Synthesis and evaluation of vinyl sulfones as caspase-3 inhibitors. A structure-activity study

Article abstract of DOI:10.1016/j.ejmech.2010.05.039

The first structure-activity relationship study of vinyl sulfones as caspase-3 inhibitors is reported. A series of 12 vinyl sulfones was synthesized and evaluated for two downstream caspases (caspases-3 and -7). Dipeptidyl derivatives were significantly superior to their counterparts containing only Asp at P₁, as caspase-3 inhibitors. Fmoc-Val-Asp-trans-CHCH-SO ₂Me was the most potent inhibitor of caspase-3 in the series, with a IC₅₀ of 29 μM and a second-order rate constant of inactivation, k_inact/K_i, of 1.5 M^-1 s^-1. Computational studies suggest that the second amino acid occupies position S₃ of the enzyme. In addition, Fmoc-Val-Asp-trans-CHCH-SO ₂Ph was inactive for caspase-7 for the tested concentrations. The first structure-activity relationship study of vinyl sulfones as caspase-3 inhibitors is reported. A series of 12 vinyl sulfones was synthesized and evaluated for caspase-3 inhibition.

Full text of DOI:10.1016/j.ejmech.2010.05.039

European Journal of Medicinal Chemistry 45 (2010) 3858e3863
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of vinyl sulfones as caspase-3 inhibitors.
A structureeactivity study
Ana S. Newton 1, Paulo M.C. Glória 1, Lídia M. Gonçalves, Daniel J.V.A. dos Santos, Rui Moreira,
*
Rita C. Guedes, Maria M.M. Santos
Medicinal Chemistry, iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-019 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The first structureeactivity relationship study of vinyl sulfones as caspase-3 inhibitors is reported.
A series of 12 vinyl sulfones was synthesized and evaluated for two downstream caspases (caspases-3
and -7). Dipeptidyl derivatives were significantly superior to their counterparts containing only Asp at P1,
as caspase-3 inhibitors. Fmoc-Val-Asp-trans-CH]CHeSO2Me was the most potent inhibitor of caspase-3
Received 27 January 2010
Received in revised form
18 May 2010
Accepted 19 May 2010
Available online 24 May 2010
in the series, with a IC50 of 29 m .
M and a second-order rate constant of inactivation, kinact/Ki, of 1.5 Mꢀ1 sꢀ1
Computational studies suggest that the second amino acid occupies position S3 of the enzyme. In
addition, Fmoc-Val-Asp-trans-CH]CHeSO2Ph was inactive for caspase-7 for the tested concentrations.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Vinyl sulfone
Caspase-3 inhibitor
Michael acceptor
Irreversible inhibitor
1. Introduction
sequences are optimal sequences for caspase-3 and caspase-8,
respectively [1]. However, a truncated sequence (e.g. AD or VD) is
Caspases are a family of cysteine endoproteases that are
involved in cytokine maturation and apoptosis [1e3]. Excessive
neuronal apoptosis leads to a variety of diseases such as stroke,
Alzheimer’s disease, Huntington’s disease and Parkinson’s disease
[4,5]. In consequence, caspases are recognized as novel therapeutic
targets for central nervous diseases in which cell death occurs
mainly by an apoptosis mechanism.
generally enough to obtain selective and potent inhibitors of cas-
pases by coupling a reactive functionality to these recognition
moieties (e.g. activated ketones) [8,9]. With this in mind, vinyl
sulfones 3aed and 6aeh (Scheme 1) containing Asp at P1 were
designed to bind in the S1 and S2 subsites of the enzyme.
2. Results and discussion
One type of irreversible cysteine proteases inhibitors that has
received special attention in the last few years are the ones 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 including papain, cathepsins B, L,
S, and K, calpains, and cruzain [6]. In contrast, there are almost no
reports of vinyl sulfones as inhibitors of cysteine proteases from the
clan CD, to which caspases belong to (e.g. VSB-C11, Fig. 1) [7]. The
lack of adequate activityestructure relationships regarding caspase
inhibition by vinyl sulfones has precluded the optimization of this
scaffold.
2.1. Chemistry
Vinyl sulfones 3aed derivatized with N-protected aspartic acid
were prepared by HornereWadswortheEmmons condensation
reaction. Aldehydes 1aeb were obtained from the appropriate
N-Cbz and N-Fmoc aspartic acids using Weinreb chemistry [10].
Acid deprotection with TFA afforded compounds 3aed with yields
of 32e56% from the correspondent aldehydes (Scheme 1). To
improve the potency of the inhibitors, we decided to extend the
recognition structure by incorporating a second amino acid. The
side chains at the R2 position were chosen based upon literature
precedent, which suggest that Ala and Val are preferred at the S2
subsite. In order to compare the effect on caspase-3 inhibition,
a third amino acid, Ile, was also used. The first approach to obtain
the dipeptidyl vinyl sulfones evolved the deprotection of the
nitrogen atom of the vinyl sulfones 2, in order to couple the second
One of the most striking features of caspases is their stringency
for Asp at the P1 residue. For example, the DEVD and LETD
* Corresponding author. Tel.: þ 351 21 794 6400; fax: þ 351 21 794 6470.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
1
Ana S. Newton and Paulo M.C. Glória contributed equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.039

