N-nitrosoanilines: A new class of caspase-3 inhibitors

Article abstract of DOI:10.1016/S0968-0896(00)00222-4

Caspases are a family of cysteine proteases activated during apoptosis. In cultured human endothelial cells, physiological levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. Previous studies have demonstrated that NO

Full text of DOI:10.1016/S0968-0896(00)00222-4

Bioorganic & Medicinal Chemistry 9 (2001) 99±106
N-Nitrosoanilines: A New Class of Caspase-3 Inhibitors
Zhengmao Guo, Ming Xian, Wei Zhang, Andrea McGill and Peng George Wang*
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Received 26 April 2000; accepted 11 August 2000
AbstractÐCaspases are a family of cysteine proteases activated during apoptosis. In cultured human endothelial cells, physiological
levels of NO prevent apoptosis and interfere with the activation of the caspase cascade. Previous studies have demonstrated that
NO inhibits the activity of caspase-3 by S-nitrosylation of the enzyme. In this study, the inhibitory e€ect of a new class of NO
donors, N-nitrosoaniline derivatives, were examined against caspase-3. Initially eight small molecule inhibitors bearing N-nitroso
moieties were assayed. It was found that the presence of an electron-donating group on the phenyl ring led to better inhibitory
potency, a trend consistent with the results from the previous papain studies. Based on the analysis of the enzyme and substrates'
structures, two peptidyl N-nitrosoaniline inhibitors [Ac-DVAD-NNO (1) and Ac-DV-AMO (2)] were designed and synthesized.
Both compounds exhibited enhanced inhibitory potency against caspase-3. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
esting observation, a series of dephostatin analogues,
substituted N-methyl-N-nitrosoanilines, were subse-
quently synthesized and studied for enzyme inhibition.
Compounds substituted at the para position by hydroxyl
and N,N-dimethylamino groups were shown to be more
potent than unsubstituted N-methyl-N-nitrosoaniline in
the inactivation. Furthermore, in our recent study on the
inactivation of cysteine proteases by peptidyl N-nitro-
soanilines,18 we have demonstrated the inhibition was
e€ected through S-nitrosylation of the proteins' active
site thiol groups by the N-nitroso compounds. This kind
of direct regulation of protein function by NO, inde-
pendent of the activation of the soluble guanylyl cyclase
and subsequent production of cGMP, has been proposed
as an alternative pathway where NO may directly play a
critical role in many processes, such as blood pressure
regulation, host defense, and neurotransmission.19 21
Nitric oxide (NO), a small potentially toxic diatomic
free-radical, has become one of the most studied entities
in biological chemistry in the past few years.1 As a bio-
logical messenger, NO was found to be involved in
diverse physiological processes such as vasodilatory and
antiplatelet e€ects, macrophage-induced cytotoxicity,
and neurotransmission.2 9 However, the half-life time of
NO in vivo is very short. It easily reacts with molecular
oxygen and other reactive oxygen species as well as free
thiols, such as glutathione and proteins, to form nitro-
sothiols. Nitrosothiols functionally mimic NO and are
thought to play important roles in vivo.10,11 Many stu-
dies on cysteine containing enzymes, such as ornithine
decarboxylase,12 vacuolar H+-ATPase,13 and protein
tyrosine phosphatase (PTPase),14,15 indicate that thiol
residues on these enzymes could be modi®ed by NO
donors through an S-nitrosylation mechanism and thus
in¯uence cellular functions.
In connection with our e€orts to develop the mechanism
based cysteine protease inhibitors mediated by S-nitro-
sylation, caspase-3 was chosen as a target enzyme in the
present study. Caspases play a crucial role in the execu-
tion of apoptosis. Caspase-3 represents the execution
enzyme of the caspase cascade that cleaves the DNAse
inhibitor ICAD (Inhibitor of Caspase-Activated Deoxy-
ribonuclease) to activate DNA-degrading DNAses.22
Within the molecular structure of caspase-3, the catalytic
cysteine group that accounts for the proteolytic activity
of the enzyme is located at position 163 of the p17 sub-
unit.23 Recently, the possibility of employing NO to
attempt to modulate the activity of the caspase family of
cysteine proteases has attracted increasing attention.24 28
Dephostatin, a PTPase inhibitor isolated from the cul-
ture broth of Streptomyces sp., was recently synthesized
in our laboratory, along with its unsubstituted pre-
cursor, N-methyl-N-nitrosoaniline.16 Besides their com-
petitive inhibition against PTPases,16 both compounds
were also found to be good inhibitors of cysteine pro-
tease papain.17 For a better understanding of this inter-
*Corresponding author. Tel.: +1-313-933-6759; fax: +1-313-577-5831;
e-mail: pwang@chem.wayne.edu
0968-0896/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00222-4

100
Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
Several studies suggested that NO may inhibit apoptosis
via interacting with caspase cysteine protease.29 31 As a
general feature of its biochemical properties, NO is
known to be capable of modifying proteins that contain
cysteine residues by S-nitrosation of the sulfhydryl
group of the respective cysteine.21 Indeed, the decrease
in caspase-3-like enzyme activity by incubation of the
enzyme with NO donors could be speci®cally assigned
to S-nitrosylation of the essential cysteine residue at the
active site of caspase-3 both in vitro26,27 and in vivo.29
This could represent a potential molecular mechanism
underlying the functional relationship between NO and
inhibition of apoptosis signaling observed in cell cul-
ture. The NO donors previously used as the inhibitors
of caspase-3 include NONOate, NOBF4, sodium nitro-
prusside, and S-nitrosoglutathione. However, they are
normally unstable, especially in solution, and are photo-
sensitive. This not only makes the delivery and handling of
these compounds very inconvenient, but also directly
a€ects the accuracy and reliability of NO research results.
