Aspartic vinyl sulfones: Inhibitors of a caspase-3-dependent pathway

Article abstract of DOI:10.1016/j.ejmech.2011.02.067

In this article we describe an expanded structure-activity relationship study for vinyl sulfones as caspase-3 inhibitors, a topic virtually unexplored in the existing literature. Most remarkably, and to our surprise, tripeptidyl vinyl sulfones were not active for caspase-3, opposite to other examples described in literature for peptidyl vinyl sulfones as potent cysteine protease inhibitors of clan CA. Moreover, the caspase-3 inhibitory activity of vinyl sulfones using an in vitro assay was then confirmed using a yeast cell-based assay. The results show that Fmoc-protected vinyl sulfones containing only the Asp moiety are inhibitors of a caspase-3-dependent pathway and the IC ₅₀ values obtained in the yeast assay are in the same order of magnitude of that obtained with the caspase-3 inhibitor tetrapeptidyl chloromethyl ketone, Ac-DEVD-CMK. This observation is consistent with appropriate cell permeability properties displayed by the vinyl sulfone inhibitors, as reflected by log P values ranging from 1.1 to 3.4. Overall, these results suggest that vinyl sulfones containing Asp at P₁ should be considered for further optimization as caspase inhibitors and modulators of caspase-3-dependent pathways.

Full text of DOI:10.1016/j.ejmech.2011.02.067

European Journal of Medicinal Chemistry 46 (2011) 2141e2146
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Aspartic vinyl sulfones: Inhibitors of a caspase-3-dependent pathway
Paulo M.C. Glória a, Isabel Coutinho b, Lídia M. Gonçalves a, Cristina Baptista b, Joana Soares b,
,
Ana S. Newton a, Rui Moreira a, Lucília Saraiva b, Maria M.M. Santos a
*
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
b REQUIMTE/CEQUP, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 4050-030 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
In this article we describe an expanded structureeactivity relationship study for vinyl sulfones as cas-
pase-3 inhibitors, a topic virtually unexplored in the existing literature. Most remarkably, and to our
surprise, tripeptidyl vinyl sulfones were not active for caspase-3, opposite to other examples described in
literature for peptidyl vinyl sulfones as potent cysteine protease inhibitors of clan CA. Moreover, the
caspase-3 inhibitory activity of vinyl sulfones using an in vitro assay was then confirmed using a yeast
cell-based assay. The results show that Fmoc-protected vinyl sulfones containing only the Asp moiety are
inhibitors of a caspase-3-dependent pathway and the IC50 values obtained in the yeast assay are in the
same order of magnitude of that obtained with the caspase-3 inhibitor tetrapeptidyl chloromethyl
ketone, Ac-DEVD-CMK. This observation is consistent with appropriate cell permeability properties
displayed by the vinyl sulfone inhibitors, as reflected by log P values ranging from 1.1 to 3.4. Overall, these
results suggest that vinyl sulfones containing Asp at P1 should be considered for further optimization as
caspase inhibitors and modulators of caspase-3-dependent pathways.
Received 11 January 2011
Received in revised form
16 February 2011
Accepted 27 February 2011
Available online 5 March 2011
Keywords:
Vinyl sulfone
Caspase-3 inhibitor
Michael acceptor
Irreversible inhibitor
Yeast cell-based assay
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
as caspase-3 inhibitors, enzymes that belong to clan CD [8]. Vinyl
sulfones containing Asp at P11aed and 2aeh (Fig. 1) were shown to
Caspases are a family of cysteine endoproteases involved in
cytokine maturation and apoptosis. They represent one of the most
specific protease families yet described, since they have an almost
absolute requirement for an aspartic acid residue in the P1 position
of their substrate [1e3]. To date, only a few caspase inhibitors have
entered preclinical studies with animal models of human diseases
and their major disadvantage is the lack of selectivity. As a conse-
quence, the search for effective caspase inhibitors as possible
therapeutic agents against different diseases caused by excessive
apoptosis, such as neurodegenerative disorders, is an important
area of research [4e6].
One type of irreversible cysteine proteases inhibitors that has
received special attention in the last few years are those based on
Michael acceptor scaffolds. This class of inhibitors includes vinyl
sulfones, which have been developed as highly potent inhibitors of
many clan CA cysteine proteases [7]. Recently, we described the
first structureeactivity relationship study of peptidyl vinyl sulfones
be only moderate inhibitors of caspase-3, with IC50 values in the mM
range. Dipeptidyl vinyl sulfones displayed improved activity over
their counterparts containing Asp as the single amino acid residue,
with this effect being particularly noticeably for the Fmoc-pro-
tected compounds. The most active compound, Fmoc-VD-VSMe 2g,
presented an IC50 value of 29
over caspase-7 [8].
mM and was selective for caspase-3
In this study, in order to expand the structureeactivity rela-
tionships (SARs) and to improve the inhibitory potency of vinyl
sulfones, we decided to extend the recognition structure by incor-
porating a third amino acid to derivatives 2. The in vitro caspase-3
inhibition for other Fmoc dipeptidyl vinyl sulfones was also studied.
Finally, the caspase-3 inhibitory effect of the vinyl sulfones active in
vitro was also analysed using a yeast cell-based assay.
In order to search for modulators of caspase-3, an independent
analysis of this caspase is required. However, the identification of at
least 14 caspase family members, coupled with the complex patterns
of caspase gene expression, the tightly synchronized cascade of
activation and the extensive cross-talk amongst numerous signal-
ling pathways in mammalian cells have hampered the pharmaco-
logical analysis of individual caspases in a cell environment [9].
