Modulation of cellular apoptosis with Apoptotic protease-activating factor 1 (Apaf-1) inhibitors

Article abstract of DOI:10.1021/jm701195j

The programmed cell death or apoptosis plays both physiological and pathological roles in biology. Anomalous activation of apoptosis has been associated with malignancies. The intrinsic mitochondrial pathway of apoptosis activation occurs through a multiprotein complex named the apoptosome. We have discovered molecules that bind to a central protein component of the apoptosome, Apaf-1, and inhibits its activity. These new first-in-class apoptosome inhibitors have been further improved by modifications directed to enhance their cellular penetration to yield compounds that decrease cell death, both in cellular models of apoptosis and in neonatal rat cardiomyocytes under hypoxic conditions.

Full text of DOI:10.1021/jm701195j

J. Med. Chem. 2008, 51, 521–529
521
Modulation of Cellular Apoptosis with Apoptotic Protease-Activating Factor 1 (Apaf-1)
Inhibitors
,§
L. Mondragón,† M. Orzáez,† G. Sanclimens,‡ A. Moure,‡ A. Armiñán,§ P. Sepúlveda, A. Messeguer,‡ M. J. Vicent,*,† and
E. Pérez-Payá*,#
Department of Medicinal Chemistry, Centro de InVestigación Príncipe Felipe, Valencia, Spain, Department of Biological Organic Chemistry,
IIQAB, CSIC, Barcelona, Spain, Cardioregeneration Unit, Centro de InVestigación Príncipe Felipe, Valencia, Spain, Fundación Hospital
General UniVersitario de Valencia, Consorcio Hospital General UniVersitario de Valencia, Valencia, Spain, and Instituto de Biomedicina de
Valencia, CSIC, Valencia, Spain
ReceiVed September 24, 2007
The programmed cell death or apoptosis plays both physiological and pathological roles in biology. Anomalous
activation of apoptosis has been associated with malignancies. The intrinsic mitochondrial pathway of
apoptosis activation occurs through a multiprotein complex named the apoptosome. We have discovered
molecules that bind to a central protein component of the apoptosome, Apaf-1, and inhibits its activity.
These new first-in-class apoptosome inhibitors have been further improved by modifications directed to
enhance their cellular penetration to yield compounds that decrease cell death, both in cellular models of
apoptosis and in neonatal rat cardiomyocytes under hypoxic conditions.
Introduction
its suppression by biological tools provided resistance to
apoptosis induction.11,12 Inactivation of the apoptosome might
provide a therapeutic avenue for treating not only neurodegen-
erative but other diseases such as ischemia and cardiac and renal
failure.
Apoptosis is a fundamental mechanism of programmed cell
death that is regulated physiologically and genetically and plays
a central role in development, normal cell turnover, and immune
system function. Moreover, abnormal apoptotic processes are
important and influence the severity of disease progression in a
number of pathologies. Defects in appropriate suppression of
apoptosis are observed in cancer pathogenesis1 and autoimmune
disorders,2 while anomalous induced apoptosis plays an impor-
tant role in acquired immunodeficiency disease (AIDS),3 and
neurodegenerative and heart diseases.4,5 The mechanism of
apoptosis is executed by a family of highly conserved proteases
known as caspases,6 which in a cascade of sequential initiator
and effector members dismantle the cell. Different cell death
stimuli can initiate the mechanism. In particular, defined
apoptotic signals activate the mitochondria-mediated or intrinsic
pathway that utilizes caspase-9 as its initiator. Caspase-9
activation is triggered by the release to the cytoplasm of
proapoptotic proteins from the mitochondrial intermembrane
space, in particular cytochrome c and Smac/Diablo.7,8 The
formation of the macromolecular complex named apoptosome
is a key event in this pathway. The apoptosome is a holoen-
zyme multiprotein complex formed by cytochrome c activated
Apaf-1a (apoptosis protease-activating factor), dATP, and
procaspase-9.9 Understanding the mechanisms of functional activa-
tion of the apoptosome has helped to define prospective targets
for treating deregulated apoptosis that is associated with human
pathologies.10 However, inappropriate apoptosome activity has
been shown to play a role in neurodegenerative disorders and
We have early developed a new structural class of Apaf-1
ligands as apoptosome inhibitors.13 From this family of N-
alkylglycine inhibitors, the most potent in vitro was peptoid 1
(Chart 1). However, this hit exhibited low membrane perme-
ability and modest efficacy arresting apoptosis in cells. We have
shown that fusion of peptoid 1 to known cell penetrating
peptides14 facilitates cell permeation in defined cellular models
of apoptosis. Furthermore, the conjugation of peptoid 1 to a
polymeric carrier (PGA-1) demonstrated an enhanced efficacy
probably related to an efficient lysosomotropic drug release on
the cytosol.15 Here, we have extended these studies by exploring
the activity of PGA-1 in different cellular models of apoptosis,
such as human cervix adenocarcinoma (HeLa), human histio-
cytic lymphoma (U937), human osteosarcoma (U-2 OS), human
lung carcinoma (A549), and human osteogenic Sarcoma (Saos-
2) with inducible expression of Bax. In addition, we have also
explored how restriction of the conformational mobility of
peptoid 1 through backbone cyclization decreased the unspecific
toxicity of 1 and increased its antiapoptotic activity. A systematic
comparative study of these new first-in-class apoptosome inhibitors
has been developed, and the results suggested that inhibition of
the apoptosome resulted in increased cell recovery, when compared
to control cells, after the apoptotic insult. Finally, we carried out
studies directed to explore the use of apoptosome inhibitors as
agents that could decrease the degree of apoptosis in neonatal rat
cardiomyocytes subjected to hypoxic conditions.
* To whom correspondence should be addressed. For M.J.V.: phone,
+34-963289680; fax, +34-963289701; e-mail, mjvicent@cipf.es. For E.P.-
P.: address, Centro de Investigación Príncipe Felipe, Av. Autopista del Saler,
16, E-46013 Valencia, Spain; phone, +34-963289680; fax, +34-963289701;
e-mail, eperez@cipf.es.