In contrast, our peptidyl N-nitrosoaniline derivatives are
quite stable in physiological systems;18 therefore, they
provide a good platform for designing new agents to
speci®cally deliver NO to the cysteine groups of protein.
In this paper, eight small N-nitrosoaniline derivatives
and two peptidyl N-nitrosoanilines were synthesized and
their inhibitory abilities against caspase-3 were reported.
group in a small molecule could help enhance inhibitory
potency. The protocol used previously in the papain
assay was followed to determine the pseudo-®rst order
rate constant.18 The activity versus time relationship is
shown in Figure 2.
Compound 4 possesses the strongest inhibition potency
with a pseudo-®rst order rate constant (kobsd) of 0.195
min 1, followed by 3 (0.129 min 1) and 5 (0.109 min 1).
Compounds 6±10 exhibited weak inhibition with kobsd
1
ranging from 0.009 min to 0.076 min 1. The inhibi-
tion constants observed in this assay were very similar
to those in the papain assay.17 Although compound 4
showed better inhibition than other carboxylate-con-
taining compounds in this series, its inhibition potency
is not comparable to that of peptide based inhibitor for
caspase-3.37 Furthermore, it was found that the car-
boxylate group in the small molecules did not play an
important role in the binding process which is con-
sidered to be important in the initial long range inter-
action between the inhibitor and the enzyme.
Design and synthesis of peptidyl N-nitrosoaniline inhibitors
Inhibitors of the caspase enzymes are now becoming a
popular area of study. In general, good substrates of the
caspase enzymes are tetrapeptide molecules (except for
caspase-2 inhibitors). The enzyme cleaves its substrate at
the C-terminus of the aspartic acid residue. This speci®city
is what distinguishes caspase from other proteases. The P4
speci®city distinguishes the members of the di€erent cas-
pase groups from each other. An aspartic acid residue is
required at the substrate's P1 position as is abbreviated in
the name given to this family of enzymes.32,33
Results and Discussion
Bioassay of small molecular N-nitrosoaniline derivatives
Eight small molecule N-nitroso compounds (Fig. 1)
were used in the initial enzymatic assay against caspase-
3. Of those eight non-peptide inhibitors, ®ve (3, 4, 7±9)
have been previously tested against papain and other
plant cysteine proteases.17 Compounds 5, 6 and 10 were
originally synthesized to target protein tyrosine phos-
phatase, which prefers a substrate with a carboxylate
group to facilitate the binding process. They were used
in this caspase-3 assay to determine if a carboxylate
The active site of caspase-3 has a catalytic dyad, consist-
ing of Cys 163 and His 121. This enzyme has a preference
for substrates with an aspartic acid residue at the P4
position.32,33 C-terminal aldehydes, nitriles, and ketones
have been found to be potent inhibitors.33,34 The enzy-
matic pockets for residues P2 and P3 in caspase-3 are
relatively open and solvent exposed while the P4 residue
has a de®ned pocket.35 A crystal structure of the enzyme
Figure 2. Inactivation of caspase-3 (250 ng/mL, 200 mL) with N-
nitrosoanilines (5 mM, 200 mL) in 10 mM Tris bu€er (pH 7.5, 1 mM
EDTA). Pseudo-®rst order rate constants (min 1): 3 (^ 0.129); 4 (&
0.195); 5 (~ 0.109); 6 (Â 0.076); 7 (Ã 0.038); 8 (* 0.023); 9 (+ 0.009);
10 ( 0.023). Trendline of 10 overlaps with that of 8.
Figure 1. Non-peptide based N-nitroso compounds.

Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
101
with the substrate in the active site revealed that the P4
aspartic acid has a polar interaction with an amide
nitrogen of the enzyme at the Phe 350 residue and the
gamma2 nitrogen of the Asn 208 residue.
concentration and much more slowly. Tetrapeptide
inhibitors designed and synthesized on the basis of
sequences at the precursor of Interleukin-1b and PARP
cleavage sites have been used to inhibit caspase-3. Ac-
DEVD-CHO, based on PARP cleavage sequence, is
very potent against caspase-3 with a Ki of 0.52 nM.37
Synthetic inhibitors of the caspase enzymes map the
tetrapeptide sequence of known substrates and replace
the C-terminal carboxylic acid, for instance, with an
aldehyde. Inhibition operates by the enzyme undergoing
Poly-(ADP-ribose) polymerase (PARP), a DNA repair
enzyme, has been implicated as a substrate for caspase-3
during apoptosis as it is cleaved into two fragments at
the onset of apoptosis.36 Other caspase homologs
including caspase-1 cleave PARP at much higher protein
Figure 3. Structures of Ac-DVAD-NNO (1) and Ac-DV-AMO (2).
Figure 4. Synthetic approach toward inhibitor 1. (a) (i) EDC, HOBt/CH2Cl2; (ii) Et2NH, DMF; (b) (i) EDC, HOBt/CH2Cl2; (ii) TFA, CH2Cl2; (c)
(i) t-BuOCOCl, NMM, TFA; (ii) NaBH4, MeOH, THF; (d) (COCl)2, DMSO, Et3N, CH2Cl2; (e) (i) 4-aminophenol, NaCNBH3, MeOH; (ii) TFA,
DCM; (f) (i) EDC, Et3N, CH2Cl2; (ii) H2/Pd±C; (g) NaNO2, HCl/H2O.