To address this issue, yeast expressing human caspase-3 can be used
to analyse the caspase-3 inhibitory effect of small molecules in
* Corresponding author. Medicinal Chemistry Group, Research Institute for
Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University
of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Tel.: þ351 21 794 6400;
fax: þ351 21 794 6470.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.067

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P.M.C. Glória et al. / European Journal of Medicinal Chemistry 46 (2011) 2141e2146
All of the proposed structures were established by NMR (1H, 13C,
CO2H
SO2R1
CO2H
SO2R1
O
COSY and HMQC), IR, and MS. The stereochemistry around the
double bond was established using the corresponding 1H NMR
coupling constant, where a double doublet (J ¼ 16.0 Hz and 4 Hz)
PGHN
N
H
PGHN
R2
and a doublet (J ¼ 16.0 Hz) are observed for the
b and a proton
1a PG=Cbz, R1=Me
1b PG=Cbz, R1=Ph
1c PG=Fmoc, R1=Me
1d PG=Fmoc, R1=Ph
2b PG=Cbz, R2=Me, R1=Ph
relative to the sulfone group, respectively, confirming the presence
of the E isomer on vinyl sulfones 2iem and 7a, 7b.
2d PG=Cbz, R2=CHMe2, R1=Ph
2e PG=Cbz, R2=CHMeEt, R1=Me
2f PG=Cbz, R2=CHMeEt, R1=Ph
2g PG=Fmoc, R2=CHMe2, R1=Me
2h PG=Fmoc, R2=CHMe2, R1=Ph
2.2. Biological activity
CO2H
The vinyl sulfones synthesized were examined for their ability
to inhibit the activity of recombinant human effector caspase-3.
The IC50 values were determined using a fluorometric assay and the
tetrapeptidyl chloromethyl ketone Ac-DEVD-CMK was used as
positive control. Fmoc dipeptidyl vinyl sulfones with Ala at position
P2 2i and 2j were inactive against caspase-3 for the tested
O
SO2Me
PGHN
N
H
R2
2a PG=Cbz, R2=Me, R1=Me
2c PG=Cbz, R2=CHMe2, R1=Me
concentrations (<200 mM), as we had observed previously for the
Cbz-Ala-Asp vinyl sulfones 2a and 2b counterparts.
Fig. 1. Vinyl sulfone caspase-3 inhibitors.
Surprisingly, the presence of Ile on vinyl sulfones 2l and 2m did
not improve the activity related to the Fmoc-Val-Asp vinyl sulfones
2g and 2h, as was observed before for the Cbz dipeptidyl series.
Most remarkably, and to our surprise, tripeptidyl vinyl sulfones
were not active for caspase-3, opposite to other examples described
in literature for peptidyl vinyl sulfones as potent cysteine protease
inhibitors of clan CA [7]. In fact, the presence of a third amino acid
on vinyl sulfones 2g and 2h also resulted in lost of activity against
caspase-3, as observed for the Fmoc-tripeptidyl vinyl sulfones 7a
and 7b.
The vinyl sulfones that were active in vitro were then studied
using a yeast cell-based assay (Table 1). In this assay, inhibitors of
caspase-3 are those compounds that stimulate the growth of yeast
expressing human caspase-3 without interfering with the growth
of control yeast (transformed with the vector without the cDNA
encoding human caspase-3; pGALL).
To implement this assay, we firstly confirmed that, as previously
reported by other authors [10e17], yeast expression of human
reverse caspase-3, an active form of human caspase-3 (Fig. 2)
caused a marked growth inhibition, when compared to control
yeast (pGALL) (Fig. 3). Additionally, the reduction of this caspase-3-
induced yeast growth inhibition by the commercial caspase-3
inhibitor, Ac-DEVD-CMK (Fig. 3) corroborated the correlation,
between stimulation of growth of yeast expressing caspase-3 and
caspase-3 inhibition, and validated this assay to search for small
molecule inhibitors of caspase-3.
a simpler eukaryotic cell system. In fact, many researchers have used
yeast expressing human caspase-3 to uncover the mechanisms of
endogenous regulation of this caspase [10e17].
Indeed, the high degree of conservation of many pathways and
cellular processes among yeast and human cells led several
researchers to use yeast as a valuable cell model for functional
studies of human proteins and as a drug screening tool [18e24].
Additionally, though yeast has a metacaspase (Yca1p), it was shown
that the activity of human caspase-3 in yeast is independent of
Yca1p activity [17]. Finally, it was shown that expression of an
active form of caspase-3 in yeast caused a marked growth inhibi-
tion [10e17], which was reverted by endogenous inhibitors of
caspase-3, such as mammalian IAPs (inhibitors of apoptosis)
homologues (MIH) MIHA, MIHB and MIHC, and baculoviral caspase
inhibitors p35 and p49 [12e16]. These studies established a corre-
lation between yeast cell growth and caspase-3 activity.
2. Results and discussion
2.1. Chemistry
Dipeptidyl aldehydes 3a and 3b were synthesized as reported
before [8] from the corresponding dipeptide acids using Weinreb
chemistry and used without further purification. After Hor-
nereWadswortheEmmons reaction between the dipeptidyl alde-
hydes 3a and 3b with the corresponding phosphonate, vinyl
sulfones 4aed were obtained with yields of 36e53%. Deprotection
of vinyl sulfones 4aed with TFA, afforded vinyl sulfones 2iem with
yields of 88e93% (Scheme 1).