Results and Discussion
To increase the cellular activity of the originally identified
apoptosome inhibitor peptoid 1,13 structural modifications of
†
Department of Medicinal Chemistry, Centro de Investigación Príncipe
a Abbreviations: Apaf-1, apoptotic protease-activating factor; DEVDase,
hydrolysis of Ac-DEVD-afc peptide; DTT, dithiothreitol; FCS, fetal-calf
serum; MEFs, mouse embryo fibroblasts; MMP, mitochondrial membrane
potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide; Ni-NTA, (Ni2+ nitrilotriacetate)–agarose; PBS, phosphate buffered
saline; PGA, poly-L-glutamic acid); rApaf-1, recombinant Apaf-1.
Felipe.
‡
IIQAB, CSIC.
§
Cardioregeneration Unit, Centro de Investigación Príncipe Felipe.
Consorcio Hospital General Universitario de Valencia.
Instituto de Biomedicina de Valencia, CSIC.
#
10.1021/jm701195j CCC: $40.75
2008 American Chemical Society
Published on Web 01/16/2008

522 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Mondragón et al.
Chart 1. Chemical Structure of 1 and Its Derivatives Studied Here, Namely, Peptide-Peptoid Hybrids (As Example, PEN-1a),
PGA-peptoid Conjugate, and the Cyclic Derivative, 2
a One-letter code for the amino acids has been used to describe the peptidic part of the molecule.
Figure 1. MTT cell viability measurements of peptoid 1 derivatives. (A) Cytotoxicity of peptoid 1 ([), the different peptide/peptoid hybrids (0
PEN-1 and O TAT-1), compound 2 (]), and PGA-1 (9) against U937 cells measured after 24 h of incubation. Data are expressed as the mean
( SE (n > 3). (B) Evaluation of compound-dependent cell recovery after doxorubicin-induced cell death in U-2 OS cells. The peptoid derivatives
and compound 2 were evaluated at different concentrations (10, 5, and 1 µM (black, gray, and white bars, respectively)) after 24, 48, and 72 h of
incubation. In all cases, SE < 10%. Data are expressed as the mean ( SE (n > 3).
this compound had to be done because of its poor solubility
and low membrane permeability. The potential production of
undesirable side effects due to the conformational freedom of
peptoids was also a matter of concern. Thus, we focused our
studies on delivery systems by means of adequate carriers or
on chemical modifications that could diminish the conforma-
tional mobility of peptoid 1. These goals were addressed in three
different ways: (i) by the design of hybrid peptide/peptoid
conjugates where the parent peptoid 1 was fused to well
characterized cell penetrating peptides (CPPs) such as penetratin
(PEN) and Tat HIV-1 (TAT) peptides, yielding compounds
PEN-1 and TAT-1,14 respectively; (ii) by means of backbone
cyclization; (iii) by conjugation to a water soluble polymeric
carriersuchaspoly-L-glutamicacid(PGA),obtainingPGA-peptoid
conjugates.15
Cytotoxicity and Cell Recovery Capability of Peptoid 1
Derivatives. Before proceeding with cellular models of apop-
tosis inhibition, the unspecific toxicity of the compounds in our
cell panel was analyzed by means of MTT assays (see
Experimental Section for full details on cell lines) (Figure 1A).
TAT-1 and PEN-1 were found to be highly and slightly
toxic, respectively, for the cells. In contrast, both 2 and PGA-1
were shown to be devoid of unspecific cell toxicity. The
enhancement of peptoid 1 cytotoxicity after fusion with TAT
peptide could be related to a cargo-induced restriction of the
conformational space available for the CPPs that could be
necessary for an effective and safe cellular membrane perme-
ation.14 A preliminary analysis of the antiapoptotic activity of
peptoid 1 derivatives was performed in osteosarcoma U-2 OS
cells where the cytotoxic compound doxorubicin induces cell
death in a time and concentration dependent manner. Doxoru-
bicin induces apoptosis through DNA damage that is transduced
to the mitochondria, disturbing the mitochondrial membrane
potential (MMP) and activating executioner caspases through
involvement of the apoptosome. After 12 h, 2 µM doxorubicin-
treated cells demonstrated staining for annexin V-PE (which
specifically binds to exposed phosphatidylserine) and loss of
MMP, both being feature markers of an apoptotic cellular
phenotype.13 When cells were treated with doxorubicin in the
presence of 2 and PGA-1, the extent of apoptosis was
remarkably diminished as determined by the lowered percentage
of cells showing apoptotic phenotype (Figure 1B). In contrast,
as expected from the previous unspecific cell toxicity evaluation,
TAT-1 did not prevent cellular death and PEN-1 showed only
partial protection at low concentration, evidencing a compromise
between the protective effect of the peptoid 1 and residual

Inhibiting Apoptosis with Apaf-1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 523
the U937 cell line when a 50 µM drug equivalent of PGA-1
was used (p < 0.05)). It is important to note that PGA conjugates
(i.e., like the PGA-1 analyzed in the present study) are usually
active at concentrations 10-fold higher than the parent low
molecular weight drugs because of the different pharmacoki-
netics.18
It is well established that inhibition of apoptotic cell death
should correlate with inhibition of executioner caspases. Then
we were willing to evaluate caspase-3 activity in extracts derived
from cells that were treated with both the apoptosis inducer and
the inhibitors and compare the results to those obtained from
cell extracts only treated with the inducers (Figure 3). Initially,
the compounds PEN-1, 2, and PGA-1 were evaluated in a
common cell line, Saos-2. At this point, for comparative
purposes, we decided to include in our study the diarylurea-
based compound 3 previously described as apoptosome inhibi-
tor.19 PEN-1 and 3 showed low caspase-3 inhibitory activity
in these experimental conditions. However, the inhibition of the
apoptosome activity by the treatment of the cells with 2 and
PGA-1 resulted in inhibition of caspase-3 activity. 2 was
effective when the cell extract was obtained after 24 h of
treatment, while PGA-1 showed increased activity at longer
times (p < 0.001) (Figure 3A). The delay on time to reach the
activity obtained for PGA-1 when compared to 2 is probably
due to the mechanism of action and cellular pharmacokinetics
of polymeric drugs. Effective biological activity of polymer-drug
conjugates relies on lysosomal enzyme cleavage to release active
drug in a specific place after cellular uptake by endocytosis.15
Interestingly, PGA-1 reaches values up to 100% of caspase-3
inhibition at 50 µM drug equivalent (HeLa at 48 h and Saos-2
at 72 h) (p < 0.05) (Figure 3B), whereas 2 at 5 µM reaches
inhibitory percentages of 50-60% (U-2 OS at 48 h and HeLa
at 48 h, respectively) (p < 0.05) (Figure 3C). The good
correlation found in our analysis of caspase-3 inhibition and
cell recovery could suggest that the compounds are direct
inhibitors of the enzymatic activity of caspase-3; however, this
is not the case. In fact, none of the compounds here analyzed
was able to inhibit caspase-3 using a recombinant-protein based
assay (results not shown). In contrast, when we directly activated
the apoptosome with an apoptosome activator (compound 4)
recently described by Nguyen and Wells,20 we obtained cell
protection when 4 was provided to the cells in the presence of
the apoptosome inhibitor PGA-1 (p < 0.001) (Figure 3D).