102
Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
Figure 5. Synthetic approach toward inhibitor 2. (a) 1.1 equiv (COCF3)2O; (b) BrCH2COOCH3, NaH, DMF; (c) RaNi, 2-propanol; (d) (i) EDC,
CH2Cl2; (ii) DMAP, DMF; (iii) 3 M HCl in EtOAc; (e) Ac-Asp-b-(OBn), EDC, Pyr; (f) (i) K2CO3, MeOH/H2O; (ii) KOH; (g) NaNO2, HCl/H2O.
a nucleophilic attack at a carboxylic position or at a
position alpha to the carboxylic group when a leaving
group can be released from the site.38
Table 1. Kinetic parameters for the inactivation of caspase-3
Inhibitor
kinact
(min
KI
kinact=KI
1
1
1
)
(M
s )
1
2
0.039
0.101
Ð
0.002 mM
0.022 mM
0.52 nM
322
78
Ð
Based on the preceding analysis, two peptide based cas-
pase-3 inhibitors were designed and their structures are
shown in Figure 3. Both compounds were designed to
incorporate our N-nitrosoaniline moiety into a peptido-
mimetic structure. Since the PARP-fragment sequence is
known to be an e€ective inhibitor, our compounds were
designed to keep a similar spatial arrangement. Com-
pound 1, Ac-DVAD-NNO, uses a tetrapeptide template
to which an N-nitrosoaniline fragment is attached on
the C-terminus by a C N bond. The P4 Asp residue is
required for molecular recognition and binding of the
inhibitor or substrate. The P3 position, which is believed
to be open to solvent in the caspase-3 active site, was
designated as a valine residue since only valine or glu-
tamic acid were experimentally shown to be favored at
that site for the caspase-3 enzyme.35 As discussed pre-
viously, an aspartic acid residue is required at the P1
position for this class of enzymes. Since an electron
donating para-substituted molecule makes NO transfer
easier, a hydroxyl group was introduced into the ben-
zene ring. For compound 2, Ac-DV-AMO, an N-nitro-
soaniline structure was con®gured with a similar spatial
arrangement to replace the P2 and P1 amino acids. It
was designed to retain two residues, Asp and Val, from
the N-terminus of the tetrapeptide, which is coupled via
an amide bond to the aromatic ring of N-nitroso-N-
phenylglycine. In the design of compound 2, the b-acid
of aspartic acid was preserved through an N-acetic acid
moiety. The pheny ring in structure 1 is necessary for the
susceptibility of the N NˆO motif, but is too bulky for
Ac-DEVE-CHO37
the S0 subsites. This same group in 2 replaced the Ala
residue in the tetrapeptide, resulting in a reduction in the
number of rotatable bonds. As in all of the previous
inhibitors, the NO is attached to the amino acid residue.
If the molecule described here is a good mimetic, clea-
vage should take place at the nitric oxide position.
Synthetic approach to inhibitor 1 is illustrated in Figure 4.
It was synthesized in a convergent manner from both C-
and N-terminals with a ®nal coupling reaction between
a tripeptide fragment 12 and aniline 15. Synthesis of the
tripeptide portion began with a coupling between an
Fmoc-protected Val and an alanine t-butyl ester by
using standard solution phase peptide coupling method.
The free amino group of the dipeptide 11 thus obtained
was subsequently coupled with b-benzyl protected
aspartic acid to yield a tripeptide 12. The synthesis of
the aniline fragement 15 started with an in situ activa-
tion of the commercially available compound N-Boc-l-
aspartic acid b-benzyl ester with isobutyl chloroformate
at 20 ꢀC to give a mixed anhydride intermediate which
was immediately treated with a suspension of sodium
borohydride in THF/methanol at 78 ꢀC. The reduc-
tion product 13 was then subjected to a Swern oxidation
to give an aldehyde 14, which underwent a reductive

Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
103
amination with a primary amine 4-aminophenol, then
deprotection of the Boc group yielded a secondary
amine product 15. Fragments 12 and 15 were then cou-
pled to give a tetrapeptide 16. N-Nitrosation of 16 in
acidic conditions gave the ®nal inhibitor 1.
derivatives are potent inhibitors of caspase-3. Taken
together with our previous studies on the inhibition of
papain and protein tyrosine phosphatase by N-nitroso-
aniline derivatives,16 18 it has been shown that this class
of compounds seems to be general inhibitors of cysteine
dependent enzymes. These NO donating compounds
provide a novel platform for the design of more potent
inhibitors to speci®cally deliver NO to the enzyme.
The designed inhibitor 2 Ac-DV-AMO was synthesized
according to Figure 5, in which m-amino-N-tri¯uoro-
acetyl-N-phenylglycine methyl ester 19 was used as a
precursor to N-nitroso substructure. Starting with 3-
nitroaniline, the amino group was ®rst protected using
tri¯uoroacetic anhydride. The resulting N-protected
nitroaniline 17 was then treated with methyl bromoace-
tate and sodium hydride to form the N-methyl acetate
moiety 18. This compound was subsequently reduced in a
solution of Raney nickel in 2-propanol, yielding 19. Ani-
line derivative 19 was coupled to the N-Boc protected
valine through an in situ generation of N-Boc-l-valine
anhydride, synthesized from the free acid N-Boc-l-
valine. After deprotection using 3 M HCl in ethyl ace-
tate, the resulting compound 20 was then coupled to N-
Ac-l-aspartic acid b-benzyl ester to form 21. Com-
pound 21 was then stepwise deprotected followed by
nitrosation using sodium nitrite in HCl to a€ord inhi-
bitor 2.