Subsequently, the effect of vinyl sulfones 1c, 1d, 2c and 2eeh on
caspase-3 activity was also analysed using the yeast caspase-3
assay. For that, concentrationeresponse curves for 5e100
sulfones and 5e50 M Ac-DEVD-CMK (positive control) were
obtained, considering the maximal growth stimulation achieved
with 25 M Ac-DEVD-CMK as 100% caspase-3 inhibition (Fig. 4A),
mM vinyl
m
m
To improve the potency of the inhibitors, we decided to extend
the molecular recognition by incorporating a third amino acid,
valine, since it has been shown experimentally to be favourable at
the P3 position [25]. We had tried before, without success, to obtain
the dipeptidyl vinyl sulfones 2 by Fmoc deprotection of the
nitrogen atom of the vinyl sulfones 5a and 5b, using several
different basic conditions (piperidine, triethylamine, DBU) [8].
However, recently this Fmoc deprotection was accomplished in the
presence of Et2NH [26]. Using the same methodology, vinyl
sulfones 6a and 6b were obtained in a one pot reaction, by
N-deprotection of vinyl sulfones 5a and 5b using Et2NH in aceto-
nitrile, followed by coupling with the dipeptide FmocValValOSu in
DMF with yields of 54e57%. Deprotection of vinyl sulfones 6a and
6b with TFA, afforded vinyl sulfones 7a and 7b with yields of
69e73% (Scheme 2).
and the IC50 value (lowest concentration of compound that caused
50% caspase-3 inhibition) was determined for each compound. The
results obtained showed that all vinyl sulfones active in vitro
behaved as inhibitors of a caspase-3 pathway in the cell-based
assay (Table 1). In fact, vinyl sulfones 1c, 1d, 2c and 2eeh, did not
significantly interfere with the growth of control yeast. This indi-
cates that these vinyl sulfones had no effect on endogenous yeast
proteins, such as yeast caspase-like. However, they inhibited
human caspase-3-induced yeast growth inhibition. This reveals
that they inhibited caspase-3 directly or indirectly by inhibition of
a caspase-3-dependent pathway.
Based on Table 1, it was observed that the IC50 values for vinyl
sulfones 1c, 1d, 2c and 2eeh determined in the yeast assay ranged
from 16 to 35
mM and were within the same order of magnitude of
that determined for Ac-DEVD-CMK (13
mM e Table 1). A remarkable

P.M.C. Glória et al. / European Journal of Medicinal Chemistry 46 (2011) 2141e2146
2143
O
CO2t-Bu
CHO
CO2t-Bu
SO2R1
CO2H
SO2R1
NaH, R1O2S
P
OEt
OEt
O
O
O
TFA, CH2Cl2
1h 0ºC
FmocHN
FmocHN
FmocHN
N
H
N
H
N
H
THF, 2h rt
R2
R2
R2
3a R2=Me
4a R2=Me, R1=Me, 36%
2i R2=Me, R1=Me, 93%
2j R2=Me, R1=Ph, 91%
3b R2=CHMeEt
4b R2=Me, R1=Ph, 44%
4c R2=CHMeEt, R1=Me, 53%
4d R2=CHMeEt, R1=Ph, 38%
2l R2=CHMeEt, R1=Me, 89%
2m R2=CHMeEt, R1=Ph, 88%
Scheme 1. Synthesis of Fmoc dipeptidyl vinyl sulfones 2iem.
increase in the activity of some vinyl sulfones, identified as weak
inhibitors of caspase-3 in the in vitro assay, was observed in the
yeast cell-based assay. In particular, vinyl sulfone 1d, exhibited
a proliferative effect (reduction of caspase-3-induced growth
inhibition) in the same order of magnitude of the commercial
caspase-3 inhibitor, Ac-DEVD-CMK (Fig. 4A,B and Table 1). These
results indicate that, in spite of their moderate activity as inhibitors
of caspase-3 [8], vinyl sulfones 1c, 1d, 2c, 2eef and 2h are inhibitors
of a caspase-3-dependent pathway. In fact, since these compounds
have a lower potency in vitro than in the yeast cell-based assay, we
hypothesize that although they inhibit caspase-3 their main effect
can be ascribed to the inhibition of a caspase-3-dependent pathway
by modulation of other proteins of its pathway that indirectly
inhibit caspase-3. In fact, the lower selectivity displayed by Fmoc-
Asp vinyl sulfones 1c and 1d towards caspase-3 can be ascribed to
the less extended recognition motif when compared to that of their
dipeptide counterparts.
Overall, the results obtained from the yeast cell-based assay
strongly suggest that the vinyl sulfones reported in the present
study are inhibitors of a caspase-3-dependent pathway. Impor-
tantly, the calc log P values [27] for vinyl sulfones range from 1.1 to
3.4 (Table 2), i.e. significantly higher than that of Ac-DEVD-CMK
(calc log P ¼ ꢀ0.6), suggesting that they present appropriate cell
permeability properties.
Additionally, they can be used as lead compounds for the devel-
opment of more potent and selective caspase-3 inhibitors. Further
work must be carried out in order to identify the molecules of the
caspase-3 pathway also modulated by these compounds.
4. Experimental protocols
4.1. Chemistry
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Melting
points were determined using a Kofler camera Bock monoscope M
and are uncorrected. The infrared spectrawere collected on a Nicolet
Impact 400 FTIR infrared spectrophotometer. High resolution mass
spectra (HMRS) were performed in Unidade de Espectrometria de
Masas, Santiago de Compostela. Elemental analyses were carried
out on a C. Erba Model 1106 (Elemental Analyzer for C, H and N) and
the results are within ꢁ0.4% of the theoretical values. Merck Silica
Gel 60 F254 plates were used as analytical TLC; flash column chro-
matography was performed on Merck Silica Gel (200e400 mesh).