Compound 2 Analogues. As explained above, the cyclic
derivative 2 was one of the most active peptoid 1 derivatives
together with PGA-1 conjugate. In order to seek further
optimization, several structural modifications of 2 were carried
out. Initially we focused our attention on the 2′,4′-dichlorophe-
nylethyl substituent attached to the perhydro-1,4-diazepine-2,5-
dione ring (I, Chart 2). This substituent turned out to be essential
for activity, and in our hands, any attempt at modification
resulted in a loss of antiapoptotic activity. Similar results were
found in early attempts at modification of the 3,3-diphenylpropyl
moiety.13 Then we addressed our attention to the R1 moiety.
As shown in Table 1, a series of 8 derivatives (2a-h) were
designed and their preparation was carried out by using a
practical approach. Thus, according both to the nature of the
R1 moiety and to the expected behavior of the mixtures in
preparative HPLC, these analogues were separated into three
subsets containing two, three, and three compounds (2a and
2b, 2c-e, and 2f-h, respectively). Then each subset of
compounds was prepared by using a synthetic pathway based
on solid-phase and microwave activation and using the positional
scanning approach for construction of controlled mixtures. By
Figure 2. Evaluation of cell death inhibition in different cell lines by
2 (A) and PGA-1 (B). 2 was evaluated at 10, 5, and 1 µM (black,
gray, and white bars, respectively) and PGA-1 at 100, 50, and 20 µM
drug equivalent (black, gray, and white bars, respectively) after 24,
48, and 72 h of incubation. Data are expressed as the mean ( SE (n >
3). In all cases, SE < 10%.
unspecific toxicity associated with the whole molecule (Figure
1B). Compound 2 and PGA-1 were then selected for a
systematic study as cell death inhibitors using different cell lines
and different cell death inducers.
To prove the extent of protection against apoptosis of
compounds 2 and PGA-1, they were evaluated in a panel of
different cell models (U937 histiocytic lymphoma, A549 lung
carcinoma, HeLa adenocarcinoma, and U-2 OS osteosarcoma)
using doxorubicin as apoptosis inducer. In addition, we also
evaluated a more well defined model of “only intrinsic”
apoptosis using Saos-2 cells were apoptosis is induced by the
doxycyclin-dependent conditional expression of Bax through
the tet-ON system (Clontech). Bax is a proapoptotic member
of the Bcl-2 family that induces apoptosis by the release of
cytochrome c from the mitochondria;16,17 thus, activation of
apoptosis solely relies on the mitochondrial pathway. Figure 2
summarizes the cell death inhibitory activity of 2 (Figure 2A)
and PGA-1 (Figure 2B) in the above-mentioned cell panel.
The two compounds, 2 and PGA-1, substantially decreased
apoptosis in both doxorubicin-induced (U937, U-2 OS, HeLa,
and A549 cell lines) and doxycyclin-induced (Saos-2 cells) cell
death when cell viability was evaluated at 24 h after treatment
(up to 60% of cell death inhibition in the Saos-2 cell line was
obtained when using a 1 µM 2 (p < 0.001) and up to 100% in

524 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Mondragón et al.
Chart 2. Chemical Structure of 2 and Its Derivatives Here
Studied
doxycyclin, in the presence or absence of the compound
subjected to evaluation. As expected from previous results,
replacement of the 2′,4′-dichlorophenylethyl moiety at R1 by a
nonaromatic moiety resulted in a decrease of potency (com-
pounds 2c, 2d, 2e, 2g). However, the behavior of compound
2c was a bit off provided that the inhibition of caspase-3 activity
was systematically close to 50% while the cell recovery ability
was 2%. Also, the activity in the cell recovery experiment for
this compound was slightly higher at 1 µM (not shown) than at
5 µM. It is mentioned that in certain occasions, biological assays
developed with cell extracts and whole cell systems provide
results that are more difficult to rationalize when compared to
those obtained from in vitro experiments. Furthermore, parallel
studies to analyze the unspecific toxicity of the compounds
suggested that only compound 2d was toxic. Different electro-
negative atoms (such as O or F) in several positions (para, meta,
or ortho) were also used in order to evaluate the effect of the
electronic density changes on this family of apoptosome
inhibitors (namely, 2a, 2e, 2g, and 2h). In general, although all
these analogues exhibited the capability of reducing the apop-
tosis induced in Saos-2 cell line, none of them showed a
significant improvement when compared to compound 2. Similar
results were obtained in the other models of our cell panel
mentioned above (HeLa, A549, U-2 OS, and U-937 cells; data
not shown). Taken altogether, it is not easy to make an
appropriate SAR because no clear trend was obtained. However,
it would be possible to suggest that the presence of a second
2′,4′-dichlorophenylethyl substituent is not essential but impor-
tant for the activity, probably because of the presence of the
two electronegative atoms and, certainly, of the aromatic ring.