Experimental
Amino acids, amino acid derivatives and other chemical
reagents were purchased from Sigma or Aldrich, and
used without further puri®cation unless otherwise
1
noted. H and 13C NMR spectra were recorded on a
Mercury 400 NMR spectrometer. Mass spectra were
obtained from a Kratos MS 80 spectrometer using elec-
tron impact mode or a Kratos MS 50 spectrometer
using fast atomic bombardment. Silica gel plates (Merck
F254) and silica gel 60 (Merck; 200±240 mesh) were
used in analytical thin-layer chromatography (TLC)
and column chromatography, respectively. Small mole-
cule N-nitroso compounds (3±10) were synthesized as in
the previous report.16 18
L-Valinyl-L-alanine t-butyl ester (11). The title com-
pound was obtained by coupling an Fmoc-Val and an
alanine t-butyl ester using standard solution phase pep-
tide coupling method (87% yield). 1H NMR (CD3OD) d
0.922 (d, 3H, CH(CH3)CH3, J=6.4 Hz), 0.992 (d, 3H,
CH(CH3)CH3, J=6.4 Hz), 1.44 (d, 3H, CH(CH3), J=
7.2 Hz), 1.47 (s, 9H, (CH3)3CO), 4.34 (m, 2H, CaH);
EIMS calcd for C12H24N2O+3 (M+) 244, found 245
(M+H).
Caspase-3 assay of inhibitors 1 and 2
Following the enzymatic assay for eight small mole-
cules, two peptide based N-nitroso compounds designed
and synthesized in this study were assayed against cas-
pase-3. The stability of each peptidyl N-nitrosoaniline
compound in aqueous solution was examined before
being used in the enzymatic assay. No decomposition of
compounds 1 and 2 was detected over a 10-h incubation
period in the assay bu€er (data not shown; the method
was the same as described in our previous report18).
Incubation of caspase-3 with each inhibitor resulted in a
time- and concentration-dependent loss of enzymatic
activity. The apparent ®rst order inactivation constant
(kapp) can be calculated by plotting the residual activity
versus time and ®tting a linear equation.18 The replots
of kapp as a function of inhibitor concentration gave a
Kitz±Wilson plot.18,39 The calculated kinetic parameters
are shown in Table 1.
N-Acetyl-L-aspartyl-L-valinyl-L-alanine ꢀ-benzyl ester (12).
This compound was obtained by coupling 11 and N-
acetyl-l-aspartic acid b-benzyl ester using standard
solution phase peptide coupling method (75% yield). 1H
NMR (CD3OD) d 0.922 (d, 3H, CH(CH3)CH3, J=6.4
Hz), 0.992 (d, 3H, CH(CH3)CH3, J=6.4 Hz), 1.44 (d,
3H, CH(CH3), J=7.2 Hz), 1.98 (s, 3H, CH3CO), 2.64
(AB, 1H, CH2CO, JAB=13.6 Hz, JHa-B=9.6 Hz), 2.80
(AB, 1H, CH2CO, JAB=13.6 Hz, JHa-A=6 Hz), 4.27 (d,
1H, CHCH(CH3)2, J=6.2 Hz); 4.45 (d, 1H, CHCH2,
J=7.2 Hz), 5.19 (s, 2H, CH2Ph), 7.39 (m, 5H, CH2Ph);
FABMS calcd for C21H29N3O+7 (M+) 435, found 435.
Assay results showed that the designed compounds are
e€ective against caspase-3 with second order rate con-
1
1
1
1
stants of 322 M
s
for 1 and 78 M
s
for 2. The
KI of 1 is lower than that of 2, which suggests that 1 has
a tighter binding than 2, whereas the second step of
inactivation by 1, as characterized by kinact, is almost
three-fold slower than that of 2.
N-Boc-L-aspartanol ꢀ-benzyl ester (13). To a solution of
N-Boc aspartic acid b-benzyl ester (1 g, 3.10 mmol) in 50
mL of THF at 20 ꢀC was added N-methyl morpholine
(1.36 mL, 12.3 mmol) followed by isobutyl chloro-
formate (460 mL, 3.30 mmol). After 10 min, the mixture
was added to a suspension of sodium borohydride (200
mg, 5.29 mmol) in 20 mL of THF and 5 mL of metha-
nol at 78 ꢀC. After being kept at 78 ꢀC for 2 h, the
mixture was quenched with hydrochloric acid, diluted
with ethyl acetate, and washed with sodium bicarbo-
nate. The organic layers were pooled and dried over
anhydrous sodium sulfate, ®ltered, and concentrated. The
residue was chromatographed (EtOAc:hexane, 1:1) to
give 13 as a colorless oil (58% yield): 1H NMR (CD3OD)
Conclusions
N-Nitroso compounds have been extensively studied in
the past few years due to their carcinogenic and muta-
genic properties,40 while substituted N-methyl-N-nitro-
soanilines normally exhibit weak carcinogenicity and
almost no mutagenicity to several di€erent animal spe-
cies.41 We showed in this study that N-nitrosoaniline

104
Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
d 1.47 (s, 9H, (CH3)3CO), 2.50 (AB, 1H, CH2CO,
JAB=13.6 Hz, JHa-B=9.6 Hz), 2.65 (AB, 1H, CH2CO,
JAB=13.6 Hz, JHa-A=6 Hz), 3.42 (AB, 1H, CH2OH,
JAB=12.6 Hz, JHa-B=9.6 Hz), 3.55 (AB, 1H, CH2OH,
JAB=12.6 Hz, JHꢀ-A=6 Hz), 3.98 (m, 1H, CaH), 5.16 (s,
1H, CH2Ph), 7.37 (m, 5H, CH2Ph). EIMS calcd for
C16H23NO+5 (M+) 309, found 310 (M+H).