A.S. Newton et al. / European Journal of Medicinal Chemistry 45 (2010) 3858e3863
3859
d
6.8 ppm. Interestingly, deprotection of the Cbz-Ala and Cbz-Val
CO2H
O
methyl vinyl sulfones 5dee led only to the formation of the Z
isomer, while Cbz-Ile methyl vinyl sulfone 5f led to a mixture of Z/E
isomers in a 1:3 ratio.
O
N
N
N
N
H
S
HN
O
O
2.2. Biological activity
The vinyl sulfones synthesized were examined for their ability
to inhibit the activity of recombinant human effector caspases-3
and -7. The IC50 values were determined for compounds 3aed and
6aeh using a fluorimetric assay. The tetrapeptidyl aldehyde Ac-
DEVD-CHO was used as positive control. All vinyl sulfones dis-
VSB-C11
C7H15
Fig. 1. Vinyl sulfone caspase inhibitor.
played IC50 values in the
mM range against caspase-3 (Table 1).
These values are in the same range of caspase-7 inhibition observed
for the vinyl sulfone VSB-C11 [7]. In contrast, compounds 3 and 6
showed to be inactive against caspase-7, thus indicating that our
vinyl sulfones are selective for caspase-3.
Inspection of the data in Table 1, allows the following observa-
tions to be made:
amino acid. However, deprotection of the Cbz group, in the pres-
ence of H2/Pd, did not result. Instead, the double bond of the
Michael acceptor was reduced.
Deprotection of the Fmoc group using several different basic
conditions (piperidine, triethylamine, DBU) was also not successful
leading to yields of 4% of unprotected product. We then decided to
use as starting material the dipeptidyl aldehydes 4aed synthesized
from the correspondent dipeptide acids [11] using Weinreb
chemistry [10]. Deprotection of vinyl sulfones 5aeh with TFA,
afforded vinyl sulfones 6aeh with yields of 34e75% from the
correspondent aldehydes (Scheme 1).
1. For inhibitors containing only the P1 residue (i.e. Asp), the
presence of a more bulky protective group, such as Fmoc seems
to improve the inhibitory activity against caspase-3 (e.g. Fmoc
vinyl sulfones 3c and 3d versus their Cbz-protected counter-
parts 3a and 3b). The same trend was observed for the dipep-
tidyl vinyl sulfones (vinyl sulfones 6geh versus compounds
6aef).
2. The presence of a P2 amino acid residue, i.e. vinyl sulfones
6aeh, improves, but not dramatically, the inhibitory activity
against caspase-3, when compared to the compounds in which
the P2 residue is lacking, i.e. 3aed. For example, the inhibitory
activity of the N-Cbz-Ile-Asp vinyl sulfones 6c and 6f are ca 2-
and 4-fold higher than that of its truncated counterpart, the
N-Cbz-Asp vinyl sulfone 3a and 3b, respectively, while the
All of the proposed structures were established by NMR (1H, 13C,
COSY and HMQC), IR, and MS. The stereochemistry around the
double bond was established using the corresponding 1H NMR
coupling constant. A double doublet (J ¼ 4.0, 15.0 Hz) and a doublet
(J ¼ 15.0 Hz) or double doublet (J ¼ 1.5, 15.0 Hz) at
d 6.5e7.0 ppm
are observed for the vinyl sulfones 3aed, 6aec and 6feh con-
firming the presence of E isomer. Surprisingly, deprotection with
TFA of N-Cbz methyl vinyl sulfones 5eef led to isomerization of the
double bond, as vinyl sulfones 6eef show a singlet for 2 protons at
O
CO2t-Bu
CO2H
R1O2S
P
OEt
OEt
CO2t-Bu
NaH,
TFA
1h 0ºC
PGHN
SO2R1
PGHN
CHO
PGHN
SO2R1
THF, 2h rt
2a PG=Cbz, R1=Ph, 46%
2b PG=Cbz, R1=Me, 55%
2c PG=Fmoc, R1=Ph, 56%
2d PG=Fmoc, R1=Me, 32%
3a-d, 100%
1a PG=Cbz
1b PG=Fmoc
CO2H
O
PGHN
N
H
SO2R1
O
P
R2
TFA
CO2t-Bu
1h 0ºC
CO2t-Bu
CHO
R1O2S
OEt
OEt
O
NaH,
O
6a-c, 100%
6g-h, 100%
PGHN
PGHN
N
H
SO2R1
N
H
THF, 2h rt
R2
R2
CO2H
TFA
1h 0ºC
O
SO2Me
4a PG=Cbz, R2=Me
PGHN
5a PG=Cbz, R2=Me, R1=Ph, 53%
N
H
4b PG=Cbz, R2=CHMe2
4c PG=Cbz, R2=CHMeEt
4d PG=Fmoc, R2=CHMe2
5b PG=Cbz, R2=CHMe2, R1=Ph, 73%
5c PG=Cbz, R2=CHMeEt, R1=Ph, 61%
5d PG=Cbz, R2=Me, R1=Me, 58%
R2
6d-e, 100%
5e PG=Cbz, R2=CHMe2, R1=Me, 75%
5f PG=Cbz, R2=CHMeEt, R1=Me, 47%
5g PG=Fmoc, R2=CHMe2, R1=Ph, 34%
5h PG=Fmoc, R2=CHMe2, R1=Me, 36%
Scheme 1. Synthesis of vinyl sulfones 3aed and 6aeh.