This is still an ongoing research, and new efforts are currently
being devoted to modify the 1,3-diphenylpropyl substituent as
well as to explore other heterocyclic scaffolds.
Membrane Potential Recovery after PGA-1 Treatment.
Mitochondria play a prominent role in cell death as a central
organelle involved in the signal transduction and amplification
of the apoptotic response.21,22 Mitochondrial dysfunction is an
early event characterized by an increase in mitochondrial
membrane permeability. Thus, cells undergoing apoptosis are
characterized by a low mitochondrial membrane potential
(∆Ψmlow). In fact, when Saos-2 and U937 cells were subjected
to an apoptotic insult (Saos-2 treated with doxycycline and U937
with doxorubicin), the number of cells with ∆Ψmlow increased
when compared to untreated cells (p < 0.05) (Figure 4).
However, the effect was clearly attenuated when cells were
subjected to the insult in the presence of PGA-1. This result
suggests that PGA-1 treated cells could probably overcome
apoptosis at this postmitochondrial level of the pathway.
Although still speculative, this class of compounds could inhibit
processes downstream from the mitochondrial depolarization
event. Then damaged cells, at early stages of depolarization,
could still be in a reversible step of the apoptotic process, and
inhibition of Apaf-1 could result in a recovery of mitochondrial
potential. Another possibility is that the recovery of membrane
Figure 3. Caspase-3 (C3) activity in cell extracts measured by the
fluorimetric DEVDase assay at 24, 48, and 72 h incubation times (black,
gray, and white bars, respectively): (A) comparison of C3 activity in
Saos-2 cell extracts after incubation with the different peptoid 1
derivatives (all compounds at 5 µM concentration except PGA-1,
which was used at 50 µM drug equivalent); C3 activity in different
cell lines after incubation with PGA-1 at 50 µM drug equivalent (B)
and 2 at 5 µM concentration (C); (D) C3 activity in U-937 cells after
direct activation of the apoptosome assembly20 with compound 4 in
the presence and absence of PGA-1 at 50 µM drug equivalent. Data
are expressed as the mean ( SE (n > 3).
this procedure, three HPLC purifications were only required for
isolating all compounds in purities over 98% and satisfactory
overall yields.
Table 1 summarizes the caspase-3 inhibitory activity deter-
mined in cell extracts and the observed cell recovery when
apoptosis was induced in Saos-2 cells upon treatment with

Inhibiting Apoptosis with Apaf-1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 525
Table 1. Percentage of Cell Recovery and Inhibition of Caspase-3 Activity Obtained with Compound 2 Analogues in Bax-Induced Apoptosis in Saos-2
Cell Linea
a Cell recovery and caspase-3 activity were analyzed after 24 h of treatment with the respective compounds at 5 µM concentration. Data are expressed
as the mean ( SE (n > 3). ND: not determined.
Figure 4. Evaluation of apoptosis by flow cytometry as the percentage of cells with low membrane potential (∆Ψmlow): flow cytometry analysis
of ∆Ψm of Saos-2 and U-937 cells untreated (black bars); subjected to an apoptotic insult by treatment with doxycyclin and doxorubicin, respectively
(gray bars); when the apoptotic insult was applied in the presence of PGA-1 at 50 µM drug equivalent (white bars). Data are expressed as the
mean ( SE (n > 3).
potential of the population would be related to a process
downstream from the mitochondria, such as the loss or not of
plasmatic membrane potential caused by a decrease in the
intracellular levels of K+ ions. Although the ability of mito-
chondrial recovery after partial depolarization is still a matter
of discussion, it seems well accepted that it could occur in
neurons,23 but it is controversial in other cell types as smooth
muscle cells or myofibroblasts.24
Inhibition of Hypoxia-Induced Apoptosis in Primary
Culture Cardiomyocytes by PGA-1. Apoptosis plays a crucial
role in ischemic-based diseases. Among them, myocardial
damage induced by ischemia-reperfusion has been shown to
be directly associated with apoptosis. Furthermore, after myo-
cardial infarction, the presence of apoptotic cells has been
demonstrated both in the infarct area and in the border zone of
the infarction.25–30 Although the signal transduction pathways
are not completely defined, it is known that mitochondrial
dysfunction is one of the most critical events associated with
myocardial ischemia-reperfusion injury.31,32 In this sense,
previous studies showed that very low levels of cardiac myocyte
apoptosis are sufficient to produce heart failure.33 In addition,
reduction of myocyte apoptosis by the potent polycaspase
inhibitor IDN-1965 improved left ventricular function and
survival of mice with an induced peripartum cardiomyopathy.34
We therefore analyzed the ability of the apoptosome inhibitors
here described, and in particular PGA-1, as potential inhibitors
of hypoxia-induced apoptosis in cardiomyocytes with an ex vivo
model of myocardial infarction. Then primary cultures of
neonatal rat cardiomyocytes were established and subjected to
hypoxic conditions. A double immunohistochemistry based
procedure clearly showed the presence of apoptotic cells and
active caspase-3 demonstrating the active role of apoptosis in
hypoxia-induced cardiomyocytes cell death (Figure 5A). The
presence of Apaf-1 protein in neonatal cardiomyocytes was also

526 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Mondragón et al.
Figure 5. PGA-1 is capable of reducing hypoxia-induced apoptosis in primary culture cardiomyocytes. (A) Apoptosis induction in primary culture
cardiomyocytes subjected to hypoxia. Double immunohistochemistry of cardiomyocytes with anti-caspase 3 (green) and anti-R-actinine (red) antibodies
was analyzed by confocal microscopy (40× magnification). Nuclei are stained with DAPI (blue). (B) Evaluation of induced cell death inhibition
by MTT assay, showing PGA-1 at different concentrations (50 and 100 µM drug equivalent; black and gray, respectively) after 24 h of incubation.