3-(S)-N-(N0-Acetyl-L-aspartyl-L-valinyl-L-alanyl)-4-N00-
(p-hydroxylphenyl)-4-N00-nitroso-butyric acid (1). To a
suspension of 16 (500 mg, 0.93 mmol) in 5 mL of H2O
was added 1 N HCl gradually until pH 3. The reaction
mixture became homogeneous. At 0 ꢀC, NaNO2 (150
mg, 2.2 mmol) was added to the solution and the pro-
1
duct (320 mg) precipitated from the solution in 1 h. H
NMR (CD3OD) d 0.922 (d, 3H, CH(CH3)CH3, J=6.4
Hz), 0.992 (d, 3H, CH(CH3)CH3, J=6.4 Hz), 1.44 (d,
3H, CH(CH3), J=7.2 Hz), 1.98 (s, 3H, CH3CO), 2.00
(s, 3H, CH3CO), 2.59 (AB, 1H, CH2CO, JAB=12.6 Hz,
JHa-B=6.6 Hz), 2.64 (AB, 1H, CH2CO, JAB=12.6 Hz,
JHa-A=6.6 Hz), 3.04 (AB, 1H, CH2NH, JAB=13.6 Hz,
JHa-B=9.4 Hz), 3.19 (AB, 1H, CH2NH, JAB=13.6 Hz,
JHa-A=9.4 Hz), 4.32 (m, 3H, CaH), 5.20 (s, 2H,
CH2Ph), 7.20 (d, 2H, CH2Ph, J=9.0 Hz), 7.39 (d, 2H,
CH2Ph, J=9.0 Hz); 13C NMR (CD3OD) 173.9, 173.0,
172.2, 171.0, 165.7, 164.4, 158.7, 138.5, 135.2, 130.2,
129.4, 127.7, 123.7, 117.0, 56.1, 45.3, 38.8, 37.2, 22.4.
FABMS calcd for C24H34N6O+10 (M+) 566, found 536
(M±NO).
N-Boc-L-aspartic aldehyde ꢀ-benzyl ester (14). At 78 ꢀC,
to a stirred solution of oxalyl chloride (1.96 mL, 3.91
mmol) in CH2Cl2 was added DMSO (0.56 mL, 7.82
mmol), followed by the addition of 13 in CH2Cl2 (500
mg, 1.6 mmol). Stirring was continued at 78 ꢀC for 10
min followed by addition of the alcohol dissolved in
CH2Cl2. The reaction mixture was stirred for 15 min,
and Et3N (2 mL, 14.3 mmol) was added with stirring at
78 C. After 30 min, water was added at room tem-
perature and stirring was continued for 10 min. The
organic layer was separated, and the aqueous phase was
re-extracted with CH2Cl2. After drying over sodium
sulfate, the solution was ®ltered and concentrated. The
residue was chromatographed (EtOAc:hexane, 1:3) to
a€ord 14 as a colorless oil (82% yield): 1H NMR
(CD3OD) d 1.47 (s, 9H, (CH3)3CO), 2.50 (AB, 1H, CH2
CO, JAB=13.6 Hz, JHa-B=9.6 Hz), 2.65 (AB, 1H,
CH2CO, JAB=13.6 Hz, JHa-A=6 Hz), 4.46 (m, 1H,
CaH), 5.16 (s, 1H, CH2Ph), 7.37 (m, 5H, CH2Ph). EIMS
calcd for C16H21NO5+ (M+) 307, found 308 (M+H).
N-Tri¯uoroacetic-3-nitroaniline (17). To a solution of 3-
nitroaniline (10 g, 72 mmol) in CH2Cl2:pyridine (1:3, v/
v, 80 mL), was added dropwise tri¯uoroacetic anhy-
dride (11 mL, 78 mmol). When the reaction was com-
plete, the solution was washed with HCl (1 N), then
brine and dried (Na2SO4). The solvent was removed
under reduced pressure a€ording quantitative yield of a
yellowish-orange, needle-like solid. 1H NMR (d-CHCl3)
d 8.52 (t, 1H, ar), 8.14 (dd, 1H, ar), 8.06 (dd, 1H, ar),
7.64 (t, 1H, ar). 13C NMR d 156.51, 149.71, 146.36,
137.47, 131.53, 127.33, 122.08, 116.68. MS calcd for
C8H5F3N2O+3 (M+) 234, found 234.