1H and 13C NMR spectra were recorded on a Bruker 400 Ultra-Shield
(400 MHz). 1H and 13C chemical shifts are expressed in
d (ppm)
referenced to the solvent used and the proton coupling constants (J)
in hertz.
3. Conclusion
4.1.1. General procedure for the preparation of vinyl sulfones 4aed
To a suspension of NaH 60% (1.8 mmol, 1.1 equiv) in THF (5 ml),
at 0 ꢂC, was added 1 equiv of the appropriate phosphonate. The
resulting solution was stirred at temperature room for 30 min.
A solution of 1 equiv of the appropriate aldehyde 3 in THF (16 ml)
was added and stirred at room temperature for 2 h. The solvent was
removed under reduced pressure, and the residue was dissolved in
CH2Cl2. The organic solution was washed with brine, dried, and
concentrated. The resulting residue was flash chromatographed.
The comprehensive SAR study of vinyl sulfones as caspase-3
inhibitors, incorporating one, two and three amino acid residues,
reveals that introduction of a third amino acid in dipeptidyl vinyl
sulfones (i.e. converting 2g and 2h into 7a and 7b, respectively), did
not improve the inhibitory activity determined using the bio-
chemical assay. For all the series of vinyl sulfones synthesized, the
compounds active against caspase-3 were then tested in a yeast cell
system expressing this enzyme. This yeast assay revealed that Fmoc
vinyl sulfones containing Asp at P1 1c and 1d were inhibitors of
a caspase-3-dependent pathway in a cell system, with IC50 values
similar to that obtained with the positive control (Ac-DEVD-CMK).
In spite of their lack of selectivity to caspase-3, they can be con-
sidered potent modulators of a caspase-3-dependent pathway
and therefore interesting compounds to be explored in vivo.
4.1.2. General procedure for the preparation of vinyl sulfones 6aed
A solution of the appropriate vinyl sulfone 5 [8] (1 mmol,
1 equiv) in a 1:1 mixture of MeCN/Et2NH (5 ml) was stirred at room
temperature for 2 h. After this time, the solvent was removed under
vacuum and the resulting residue was dissolved in DMF (3 ml). To
this solution was added the protected dipeptide (FmocValValOSu,
CO2H
SO2R1
CO2t-Bu
SO2R1
CO2Bu
SO2R1
O
O
H
N
H
TFA, CH2Cl2
1h 0ºC
a) Et2NH, MeCN
N
FmocHN
N
H
FmocHN
N
H
FmocHN
b) FmocValValOSu, DMF
O
O
7a R1=Me, 73%
7b R1=Ph, 69%
5a R1=Me
5b R1=Ph
6a R1=Me, 54%
6b R1=Ph, 57%
Scheme 2. Synthesis of Fmoc-tripeptidyl vinyl sulfones 7a and 7b.

2144
P.M.C. Glória et al. / European Journal of Medicinal Chemistry 46 (2011) 2141e2146
Table 1
100
Inhibition of human caspase-3 using
[PGeNeXeCH]CHSO2R].
a
yeast cell system by vinyl sulfones
80
Compound
PG
X
R
IC50
(m
M)a
Growth
stimulation (%)b
60
40
20
0
Cell system
(Control yeast)
1c
1d
2c
2e
2f
2g
2h
Fmoc
Fmoc
Cbz
Cbz
Cbz
Fmoc
Fmoc
e
Asp
Asp
Me
Ph
Me
Me
Ph
Me
Ph
e
23.7 ꢁ 4.5
16.2 ꢁ 2.0
35.3 ꢁ 3.3*
30.5 ꢁ 3.5*
28.3 ꢁ 8.9
34.0 ꢁ 4.3*
29.9 ꢁ 3.4*
13.4 ꢁ 2.8
1.7 ꢁ 3.2
2.8 ꢁ 2.3
ꢀ1.0 ꢁ 0.4
ꢀ3.3 ꢁ 2.1
1.9 ꢁ 2.2
ꢀ7.0 ꢁ 0.1
1.3 ꢁ 3.9
1.7 ꢁ 3.2
*
Val-Asp
Ile-Asp
Ile-Asp
Val-Asp
Val-Asp
e
pGALL
Caspase-3
Ac-DEVD-CMK
The percentage of growth stimulation was calculated considering 100% growth the
number of CFU obtained with transformed yeast incubated with DMSO only.
DMSO
Ac-DEVD-CMK
+
-
+
-
-
a
IC50 values, were obtained from concentrationeresponse curves of 5e100
M Ac-DEVD-CMK, which were obtained considering the
maximal growth stimulation obtained with 25 M Ac-DEVD-CMK as 100% caspase-3
inhibition. Data are means ꢁ s.e.m. of IC50 values obtained from 4e5 independent
experiments with replicates each. IC50 value significantly higher than that
mM
+
vinyl sulfones and 5e50
m
m
Fig. 3. Expression of human caspase-3 caused a marked yeast growth inhibition that
was significantly reduced by the commercial caspase-3 inhibitor Ac-DEVD-CMK.
Transformed yeast cells were incubated in galactose selective medium with 25 mM Ac-
DEVD-CMK or DMSO only to 0.3 OD600. Yeast growth was evaluated by CFU counts,
considering the number of CFU obtained with control yeast (pGALL) incubated only
with DMSO as 100% growth. Data are means ꢁ s.e.m. of 4 independent experiments
with 6 replicates each. Value of yeast expressing caspase-3 incubated with Ac-DEVD-
CMK significantly higher than that obtained with DMSO only, *P < 0.05.