(C) Caspase-3 activity in cell extracts measured by the fluorimetric DEVDase assay at 24 h of incubation time. Data are expressed as the mean (
SE (n > 3).
determined by Western blot (data not shown). These results
provide a validation of the cellular model selected for our ex
vivo studies. Importantly, when the neonatal cardiomyocytes
were subjected to the hypoxia conditions in the presence of
PGA-1, the percentage of cell death was clearly diminished
when compared to the control cell population (p < 0.05) (Figure
5B). Furthermore, to determine whether or not PGA-1 could
decrease caspase-3 activity in this cell system, cells from a
2-day-old established culture were subjected to the hypoxia
conditions in the presence of different concentrations of PGA-1
and caspase-3 activity was assessed (Figure 5C). The results
revealed a marked PGA-1 dependent decrease of caspase-3
activity (p < 0.001).
an appropriate in vivo model. We are currently evaluating
different experimental models of myocardial infarction. If the
compounds analyzed here further demonstrate the ability to
reduce hypoxia-induced cardiomyocyte apoptosis in vivo, it
would result in a prevention of ventricular remodeling and
improvement of damaged cardiac function. That in turn would
open new strategies to improve the therapeutic resources for
the treatment of this unfortunately very common pathology.
Experimental Section
Materials. Poly-L-glutamic acid sodium salt (PGA), N,N-
diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt),
diisopropylethylamine (DIEA), and anhydrous dimethylformamide
(DMF) were purchased from Sigma-Aldrich and used without
further purification. All solvents including HPLC grade were
obtained from Merck (Barcelona, Spain). Tissue culture grade
dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), trypan blue solution (0.4%) (cell
culture grade), bovine cathepsin B, and dithiothreitol (DTT) were
from Sigma-Aldrich. Samples of 0.25% trypsin-EDTA, fetal calf
serum (FCS), RPMI 1640 with L-glutamine, D-MEM with L-
glutamine, and collagenase type II were from Gibco BRL Life
Technologies (Paisley, U.K.). Annexin V-FITC apoptosis detection
kit I was from BD PharmingenTM (BD Bioscience, Madrid, Spain)
and 3,3′-dihexyloxacarbocyanine iodide (DIOC6(3)) from Molecular
Probes (Invitrogen, Barcelona, Spain). The fluorogenic caspase-3
substrate, Ac-DEVD-afc, was purchased from Biomol International
LP (Exeter, U.K.). Protein quantification was performed with a
bicinchoninic acid kit from Pierce Technology Corporation (NJ).
Dispase was purchased from Roche Diagnostics (Godo Shusei Co,
Conclusions
The overall data obtained suggest that apoptosome inhibitors
can decrease the extension of cell death. Two important concepts
come into view from this study. First, compound 2 and the PGA-
based derivative PGA-1 inhibit apoptosis in different cell lines.
Second, inhibition of the intrinsic cell death (or mitochondrial)
pathway by specific inhibition of Apaf-1 could result in the
recovery of the cells or in prevention of the cellular dam-
age induced by apoptotic insults. Whether or not this could have
therapeutic interest is a fundamental question. In this sense, we
have observed that PGA-1 was active inhibiting hypoxia-
induced cell death in cardiomyocytes. Although the results
shown here are derived from ex vivo studies, we are now
moving ahead to the next level of complexity that represents

Inhibiting Apoptosis with Apaf-1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 527
Ltd., Tokyo, Japan). For immunochemistry assays, rabbit polyclonal
active caspase-3 antibody was purchased from Cell Signaling
Technology (Beverly, MA) and mouse monoclonal anti-R-actinine
(sarcomeric) was purchased from Sigma-Aldrich. Antirabbit and
antimouse secondary antibodies were coupled to FITC and cyanine-
3, respectively, and were purchased from Jackson Immunoresearch
(Suffolk, England). All other reagents were of general laboratory
grade.
Chemistry. The HPLC analyses of peptoid 1 and compound 2
and its analogues were carried out with a Hewlett-Packard series
1100 (UV detector 1315A) modular system using a Kromasil 100
C8 (15 cm × 0.46 cm, 5 µm) column with CH3CN-H2O mixtures
containing 0.1% TFA at 1 mL/min as mobile phase and with
monitoring at 220 nm. Compounds were purified from the crude
reaction mixture by semipreparative HPLC using a Kromasil C8
(25 cm × 2 cm, 5 µm) column, CH3CN-H2O mixtures containing
0.1% TFA as mobile phases, and a flow rate of 5 mL/min. The
NMR spectra of peptoids were recorded in CD3OD solutions using
a Varian Inova 500 apparatus (1H NMR, 500 MHz; 13C NMR,
125 MHz). The assignments of absorptions observed for peptoid 1
and compound 2 were confirmed by gDQCOSY and gHSQC
experiments. The different conformations present in both com-
pounds led to the observation of complex absorptions. High-
resolution mass spectra (HRMS-FAB) were carried out at the Mass
Spectrometry Service of the University of Santiago de Compostela
(Spain). The synthesis and characterization of PGA-1, PEN-1,
and TAT-1 have been reported.14,15
Synthesis of [N-(2,4-Dichlorophenethyl)glycyl]-[N-(3,3-diphe-
nylpropyl)]glycyl]-N-(2,4-dichlorophenethyl)glycinamide (1).