4-N-(p-Hydroxylphenyl)-3-(S)-amino-butyric acid benzyl
ester (15). To a stirred solution of 14 (246 mg, 0.8
mmol) in anhydrous MeOH (20 mL) was added 4-ami-
nophenol (262 mg, 2.4 mmol) and NaCNBH3 (51 mg, 0.8
mmol). After the mixture being stirred overnight at room
temperature, the solvent was removed in vacuo and the
residue was chromatographed with 0±5% MeOH/EtOAc
m-Nitro-N-tri¯uoroacetyl-N-phenylglycine methyl ester
(18). To a solution of NaH (1.27 g, 53 mmol) in DMF
(25 mL) at 78 ꢀC was slowly added 17 (4.9 g, 21 mmol)
dissolved in DMF (25 mL). After addition of the ani-
line, the temperature was brought to 0 ꢀC. After
approximately 1 h, the temperature was reduced to
78 ꢀC for the addition of the methyl bromoacetate (6.4
g, 42 mmol). The temperature was then brought to
25 ꢀC. It was worked up by pouring the solution into
cold HCl (1 N). It was then extracted with EtOAc,
washed with brine then dried (Na2SO4). The solvent was
removed under reduced pressure. Puri®cation by col-
umn chromatography with hexane and hexane:EtOAc
(20:1, 15:1, 10:1 then 5:1, v/v) a€orded a bright yellow
1
to a€ord product 15 (50%): H NMR (CD3OD) d 2.50
(AB, 1H, CH2CO, JAB=13.6 Hz, JHa-B=9.6 Hz), 2.65
(AB, 1H, CH2CO, JAB=13.6 Hz, JHa-A=6 Hz), 3.04 (AB,
1H, CH2NH, JAB=13.6 Hz, JHa-B=9.4 Hz), 3.19 (AB,
1H, CH2NH, JAB=13.6 Hz, JHa-A=9.4 Hz), 4.46 (m, 1H,
CaH), 5.16 (s, 2H, CH2Ph), 6.45 (d, 2H, J=9.0 Hz, Ph),
6.63 (d, 2H, J=9.0 Hz, Ph), 7.37 (m, 5H, CH2Ph). EIMS
calcd for C17H20N2O3+ (M+) 300, found 301 (M+H).
3-(S)-N-(N0Acetyl-L-aspartyl-L-valinyl-L-alanyl)-4-N00-(p-
hydroxylphenyl)-butyric acid (16). To a stirring solution
of 12 (436 mg, 1 mmol) and 15 (300 mg, 1 mmol) in
CH2Cl2 (25 mL) were added EDC (201 mg, 1.05 mmol)
and Et3N (0.15 mL, 1.05 mmol), with stirring being
continued at room temperature for 24 h. The reaction
mixture was worked up to give 16 (65%). 1H NMR
(CD3OD) d 0.922 (d, 3H, CH(CH3)CH3, J=6.4 Hz),
0.992 (d, 3H, CH(CH3)CH3, J=6.4 Hz), 1.44 (d, 3H,
CH(CH3), J=7.2 Hz), 1.98 (s, 3H, CH3CO), 2.00 (s,
3H, CH3CO), 2.59 (AB, 1H, CH2CO, JAB=12.6 Hz,
JHa-B=6.6 Hz), 2.64 (AB, 1H, CH2CO, JAB=12.6 Hz,
JHa-A=6.6 Hz), 3.04 (AB, 1H, CH2NH, JAB=13.6 Hz,
JHa-B=9.4 Hz), 3.19 (AB, 1H, CH2NH, JAB=13.6 Hz,
JHa-A=9.4 Hz), 4.32 (m, 3H, CaH), 5.20 (s, 2H,
CH2Ph), 6.45 (d, 2H, J=9.0 Hz, Ph), 6.63 (d, 2H,
J=9.0 Hz, Ph); FABMS calcd for C24H35N5O+9 (M+)
537, found 537.
1
crystalline solid (42%). H NMR (d-CHCl3) d 8.31 (d,
2H, ar), 7.83 (d, 1H, ar), 7.67 (d, 1H, ar), 4.46 (s, 2H),
3.81 (s, 3H); 13C NMR d 168.19, 149.40, 141.14, 135.46,
131.30, 127.19, 125.26, 124.43, 116.49, 61.34, 53.43. MS
calcd for C11H9F3N2O+5 (M+) 306, found 306.
m-Amino-N-tri¯uoroacetyl-N-phenylglycine methyl ester
(19). To a slurry of Raney nickel (50% in water, 20 g) in
2-propanol (45 mL) was added compound 18 (1.99 g, 6.5
mmol). After 5 h the solution was carefully ®ltered to avoid
ignition of the metal. The solvent of the ®ltrate was
removed under reduced pressure. Puri®cation by column
chromatography with hexane and hexane:EtOAc (20:1,
10:1 then 5:1, v/v) a€orded a white, semi-crystalline

Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
105
solid (53%). The ®rst product to elute from the column
1
Hz), 2.91 (m, 1H, CHCH2), 3.30 (m, 2H, CH2NNO),
4.29 (m, 1H, CHCH), 7.20 (t, 1H, Ph), 7.52 (d, 2H, Ph,
J=9.2 Hz), 7.96 (s, 1H, Ph). FABMS calcd for
C19H25N5O+8 (M+) 451, found 421 (M NO).
was the desired product. H NMR (d-CHCl3) d 7.20 (t,
1H, ar), 6.74 (d, 2H, ar), 6.74 (s, 1H, ar), 4.41 (s, 2H),
3.80 (s, 3H), 1.27 (s, 2H, aniline); 13C NMR d 169.10,
148.61, 141.71, 131.36, 118.63, 116.92, 115.19, 54.16,
53.67, 30.83. MS calcd for C11H11F3N2O+3 (M+) 276,
found 276.
Caspase-3 assays
Recombinant human caspase-3 was purchased from
PharMingen. Assays were conducted on a 96-well
microtiter plate and contained 65 mL of assay bu€er (10
mM Tris, 1 mM EDTA, pH 7.5), 10 mL of incubation
solution, 5 mL of DMSO containing the inhibitor and 20
mL of substrate Ac-DEVD-pNA. Production of p-
nitroaniline (pNA) from reaction mixtures was mea-
sured by following the absorbance at 405 nm. Second
order rate constants for irreversible inhibitors were
determined from assays where the reaction bu€er con-
taining inhibitor and caspase-3 was incubated at 37 ꢀC,
and aliquots were used for enzyme activity determina-
tion. Kitz±Wilson plots of the kinetic data were pre-
pared to derive the second order rate constants.
m-(Val-NH)-N-tri¯uoroacetyl-N-phenylglycine methyl
ester (20). Boc-Val (3.5 g, 16.1 mmol) and EDC (2.3 g,
12 mmol) was stired in CH2Cl2 for 30 min at 0 ꢀC. This
solution was then pipetted into a solution of 19 (442 mg,
1.6 mmol) with Et3N (2 mL). DMAP (3 mmol) was then
added and the reaction mixture was stirred for 5 h.