6
obtained with Ac-DEVD-CMK, *P < 0.05.
b
Each compound was tested in control yeast at the concentration that caused the
maximal effect on yeast expressing caspase-3. Positive values indicate stimulation of
yeast growth. Data are means ꢁ s.e.m. of 4 independent experiments with 6 repli-
cates each.
7.59 (2H, d, J ¼ 8.0 Hz), 7.42 (2H, t, J ¼ 8.0 Hz), 7.28 (2H, t, J ¼ 8.0 Hz),
6.63 (1H, dd, J ¼ 16.0, 4.0 Hz, CH]CHSO2Me), 6.56 (1H, d, J ¼ 16.0 Hz,
CH]CHSO2Me), 5.50 (1H, m), 5.21 (1H, sl), 4.43 (2H, m), 4.30 (1H,
m), 4.16 (1H, m), 3.12 (3H, s), 2.51 (1H, m), 2.48 (1H, m), 1.24 (3H, d,
1 equiv) and the mixture stirred for 12 h at room temperature. The
reaction mixture was diluted with H2O, extracted with AcOEt (3ꢃ)
and the organic layers combined, dried under Na2SO4 and the
solvent removed under vacuum. The resulting residue was flash
chromatographed.
J ¼ 8.0 Hz); 13C NMR (100 MHz, CDCl3)
d 173.86, 171.90, 160.65,
158.68, 144.95, 141.31, 139.57, 128.78, 127.56, 124.34, 122.93, 66.68,
50.69, 50.33, 48.17, 39.89, 38.13, 18.44; HRMS-ESI-TOF: m/z calcd
C24H26N2O7SNa (Mþ þ Na) 509.1358, found 509.1364.
4.1.3. General procedure for the preparation of vinyl sulfones 2iem
and 7a, 7b
The appropriate vinyl sulfone was treated with TFA at 0 ꢂC for
1 h. TFA was removed under vacuum and the final products were
recrystallized from ethyl acetate/hexane as white solids.
A
125
4.1.3.1. FmocAlaAspVSMe (2i). Obtained in 93% yield. M.p. 97e98 ꢂC;
100
75
50
25
0
IR (NaCl): 3421, 3318, 3074, 2959, 1714, 1682, 1510 cmꢀ1 1H NMR
;
(400 MHz, CDCl3)
d
7.91 (1H, d, J ¼ 8.0 Hz), 7.77 (2H, d, J ¼ 8.0 Hz),
[µM]
25
5
15
B
125
100
75
50
25
0
[µM]
25
5
15
Fig. 2. Expression of human reverse caspase-3 in yeast was confirmed by Western blot
analysis. Control yeast (pGALL) and yeast expressing caspase-3 were incubated in
galactose selective medium to 0.3 OD600. Full length procaspase (32 kDa) and the
respective cleaved fragments (processed caspase-3) were detected using an anti-cas-
pase-3 polyclonal antibody. Detection of Pgk1p was used as loading control. Immu-
noblots were developed by chemiluminescence.
Fig. 4. Concentrationeresponse curves for (A) Ac-DEVD-CMK and (B) vinyl sulfone 1d.
The percentage of growth stimulation caused by compounds was calculated consid-
ering the number of CFU obtained with yeast expressing caspase-3 incubated with
DMSO only as 100% growth. Concentrationeresponse curves were obtained consid-
ering the maximal growth stimulation obtained with 25
mM Ac-DEVD-CMK as 100%
caspase-3 inhibition. Data are means ꢁ s.e.m. of 4e5 independent experiments.

P.M.C. Glória et al. / European Journal of Medicinal Chemistry 46 (2011) 2141e2146
2145
Table 2
NMR (400 MHz, CDCl3)
d
9.50 (1H, sl), 7.88 (2H, d, J ¼ 8.0 Hz), 7.79
Relevant parameters for preliminary permeation properties of vinyl sulfones 1c, 1d,
2c and 2eeh (data for Ac-DEVD-CMK are also included for comparison).
(2H, d, J ¼ 8.0 Hz), 7.71 (1H, m), 7.58 (2H, m), 7.43 (4H, m), 7.33
(2H, m), 7.16 (1H, sl), 6.50 (1H, dd, J ¼ 16.0, 4.0 Hz, CH]CHSO2Ph),
6.25 (1H, d, J ¼ 16.0 Hz, CH]CHSO2Ph), 4.70 (2H, d, J ¼ 8.0 Hz), 4.60
(1H, sl), 4.46 (1H, m), 4.39 (2H, sl), 4.15 (1H, sl), 2.83 (1H, dd, J ¼ 8.0,
4.0 Hz), 2.62 (2H, m), 2.27 (1H, m), 1.11 (6H, d, J ¼ 8.0 Hz), 0.91 (6H,
Compound
PG
X
R
log Pa
1c
1d
2c
2e
2f
2g
2h
Fmoc
Fmoc
Cbz
Cbz
Cbz
Fmoc
Fmoc
e
Asp
Asp
Me
Ph
Me
Me
Ph
Me
Ph
e
2.22
3.34
1.16
1.53
2.38
2.33
3.43
ꢀ0.63
Val-Asp
Ile-Asp
Ile-Asp
Val-Asp
Val-Asp
e
d, J ¼ 8.0 Hz); 13C NMR (100 MHz, CDCl3)
d 172.34, 172.16, 171.76,
160.73,144.81, 140.75, 139.43, 136.77, 135.44,133.03, 129.09, 128.64,
128.64, 127.42, 124.21, 122.79, 66.56, 59.13, 58.35, 50.22, 48.05,
39.78, 30.50, 19.06; HRMS-ESI-TOF: m/z calcd C36H41N3O8SNa
(Mþ þ Na) 698.2512, found 698.2516.