The synthesis of this peptoid was carried out following the method
described elsewhere.35 1H NMR: δ 7.5–7.4 (2H, CHAr), 7.25–7.35
(12H, CHAr), 7.15 (2H, CHAr), 4.2–3.8 (6H, COCH2N), 4.02 (1H,
CH), 3.58 (2H, NCH2), 3.20 (2H, NCH2CH), 3.15 (2H, NHCH2),
3.10 (2H, NHCH2CH2), 3.0–2.90 (2H, NCH2CH2), 2.4–2.1 (2H,
NCH2CH2CH). 13C NMR: δ 172.62, 172.39 (CO), 170.14, 169.87
(CO), 167.53, 166.60 (CO), 145.85 (2 × CAr), 136.0 (2 × CAr),
134.2 (2 × CAr), 133.8 (2 × CAr), 133.2, 133.1 (2 × CHAr), 129.8
(2 × CHAr), 129.5 (2 × CHAr), 128.9 (4 × CHAr), 128.8 (4 × CHAr),
127.4 (2 × CHAr), 50.4 (COCH2N), 49.5 (CH), 49.1 (NCH2), 48.9
(COCH2N), 48.2 (NCH2CH), 48.1 (COCH2N), 47.8 (HNCH2), 34.2
(NCH2CH2CH), 32.6 (NCH2CH2), 30.4 (HNCH2CH2). HRMS (M
+ 1) calcd C37H3935Cl4N4O3, 727.1747; found, 727.1751.
Synthesis of 1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophe-
nyl)ethyl]-7-[N-aminocarbonylmethyl-N-[2′-(2′′,4′′-dichlorophe-
nyl)ethyl]aminocarbonyl]perhydro-1,4-diazepine-2,5-dione (2).
The synthesis of this heterocyclic derivative was carried out
following a solid-phase general procedure with microwave activa-
tion developed in our laboratory that is briefly described below for
the preparation of compound 2 analogues and that will be reported
elsewhere. 1H NMR: δ 7.62–7.53 (2H), 7.48–7.35 (2H), 7.32–7.23
(10H), 7.16 (m, 2H), 4.70, 4.60 (1H, (CH)het), 4.21, 4.19 (d, J )
17.1 Hz, 1H, (COCH2N)het), 4.02–3.78 (1H (COCH2N)het + 2H
(COCH2N) + 1H (CH)), 4.02 (CHPh2), 3.53–3.45 (6H, NCH2),
3.00, 293 (dd, J ) 14.7 and 6.3 Hz, 1H, COCH2CH), 2.90–2.71
quently, resins were treated with a solution of bromoacetic acid
(130 mg, 5 equiv) and DIC (145 µL, 5 equiv) in DMF (3 mL).
The reaction mixtures were stirred for 2 min at 60 °C in a
microwave reactor. After they were drained and washed, a solution
of (tetrahydrofuran-2-yl)methanamine (98 µL, 5 equiv) and Et3N
(131 µL, 5 equiv) in 3 mL of DMF was added to the first resin and
the suspension was stirred for 2 min at 90 °C under microwave
activation. Another solution of butylamine (93 µL, 5 equiv) and
Et3N (131 µL, 5 equiv) in 3 mL of DMF was added to the second
resin, and the suspension was stirred for 2 min at 90 °C under
microwave activation. Finally, the third resin was treated with a
solution of 2-(2-fluorophenyl)ethanamine (123 µL, 5 equiv) and
Et3N (131 µL, 5 equiv) in 3 mL of DMF and the suspension was
stirred for 2 min at 90 °C under microwave activation. The
supernatant of the three resins was removed, and the residue was
drained and washed. Afterward, all resins were mixed into the same
syringe and the subsequent steps were carried out together. The
overall resin was treated with a solution of allyl maleate (440 mg,
5 equiv), HOBt (380 mg, 5 equiv), and DIC (436 µL, 5 equiv) in
DCM/DMF (2:1, 5 mL). The reaction mixture was stirred at room
temperature for 30 min and filtered, and the reaction was repeated
under the same conditions. After the resin was drained and washed,
a solution of 3,3-diphenylpropylamine (594 mg, 5 equiv) and Et3N
(395 µL, 5 equiv) in 6 mL of DMF was added to the resin and the
suspension was stirred for 3 h at room temperature. The reaction
was repeated overnight at the same temperature. The supernatant
was removed, and the residue was drained and washed. Then the
resin was treated with a solution of bromoacetic acid (391 mg, 5
equiv) and DIC (436 µL, 5 equiv) in DMF (6 mL). The reaction
mixture was stirred for 2 min at 60 °C in a microwave reactor.
The resin was drained and washed. A solution of 2,4-dichlorophen-
ethylamine (425 µL, 5 equiv) and Et3N (395 µL, 5 equiv) in 6 mL
of DMF was added to the resin, and the suspension was stirred for
2 min at 90 °C under microwave activation. The supernatant was
removed, and the residue was drained and washed. Then the resin
was treated with Pd(PPh3)4 (65 mg, 0.1 equiv) and PhSiH3 (693
µL, 10 equiv) in anhydrous DCM for 15 min at room temperature
under an inert atmosphere. The supernatant was removed, and the
residue was drained and washed. The final cyclization was promoted
by treatment with PyBOP (440 mg, 1.5 equiv), HOBt (114 mg,
1.5 equiv), and DIEA (300 µL, 3 equiv) in DMF (6 mL). The
reaction mixture was stirred at room temperature for 16 h and
filtered. Finally, treatment of the resin (1013 g) with a mixture of
60:40:2 TFA/DCM/water released a crude reaction mixture contain-
ing the three target compounds. Solvents from the filtrate were
removed by evaporation under reduced pressure followed by
lyophilization. The residue obtained (286 mg) was characterized
by analytical RP-HPLC and RP-HPLC-MS using aqueous aceto-
nitrile gradient (20% CH3CN f 80% CH3CN, 20 min). Retention
times of compounds were found to be 17.14 min for 2d, 18.1 min
for 2c, and 19.0 min for 2e. The crude reaction mixture was purified
by semipreparative RP-HPLC using aqueous acetonitrile gradient
(42% CH3CN f 80% CH3CN, 35 min) to give the desired products
(yield 30–47%, g98% purity).
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-[(tetrahydrofuran-2′-yl)methyl]-
aminocarbonyl]perhydro-1,4-diazepine-2,5-dione (2d):. HRMS
(M + H)+ calcd for C35H4035Cl2N4O5, 679.2449; found, 679.2443.