Then, the mixture was worked up. The crude product
was deprotected by HCl (3 M). After the reaction was
complete, the solvent was removed by rotary evapora-
tion. Water was added to the residue and it was washed
three times with ethyl ether. The aqueous solution was
then neutralized with sodium bicarbonate, then extrac-
ted with ethyl acetate. The organic solution was then
dried (Na2SO4). The solvent was removed under
reduced pressure a€ording a pale-yellow solid (59%).
1H NMR (CD3OD) d 7.82 (s, 1H, ar), 7.65 (d, 1H, ar),
7.40 (t, 1H, ar), 7.17 (d, 1H, ar), 4.46 (s, 2H), 3.76 (s,
3H), 3.23 (d, 1H), 2.03 (m, 1H), 0.99 (dd, 6H); 13C
NMR d 174.39, 168.29, 157.10, 156.74, 140.19, 139.40,
129.53, 123.27, 102.49, 119.38, 117.71, 114.86, 60.92,
52.78, 51.64, 32.38, 18.49, 16.42. MS calcd for
C16H20F3N3O+4 (M+) 375, found 375.
Acknowledgements
This work was generously supported by the research
grants from NIH (GM54074).
References and Notes
1. Culotta, E.; Koshland, D. E. Science 1992, 258, 1862.
2. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol.
Rev. 1991, 43, 109.
3. Kerwin, J. F.; Lancaster, J. R.; Feldman, P. L. J. Med.
Chem. 1995, 38, 4343.
4. Feldman, P. L.; Grith, O. W.; Stuehr, D. J. Chem. Eng.
News 1993 December 20, 26.
5. Methods in Nitric Oxide Research; Feelish, M., Stamler, J.
S., Eds.; Wiley: Chichester, 1996.
6. Nitric Oxide Part A: Sources and Detection of NO; NO
Synthase Methods in Enzymology; Packer, L., Ed.; Academic:
San Diego, 1996.
7. Nitric Oxide Part B: Physiological and Pathological Pro-
cesses Methods in Enzymology; Packer, L., Ed.; Academic: San
Diego, 1996.
8. Nitric Oxide: Biochemistry, Molecular Biology, and Ther-
apeutic Implications; Ignarro, L., Murad, F., Eds.; Academic:
San Diego, 1995.
9. Nitric Oxide: Principles and Actions; Lancaster, J., Ed.;
Academic: San Diego, 1996.
10. Stamler, J. S.; Jaraki, O.; Osborne, J.; Simon, D. I.; Kea-
ney, J.; Vita, J. A.; Singel, D.; Valeri, C. R.; Loscalzo, J. Proc.
Natl. Acad. Sci. USA 1992, 89, 7674.
11. Stamler, J. S.; Simon, D. I.; Osborne, J. A.; Mullins, M.
E.; Jaraki, O.; Michel, T.; Singel, D.; Loscalzo, J. Proc. Natl.
Acad. Sci. USA 1992, 89, 444.
12. Bauer, P. M.; Fukuto, J. M.; Buga, G. M.; Pegg, A. E.;
Ignarro, L. J. Biochem. Biophys. Res. Commun. 1999, 262, 355.
13. Forgac, M. J. Biol. Chem. 1999, 274, 1301.
14. Xian, M.; Wang, K.; Chen, X.-C.; Hou, Y.; Zhou, B.;
Cheng, J.-P.; Zhang, Z.-Y.; Wang, P. G. Biochem. Biophys.
Res. Commun. 2000, 268, 310.
15. Caselli, A.; Camici, G.; Manao, G.; Moneti, G.; Pazzagli,
L.; Cappugi, G.; Ramponi, G. J. Biol. Chem. 1994, 269, 24878.
16. Yu, L.-B.; McGill, A.; Ramirez, J.; Wang, P. G.; Zhang,
Z.-Y. Bioorg. Med. Chem. Lett. 1995, 5, 1003.
m-(Ac-L-Asp-L-Val-NH)-N-phenylglycine (22). Com-
pound 20 (188 mg, 0.5 mmol) was dissolved in CH2Cl2
(10 mL) and pyridine (2.5 mmol). Ac-Asp-b-(OBn) (186
mg, 0.7 mmol), then EDC (134 mg, 0.7 mmol) were
added. When the reaction was complete, the mixture
was worked up. The crude product was dissolved in
MeOH:H2O (1:1). K2CO3 (2 mmol) was added and,
once the tri¯uoroacetate and benzyl groups were
removed, KOH (9 mmol) was added. After the reaction
was complete, it was acidi®ed by adding Dowex 50
WX2-100 ion exchange resin (acidic). The solution was
then ®ltered and the resin rinsed with water. The solvent
was then removed under reduced pressure. A pale-yel-
1
low solid was a€orded (43%). H NMR (D2O) d 0.912
(d, 3H, CH(CH3)CH3, J=6.2 Hz), 0.982 (d, 3H, CH
(CH3)CH3, J=6.2 Hz), 2.01 (s, 3H, CH3CO), 2.09 (m,
1H, CH(CH3)2), 2.65 (d, 2H, CH2COOH J=7.0 Hz),
2.88 (m, 1H, CHCH2), 2.92 (m, 2H, CH2NH), 4.20 (m,
1H, CHCH), 7.66 (t, 1H, Ph), 7.92 (d, 2H, J=9.2 Hz,
Ph), 8.10 (s, 1H, Ph). MS calcd for C19H26N4O+7 (M+)
422, found 422.
m-(Ac-L-Asp-L-Val-NH)-N-nitroso-N-phenylglycine (2).