Ac-DEVD-CMK
a
Estimated by the ALOGPS 2.1 algorithm [27].
4.2. Caspase-3 in vitro assays
4.1.3.2. FmocAlaAspVSPh(2j). Obtained in 91% yield. M.p. 99e100 ꢂC;
IR (NaCl): 3421, 3305, 3061, 2932, 1714, 1657, 1510 cmꢀ1 1H NMR
(400 MHz, CDCl3)
Caspase-3 fluorometric assay was used, which is based on the
hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-7-
amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase-3, resulting
in the release of the fluorescent 7-amido-4-methylcoumarin (AMC)
;
d
8.02 (1H, d, J ¼ 8.0 Hz), 7.74 (2H, d, J ¼ 8.0 Hz),
7.56e7.23 (11H, m), 6.72 (1H, dd, J ¼ 16.0, 4.0 Hz, CH]CHSO2Ph),
6.66 (1H, d, J ¼ 16.0 Hz, CH]CHSO2Ph), 5.29 (1H, m), 5.22 (1H, sl),
4.43 (2H, m), 4.30 (1H, m), 4.16 (1H, m), 3.12 (3H, s), 2.81 (1H, m), 2.75
moiety. Briefly, 5
caspase-3 (caspase-3, human, recombinant, Calbiochem) was
added to 5 l of the tested inhibitors at various concentrations. The
reaction was initiated by the addition of 190 l of substrate to a final
concentration of 200 M in assay buffer (20 mM HEPES, 2 mM
ml of stock solution in assay buffer (10 U/ml) of
(1H, m), 1.25 (3H, d, J ¼ 8 Hz); 13C NMR (100 MHz, CDCl3)
d 173.72,
m
171.76, 160.51, 144.81, 140.75, 139.43, 136.77, 135.44, 133.04, 129.08,
128.63,128.14,127.41,124.20,122.79, 66.54, 50.54, 50.18, 48.03, 39.75,
18.29; HRMS-ESI-TOF: m/z calcd C29H28N2O7SNa (Mþ þ Na) 571.1515,
found 571.1523.
m
m
EDTA, 0.1% CHAPS, and 5 mM DTT, pH 7.4). Liberation of AMC was
monitored continuously at 37 ꢂC using a Tecan infinite M200
(Tecan, Switzerland) 96-well plate reader (white plates from
Greiner bio-one, Germany) using an excitation wavelength of
360 nm and an emission wavelength of 460 nm. Inhibitors stock
solutions were prepared in DMSO, and serial dilutions were made
in DMSO. Controls were performed using enzyme alone, substrate
alone, enzyme with DMSO and a positive control (Ac-DEVD-CMK,
Calbiochem).
4.1.3.3. FmocIleAspVSMe (2l). Obtained in 89% yield. M.p. 87e88 ꢂC;
IR (NaCl): 3305, 2971, 1727, 1657, 1523 cmꢀ1 1H NMR (400 MHz,
;
CDCl3)
d
8.01 (1H, d, J ¼ 8.0 Hz), 7.59 (2H, d, J ¼ 8.0 Hz), 7.45 (2H, d,
J ¼ 8.0 Hz), 7.41 (2H, t, J ¼ 8.0 Hz), 7.32 (2H, t, J ¼ 8.0 Hz), 6.73 (1H, dd,
J ¼ 16.0, 4.0 Hz, CH]CHSO2Me), 6.66 (1H, d, J ¼ 16.0 Hz, CH]
CHSO2Me), 5.50 (1H, m), 5.23 (1H, sl), 4.43 (2H, m), 4.30 (1H, m), 4.16
(1H, m), 3.02 (3H, s), 2.72 (1H, m), 2.60 (1H, m),1.51 (1H, sl),1.23 (1H,
sl), 0.97 (6H, m); 13C NMR (100 MHz, CDCl3)
d 173.20, 171.85, 160.83,
158.64, 144.91, 141.27, 139.53, 128.73, 127.51, 124.30, 122.89, 66.63,
60.57, 50.28, 48.12, 39.85, 38.08, 35.66, 25.05,16.37,11.66; HRMS-ESI-
TOF: m/z calcd C27H32N2O7SNa (Mþ þ Na) 551.1828, found 551.1835.
4.3. Plasmids, yeast strain, transformation and growth conditions
Constructed yeast expression plasmid pGALL-LEU2 encoding
human reverse caspase-3, an active form of human caspase-3, and
the respective empty vector were kindly provided by Dr. C.J.
Hawkins (Children’s Cancer Centre, Royal Children’s Hospital,
Parkville, Australia). Plasmids have a galactose-inducible GAL1/10
promoter.