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-(butyl)aminocarbonyl]perhydro-
1,4-diazepine-2,5-dione (2c):. HRMS (M + H)+ calcd for
C35H4035Cl2N4O4, 651.2499; found, 651.2524.
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-[2′-(2′′-fluorophenyl)ethyl]ami-
nocarbonyl]perhydro-1,4-diazepine-2,5-dione (2e):. HRMS (M
+ H)+ calcd for C39H3935Cl2FN4O4, 717.2405; found, 717.2396.
The same procedure was carried out for the synthesis of the
second and the third subset of compound 2 derivatives (second
subset 2f, 2g, and 2h and third subset 2a and 2b). Retention times
for compounds were 16.49 min for 2f, 20.91 min for 2g, 21.55
min for 2h, and 20.58 min for 2b. The corresponding crude reaction
(5H, 1H (COCH2CH)
+ 4H(NCH2CH2)), 2.48–2.15 (2H,
NCH2CH2CH). 13C NMR: δ 172.5 (CO), 171.5, 170.8 (CO), 170.1
(CO), 169.3 (CO), 145.4 (CAr), 145.0 (CAr), 136.3–135.4 (2 × CAr),
134.7 (2 × CAr), 133.5 (CHAr), 133.1 (CHAr), 132.9 (CHAr), 132.5
(2 × CAr), 129.4–129.0 (4 × CHAr), 128.1 (4 × CHAr), 126.8
(CHAr), 126.7 (CHAr), 56.0, 55.6 (CHhet), 54.2–49.7 (2 × COCH2N),
49.2, 49.0 (CH), 47.8, 47.7 (NCH2), 47.0 (NCH2), 46.9 (NCH2CH),
37.6, 37.1 (COCH2CH), 32.7, 32.5 (NCH2CH2CH), 31.4
(NCH2CH2), 30.8 (NCH2CH2). HRMS (M + 1) calcd for
C39H3835Cl4N4O4, 767.1720; found, 767.1735.
Synthesis of Compound 2 Analogues. The preparation of the
first set of analogues (2c, 2d, and 2e) was carried out as follows.
After all operations were carried out in solid phase, the polymer
was drained and washed with with DCM (3 × 3 mL) and DMF (3
× 3 mL). Thus, three syringes were prepared with 250 mg of Rink
amide resin (0.2 mmol) and treated separately with 3 mL of 20%
piperidine in DMF. The resins were filtered and washed. Subse-

528 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Mondragón et al.
mixtures were purified as described above to yield the expected
compounds in 30–45% yields and g98% purity).
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-(2′-acetylaminoethyl)aminocarbo-
nyl]perhydro-1,4-diazepine-2,5-dione (2f):. HRMS (M + H)+
calcd for C35H3935Cl2N5O5, 680.2401; found, 680.2376.
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-[2′-(2′′-methoxyphenyl)ethyl]ami-
nocarbonyl]perhydro-1,4-diazepine-2,5-dione (2g):. HRMS (M
+ H)+ calcd for C40H4235Cl2N4O5, 729.2605; found, 729.2613.
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-aminocarbonylmethyl-N-[2′-(4′′-chlorophenyl)ethyl]aminocar-
bonyl]perhydro-1,4-diazepine-2,5-dione (2h):. HRMS (M + H)+
calcd for C39H3935Cl3N4O4, 733.2110; found, 733.2084.
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-(cyclopropyl)aminocarbonyl]per-
hydro-1,4-diazepine-2,5-dione (2b):. HRMS (M + H)+ calcd for
C34H3635Cl2N4O4, 635.2186; found, 635.2173.
and they were allowed to set for 24 h.13 Then cells were treated
with doxycycline (2 µg/mL in PBS) for Saos-2 cells and doxoru-
bicin in the cases of HeLa, U-2 OS, and U937 at concentrations of
2, 2.5, and 0.25 µg/mL in PBS, respectively. After 30 min, PEN-1
and compound 2 derivatives at 5 µM and PGA-1 at 50 and 100
µM drug equivalents were added. Cells were harvested after 24,
48, and 72 h of incubation and pellets resuspended in 30 µL of
extraction buffer (50 mM PIPES, 50 mM KCl, 5 mM EDTA, 2
mM MgCl2, 2 mM DTT supplemented with protease inhibitor
cocktail from Sigma) and kept on ice for 5 min. Once pellets were
frozen and thawed three times, cell lysates were centrifuged at
14 000 rpm for 5 min and supernatants were collected. Quantifica-
tion of total protein concentration from these cell extracts was
performed using the bicinchoninic acid method. Aliquots were
prepared at the same concentration, and afterward they were mixed
with 200 µL of caspases assay buffer (PBS, 10% glycerol, 0.1 mM
EDTA, 2 mM DTT) containing 20 µM Ac-DEVD-afc. Caspase
activity was continuously monitored following the release of
1-(3′,3′-Diphenylpropyl)-4-[2′-(2′′,4′′-dichlorophenyl)ethyl]-7-
[N-(aminocarbonylmethyl)-N-[2′-(4′′-fluorophenyl)ethyl]ami-
nocarbonyl]perhydro-1,4-diazepine-2,5-dione (2a):. HRMS (M
+ H)+ calcd for C39H3935Cl2FN4O4, 717.2405; found, 717.2410.
Biological Assays. Cell Culture and Culture Conditions. The
U937 human histiocytic lymphoma and the U-2 OS human
osteosarcoma cell lines were obtained from the American Type
Culture Collection (Rockville, MD). The U937 cells were grown
in suspension in RPMI 1640 medium supplemented with 10% FCS
and 2 mM L-glutamine. The Saos-2 human osteosarcoma cell line
were kindly provided by Karin Vousden (Cancer Research U.K.,
Glasgow), and the HeLa human cervix adenocarcinoma cell lines
were kindly provided by Jaime Font de Mora (CIPF, Valencia,
Spain). The U-2 OS, Saos-2, HeLa, and A549 cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with 15% FCS
and 2 mM L-glutamine in all cases except in Saos-2. Cells were
maintained at 37 °C in an atmosphere of 5% carbon dioxide and
95% air and underwent passage twice weekly.
