volume
containing 50 mM HEPES pH 7.0, 150 mM NaCl, 10% sucrose,
0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA, 100 µM
Ac-DEVD-AMC (synthesized in this laboratory), and 20 µL cell
lysate. The release of AMC was measured by a Victor II (Perkin-
Elmer, excition wavelength, 360 nm, emission wavelength, 460 nm)
at room temperature for 20 min. Caspase-3 activity was measured
as fluorescence units normalized by the same concentration of
protein.
DNA Fragmentation. DNA fragments detection represents the
symbol of cell apoptosis. After treatment by compounds, cells were
disrupted in 500 µL lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM
EDTA, and 0.5% Triton-100) on ice for 20 min, and then DNA
was extracted with equal volumes of phenol and chloroform and
precipitated with 1000 µL of ethanol in the presence of 0.3 M
sodium acetate pH 5.5, at -80 °C for 1 h. After centrifugation at
12000g for 20 min, DNA pellets were washed with 70% ethanol,
air-dried, dissolved in TE buffer (10 mM Tris-HCl pH8.0, 10 mM
EDTA), and then digested with 50 µg/mL RNase at 37 °C for 30
min. The DNA fragment was identified by electrophoresis on a
1.0% agarose gel.
Method for Testing Caspase-3 Inhibitors in Focal Cerebral
Ischemic Stroke. Six compounds were selected for animal brain
ischemia studies, and compound 13f was explored further given
the property of central neural system. A widely accepted animal
model of brain ischemia is the transient middle cerebral artery
(MCA) occlusion stroke model.16 It was performed using a method
described previously,17 with minor modification. Briefly, the
bifurcation of the left common carotid artery was exposed. The
right MCA was occluded by insertion of a silicon-coated nylon
suture (USS-DG, Dermalon) through the common carotid artery.
After closure of the operative sites, the animals were temporarily
transferred to a cage with a heating lamp and the suture was gently
removed at 1.5 h of MCA occlusion. Drugs were dissolved in
DMSO and subcutaneously (sc) injected 5 min after the start of
MCA occlusion at a volume of 0.5 mL/kg.
Sacrifice was performed 24 h after transient or permanent MCA
occlusion by decapitation under halothane anesthesia. The brain
was rapidly removed and cut into 2 mm coronal sections and stained
according to the standard 2,3,5-triphenyl tetrazolium chloride (TTC)
method.22 The image of each slice was captured by using digital
camera (Nikon, COOLPIX 4300), followed by analysis by the
image system (Adobe ImageReady 7.0). The calculated infarction
areas were then compiled to obtain the infarction volume per brain
(in cubic micrometers). Infarction volume was corrected by using
an “indirect method” [area of intact contralateral (right) hemisphere
minus area of intact region of the ipsilateral (left) hemisphere] to
compensate for edema formation in the ipsilateral hemisphere.23
Animals. Male Sprague-Dawley rats, weighing between 200 and
240 g, were used. The animals had free access to solid food and
water ad libitum under standard conditions of temperature, humidity,
and light. All experiments were performed during the light phase
of the light/dark cycle.
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