4.1.3.4. FmocIleAspVSPh (2m). Obtained in 88% ꢀy1ield. M.p.
111e112 ꢂC; IR (NaCl): 3429, 2974, 1705, 1523 cm
;
1H NMR
(400 MHz, CDCl3)
d
8.00 (1H, d, J ¼ 8.0 Hz), 7.75 (2H, d, J ¼ 8.0 Hz),
7.61e7.23 (11H, m), 6.73 (1H, dd, J ¼ 16.0, 4.0 Hz, CH]CHSO2Ph),
6.63 (1H, d, J ¼ 16.0 Hz, CH]CHSO2Ph), 5.46 (1H, m), 5.24 (1H, sl),
4.49 (2H, m), 4.23 (1H, m), 4.15 (1H, m), 2.73 (1H, m), 2.58 (1H, m),
1.51 (1H, sl), 1.27 (1H, sl), 0.68 (6H, m); 13C NMR (100 MHz, CDCl3)
Saccharomyces cerevisiae CG379 (a ade5 his7-2 leu2-112 trp1-289
ura3-52 [Kil-O]; Yeast Genetic Stock Center) strain was transformed
by the lithium acetate method as described [24]. Transformed cells
were routinely grown in a minimal selective medium with 2% (w/v)
glucose, 0.67% (w/v) yeast nitrogen base without amino acids
d
173.11, 171.76, 160.73, 144.81, 140.75, 139.43, 136.77, 135.44,
133.04, 129.08, 128.63, 128.14, 127.41, 124.20, 122.79, 66.54, 60.47,
50.18, 48.03, 39.75, 35.56, 24.95, 16.27, 11.56; HRMS-ESI-TOF: m/z
calcd C32H34N2O7SNa (Mþ þ Na) 613.1984, found 613.1993.
(Difco) and all the amino acids required for yeast growth (50 mg/ml)
except leucine, at 30 ꢂC, with mechanical shaking (200 r.p.m.) to
approximately 1 optical density (OD600; Jenway 6310 Spectropho-
tometer). To induce expression of human caspase-3, yeast cells were
diluted to 0.05 OD600 in a minimal selective medium with 2% (w/v)
galactose and raffinose, instead of glucose, and 3% (v/v) glycerol,
and grown at 30 ꢂC with mechanical shaking (200 r.p.m.) to 0.3
4.1.3.5. FmocValValAspVSMe (7a). Obtained in 69% yield.ꢀ1M.p.
96e97 ꢂC; IR (NaCl): 3283, 2949, 1741, 1700, 1655, 1522 cm
;
1H
NMR (400 MHz, CDCl3)
d 9.23 (1H, sl), 8,12 (1H, sl), 7.76 (2H, d,
J ¼ 8.0 Hz), 7.41e7.33 (6H, m), 6.74 (1H, dd, J ¼ 16.0, 4.0 Hz, CH]
CHSO2Me), 6.47 (1H, d, J ¼ 16.0 Hz, CH]CHSO2Me), 4.70 (2H, d,
J ¼ 8.0 Hz), 4.60 (1H, sl), 4.54 (1H, sl), 4.47 (2H, m), 3.28 (1H, sl), 2.93
(3H, s), 2.78 (1H, dd, J ¼ 8.0, 4.0 Hz), 2.57 (1H, dd, J ¼ 8.0, 4.0 Hz), 2.03
(1H, m), 1.85 (1H, m), 1.07 (6H, d, J ¼ 8.0 Hz), 1.00 (6H, d, J ¼ 8.0 Hz);
OD600
.
4.3.1. Western blot analysis
Preparation of yeast extracts and Western blot analysis were
performed basically as described [24]. Expression of human cas-
pase-3 in yeast was detected using the anti-caspase-3 rabbit poly-
clonal antibody (1:200; Santa Cruz Biotechnology), followed by the
anti-rabbit horseradish peroxidase (HRP)-conjugated secondary
antibody (1:5000; Santa Cruz Biotechnology). For loading control,
membranes were stripped and reprobed with the anti-Pgk1p mouse
monoclonal antibody (1:5000; Molecular probes) followed by the
13C NMR (100 MHz, CDCl3)
d 172.39, 172.21, 171.81, 160.79, 158.60,
144.87, 144.87, 141.23, 139.49, 128.69, 127.47, 124.26, 122.85, 66.54,
59.11, 58.33, 50.19, 48.03, 39.76, 37.99, 30.48, 19.04; HRMS-ESI-TOF:
m/z calcd C31H39N3O8SNa (Mþ þ Na) 636.2356, found 636.2363.
4.1.3.6. FmocValValAspVSPh (7b). Obtained in 73% yield.ꢀ1M.p.
86e87 ꢂC; IR (NaCl): 3314, 2992, 1740, 1702, 1666, 1516 cm
;
1H

2146
P.M.C. Glória et al. / European Journal of Medicinal Chemistry 46 (2011) 2141e2146
anti-mouse HRP-conjugated secondary antibody (1:5000; Santa
Cruz Biotechnology). Immunoblots were developed by enhanced
chemiluminescence.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2011.02.067.
4.3.2. Yeast caspase-3 assay
To analyse the effect of compounds on the growth of yeast
expressing human caspase-3 and control yeast (transformed with
the vector without the cDNA encoding human caspase-3; pGALL),
transformed cells were incubated in galactose selective medium
References
[1] W.C. Earnshaw, L.M. Martins, S.H. Kaufmann, Annu. Rev. Biochem. 68 (1999)
383e424.
[2] J.-B. Denault, G.S. Salvesen, Chem. Rev. 102 (2002) 4489e4500.
[3] P. Fuentes-Prior, G.S. Salvesen, Biochem. J. 384 (2004) 201e232.
[4] A. Fleischer, A. Ghadiri, F. Dessauge, M. Duhamel, M.P. Rebollo, F. Alvarez-
Franco, A. Rebollo, Mol. Immunol. 43 (2006) 1065e1079.
[5] R.C. Taylor, S.P. Cullen, S.J. Martin, Nat. Rev. Mol. Cell Biol. 9 (2008) 231e241.