Culture of Neonatal Rat Cardiomyocytes and Hypoxic
Conditions. Rat neonatal cardiomyocytes were prepared from
ventricles of 1–2 days Wistar rats by a previously described
method34,36 with some modifications. Briefly, hearts were isolated,
auricles were discarded, and ventricles were minced and digested
with 2 mg/mL of collagenase type II for 1 h and subsequently with
1.2 U/mL dispase for 30 min with gently shaking. After two washes
primary cultures were preplated for 90 min to eliminate contaminat-
ing fibroblasts. Hypoxia was induced by incubating the cardiomyo-
cyte cultures in an atmosphere of 1% O2. Experiment was performed
three times with different breedings. Each procedure was repeated
twice for two identically treated culture flasks.
fluorescent afc at 37 °C using a Wallac 1420 Workstation (λexc
400 nm; λem) 508 nm). DEVDase activity was expressed as the
increase of relative fluorescence units per minute (∆FU/min).
)
Evaluation of Cellular Apoptosis by Flow Cytometry Analy-
sis. Saos-2 and U937 cells were seeded in six-well plates at a
seeding density of 2 × 105 and 4 × 105 cells per plate, respectively.
Cells were treated with doxycycline (2 µg/mL in PBS) and
doxycycline plus PGA-1 (50 µM drug equivalent) for Saos-2 or
doxorubicin (0.25 µg/mL in H2O) and doxorubicin plus PGA-1
in the case of U937 for 24, 48, and 72 h. Apoptosis was analyzed
by flow cytometry by determining the changes in cell forward/side
scatter and through the determination of phosphatidylserine (PS)
exposure and mitochondrial membrane potential (∆Ψm) loss with
Annexin V-FITC and 3,3-dihexyloxacarbocyanine iodide (Di-
OC6(3)), respectively. Necrotic cell death was distinguished from
apoptotic using propidium iodide (PI) by co-incubation either with
Annexin V-FITC or with DiOC6(3). FL1 detector was used to
analyze Annexin V-FITC binding or DiOC6(3) accumulation (λexc
) 488 nm; λem ) 530 nm), and PI staining was followed by signal
detector FL3 (λexc ) 530 nm; λem ) 617 nm). For adherent cells,
first these were gently trypsinized and washed twice with cold PBS.
Then cells were resuspended in 1× binding buffer (10 mM Hepes/
NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at a concentration
of 1 × 106 cells/mL. After the transfer of 100 µL of the cell
solutions (1 × 105 cells) to a 5 mL culture tube, two different
staining protocols were followed: (i) 5 µL of annexin V-FITC and
5 µL of PI (ready-to-use solutions from the apoptosis detection
kit) were added to the cell solutions. Cells were gently vortexed
and incubated for 15 min at room temperature in the dark. (ii) An
amount of 5 µL of 100 nM DiOC6(3) in PBS was added to the
solution, and cells were incubated for 30 min at 37 °C. Then 5 µL
of PI (ready-to-use solutions from the apoptosis detection kit) was
also added and incubated for further 15 min at room temperature
in the dark. The highly fluorescent population was considered viable
cells, and the dull population represented apoptotic cells. Prior to
flow cytometry analysis, an amount of 400 µL of 1× binding buffer
was added in all cases to each tube.
MTT Cell Viability Assays.37 Cells were cultured in sterile 96-
well microtiter plates at a seeding density of 104, 3 × 103, 2.5 ×
103, and 2 × 103 cells/well for Saos-2, U-2 OS, HeLa, and A549,
respectively, and 2.5 × 103 and 4 × 103 cells/well in the case of
U937, and they were allowed to set for 24 h. Doxycycline (2 µg/
mL in PBS) for Saos-2 cells and doxorubicin in the cases of A549,
HeLa, U-2 OS, and U937 at concentrations of 1, 1.5, 2, and 0.25
µg/mL in PBS, respectively, were then added, and after 30 min
cells were treated with compounds (0.2 µm filter sterilized) at final
concentrations of 1, 5, and 10 µM in the cases of PEN-1, TAT-1,
and 2 and its derivatives, while PGA-1 was tested at concentrations
of 20, 50, and 100 µM drug equivalents. Compounds were incubated
for 19 h, and then MTT (20 µL of a 5 mg/mL solution) was added
to each well. Cells were further incubated for 5 h (a total of 24 h
of incubation with the inhibitors was therefore studied). In the next
step, medium was removed, the precipitated formazan crystals were
dissolved in optical grade DMSO (100 µL), and plates were read
at 570 nm using a Wallac 1420 workstation.
Immunohistochemistry. Cardiomyocytes were fixed in 2%
paraformaldehyde (PFA). Immunohistochemistry was performed
with antibodies against caspase-3 (1:100) and anti-R-actinine
(sacomeric) (1:800) followed by detection with secondary antibodies
coupled to either cyanine or FITC.
Statistical Analysis. Data are expressed as the mean ( standard
error. Student t tests were performed, and mean values were
considered statistically significant when p values less than 0.05 were
obtained.
Acknowledgment. This work was supported by grants from
Spanish Ministry of Science and Education (MEC) (Grants
BIO2004-998, RYC-2006-002438, and CTQ 2005-00995) and
Fundación Centro de Investigación Príncipe Felipe (CIPF) and
by Marie Curie Reintegration Grant MERG-2004-06307. M.
Caspase Activation Assays in Cellular Models (DEVDase
Activity). Cell extracts were prepared from cells seeded in 3.5 cm
diameter plates at seeding densities of 2 × 105 cells per plate in
the case of Saos-2, 3 × 105 and 3.5 × 105 cells per plate for HeLa
and U-2 OS, and 4 × 105 and 4.5 × 105 cells per plate for U937,

Inhibiting Apoptosis with Apaf-1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 529
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Supporting Information Available: HRMS spectra of key target
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