Compound 22 (25 mg, 0.06 mmol) was dissolved in 0.1
N HCl. An aqueous solution of NaNO2 (5 mg, 0.07
mmol) was added at 0 ꢀC. One hour later, the solvent
was removed under reduced pressure. The crude mix-
ture was chromatographed on silica gel and eluted with
1
dichloromethane to a€ord a red solid (64%). H NMR
(D2O) d 0.910 (d, 3H, CH(CH3)CH3, J=6.2 Hz), 0.985 (d,
3H, CH(CH3)CH3, J=6.2 Hz), 2.04 (s, 3H, CH3CO), 2.14
(m, 1H, CH(CH3)2), 2.76 (d, 2H, CH2COOH J=7.2

106
Z. Guo et al. / Bioorg. Med. Chem. 9 (2001) 99±106
17. Guo, Z.-M.; McGill, A.; Yu, L.-B.; Li, J.; Ramirez, J.;
Wang, P. G. Bioorg. Med. Chem. Lett. 1996, 6, 573.
18. Guo, Z.-M.; Ramirez, J.; Li, J.; Wang, P. G. J. Am. Chem.
Soc. 1998, 120, 3726.
31. Kim, Y. M.; de Vera, M. E.; Watkins, S. C.; Billiar, T. R.
J. Biol. Chem. 1997, 272, 1402.
32. Talanian, R. V.; Allen, H. J., Annu. Rep. Med. Chem.
1998; 33, 273.
19. Broillet, M.-C. Cell. Mol. Life. Sci. 1999, 55, 1036.
20. Gaston, B. Biochim. Biophys. Acta 1999, 1411, 323.
21. Stamler, J. S. Cell 1994, 78, 931.
22. Enari, M.; Sakahira, H.; Yokoyama, H.; Okawa, K.;
Iwamatsu, A.; Nagata, S. Nature 1998, 391, 43.
23. Nicholson, D. W.; Ali, A.; Thornberry, N. A.; Vaillan-
court, J. P.; Ding, C. K.; Gallant, M.; Gareau, Y.; Grin, P.
R.; Labelle, M.; Lazebnik, Y. A. Nature 1995, 376, 37.
24. Zech, B.; Wilm, M.; Eldik, R.; Brune, B. J. Biol. Chem.
1999, 274, 20931.
33. Nicholson, D. W. Nature Biotech. 1996, 14, 297.
34. Mullican, M. D.; Lau€er, D. J.; Gillespie, R. J.; Matharu,
S. S.; Kay, D.; Porritt, G. M.; Evans, P. L.; Golec, J. M.;
Murcko, M. A.; Luong, Y. P.; Raybuck, S. A.; Livingston, D.
J. Bioorg. Med. Chem. Lett. 1994, 4, 2359.
35. Talanian, R. V.; Quinlan, C.; Trautz, S.; Hackett, M. C.;
Mankovich, F. A.; Banach, D.; Ghayur, T.; Brady, K. D.;
Wong, W. W. J. Biol. Chem. 1997, 272, 9677.
36. Takahashi, A.; Earnshaw, W. C. Curr. Opin. Genet. Dev.
1996, 26, 50.
25. Kim, Y. M.; Talanian, R. V.; Billiar, T. R. J. Biol. Chem.
1997, 272, 31138.
26. Li, J. R.; Billiar, T. R.; Talanian, R. V.; Kim, Y. M. Bio-
chem. Biophys. Res. Commun. 1997, 240, 419.
27. Dimmeler, S.; Haendeler, J.; Nehls, M.; Zeiher, A. M. J.
Exp. Med. 1997, 185, 601.
37. Margolin, N.; Raybuck, S. A.; Wilson, K. P.; Chen, W.;
Fox, T.; Gu, Y.; Livingston, D. J. J. Biol. Chem. 1997, 272, 7223.
38. Krantz, A.; Copp, L.; Smith, R. A.; Heard, S. B. Bio-
chemistry 1991, 30, 4678.
39. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
40. Nitrosoamines and Related N-Nitroso Compounds;
Loeppky, R. N., Michejda, C. J., Eds.; ACS Symposium Series
553, 1994.
28. Mohr, S.; Zech, B.; Lapetina, E. G.; Brune, B. Biochem.
Biophys. Res. Commun. 1997, 238, 387.
29. Rossig, L.; Fichtlscherer, B.; Breitschopf, K.; Haendeler,
J.; Zeiher, A. M.; Mulsch, A.; Dimmeler, S. J. Biol. Chem.
1999, 274, 6823.
30. Haendeler, J.; Weiland, U.; Zeiher, A. M.; Dimmeler, S.
Nitric Oxide 1997, 1, 282.
41. Lijinsky, W. Chemistry and Biology of N-Nitroso Com-
pounds; Cambridge University: Cambridge: U.K., 1992.
American Chemical Society Monograph 182: Chemical Carci-
nogens, Searle, C. E.; Ed., American Chemical Society:
Washington, DC, 1984; Vols 1 and 2.