[6] R.E. Castro, M.M.M. Santos, P.M.C. Glória, C.J.A. Ribeiro, D.M.S. Ferreira,
J.M. Xavier, R. Moreira, C.M.P. Rodrigues, Curr. Pharm. Des. 16 (2010) 2851e2864.
[7] M.M.M. Santos, R. Moreira, Mini-Rev. Med. Chem. 7 (2007) 1040e1050.
[8] A.S. Newton, P.M.C. Glória, L.M. Gonçalves, D.J.V.A. dos Santos, R. Moreira,
R.C. Guedes, M.M.M. Santos, Eur. J. Med. Chem. 45 (2010) 3858e3863.
[9] I.N. Lavrik, A. Golks, P.H. Krammer, J. Clin. Invest. 115 (2005) 2665e2672.
[10] J.J. Kang, M.D. Schaber, S.M. Srinivasula, E.S. Alnemri, G. Litwack, D.J. Hall,
M.A. Bjornsti, J. Biol. Chem. 274 (1999) 3189e3198.
with 5e100 mM vinyl sulfones, 5e50 mM Ac-DEVD-CMK (positive
control; caspase-3 inhibitor III; Calbiochem) or 0.1% DMSO only to
0.3 OD600. Cell growth was analysed by counting the number of
colony-forming units (CFU) per ml obtained after 2 days incubation,
at 30 ꢂC, on Sabouraud Dextrose Agar plates. The percentage of
growth stimulation of yeast expressing caspase-3 (proportional to
the degree of caspase-3 inhibition) caused by compounds was
calculated considering the number of CFU obtained with yeast
expressing caspase-3 incubated only with DMSO as 100% growth.
Concentrationeresponse curves for 5e100
5e50 M Ac-DEVD-CMK were obtained, considering the maximal
growth stimulation achieved with 25 M Ac-DEVD-CMK as 100%
mM vinyl sulfones and
[11] M.E. Wright, D.K. Han, L. Carter, S. Fields, S.M. Schwartz, D.M. Hockenbery,
FEBS Lett. 446 (1999) 9e14.
[12] M.E. Wright, D.K. Han, D.M. Hockenbery, FEBS Lett. 481 (2000) 13e18.
[13] C.J. Hawkins, J. Silke, A.M. Verhagen, R. Foster, P.G. Ekert, D.M. Ashley,
Apoptosis 6 (2001) 331e338.
m
m
caspase-3 inhibition, and the lowest concentration that caused 50%
caspase-3 inhibition (IC50) calculated.
[14] J. Silke, P.G. Ekert, C.L. Day, C.J. Hawkins, M. Baca, J. Chew, M. Pakusch,
A.M. Verhagen, D.L. Vaux, EMBO J. 20 (2001) 3114e3123.
[15] A.M. Jabbour, P.G. Ekert, E.J. Coulson, M.J. Knight, D.M. Ashley, C.J. Hawkins,
Cell Death Differ. 9 (2002) 1311e1320.
[16] P.K. Ho, A.M. Jabbour, P.G. Ekert, C.J. Hawkins, FEBS J. 272 (2005) 1401e1414.
[17] M.A. Puryer, C.J. Hawkins, Apoptosis 11 (2006) 509e517.
[18] A. Barberis, T. Gunde, C. Berset, S. Audetat, U. Lu, Drug Discov. Today Technol.
2 (2005) 187e192.
4.3.3. Statistical analysis
Data were analysed statistically using the SigmaStat 3.5 software.
Differences between means were tested for significance using the
unpaired Student’s t test (P < 0.05).
[19] C.M. Sturgeon, D. Kemmer, H.J. Anderson, M. Roberge, Biotechnol. J. 1 (2006)
289e298.
[20] L. Miller-Fleming, F. Giorgini, T.F. Outeiro, Biotechnol. J. 3 (2008) 325e338.
[21] D. Petranovic, J. Nielsen, Trends Biotechnol. 26 (2008) 584e590.
[22] M.T. Greenwood, P. Ludovico, Cell Death Differ. 17 (2009) 737e745.
[23] I. Coutinho, G. Pereira, M.F. Simões, M. Côrte-Real, J. Gonçalves, L. Saraiva,
Biochem. Pharmacol. 78 (2009) 449e459.
[24] L. Saraiva, R. Silva, G. Pereira, J. Gonçalves, M. Côrte-Real, J. Cell Sci. 119 (2006)
3171e3181.
[25] R.V. Talanian, C. Quinlan, S. Trautz, M.C. Hackett, J.A. Mankovich, D. Banach,
T. Ghayur, K.D. Brady, W.W. Wong, J. Biol. Chem. 272 (1997) 9677e9682.
[26] J.T. Lee, D.Y. Chen, Z. Yang, A.D. Ramos, J.J.-D. Hsieh, M. Bogyo, Bioorg. Med.
Chem. Lett. 19 (2009) 5086e5090.
Acknowledgments
This work was supported by the Fundação para a Ciência e Tec-
nologia (Lisbon, Portugal) by the award of doctoral fellowships to
I.C. (SFRH/BD/36066/2007) and A.S.N. (SFRH/BD/41276/2007) and
by the award of post doctoral fellowship to P.M.C.G. (SFRH/BPD/
22631/2005). We also thank REQUIMTE/CEQUP, FCT (I&D No 8/94),
POCTI (QCA III), FEDER and Universidade do Porto/Santander Totta
for financial support. We are grateful to Dr. Christine J. Hawkins for
providing pGALL-LEU2 encoding human reverse caspase-3.
[27] ALOGPS 2.1, Virtual Computational Chemistry Laboratory. http://www.vcclab.
org/lab/alogps/ (accessed 15.02.11).