3860
A.S. Newton et al. / European Journal of Medicinal Chemistry 45 (2010) 3858e3863
Table 1
reactive towards nucleophiles than vinyl esters [14,15]. However,
the poor to modest inhibition of caspases displayed by vinyl
sulfones 3 and 6 is line to that reported for VSB-C11 (Fig. 1), and
may reflect a lower intrinsinc reactivity of vinyl sulfones containing
Asp at P1 when compared to related systems based on other amino
acids.
Inhibition of human caspases-3 and -7 by vinyl sulfones [PG-N-X-CH]CHSO2R].
Compound
PG
X
R
IC50 (mM)
Caspase-3
Caspase-7
3a
3b
3c
3d
6a
6b
6c
6d
6e
6f
6g
6h
Cbz
Cbz
Fmoc
Fmoc
Cbz
Cbz
Cbz
Cbz
Cbz
Asp
Asp
Asp
Asp
AlaeAsp
ValeAsp
IleeAsp
AlaeAsp
ValeAsp
IleeAsp
ValeAsp
ValeAsp
e
Ph
Me
Ph
Me
Ph
Ph
Ph
Me
Me
Me
Ph
Me
e
453.0 ꢁ 78.4
434.6 ꢁ 92.3
228.0 ꢁ 64.4
163.2 ꢁ 41.1
343.0 ꢁ 65.6
387.9 ꢁ 4.1
185.4 ꢁ 17.5
655.8 ꢁ 204.6
133.4 ꢁ 27.5
94.6 ꢁ 29.5
69.1 ꢁ 8.1
ND
ND
>200
>200
ND
ND
>200
ND
>200
>200
>200
>200
59.1 ꢁ 6.0 nM
2.3. Docking studies
To get some insight to the binding mode of vinyl sulfones,
compounds 3aed and 6aeh were docked into the active site of
caspase-3 using a covalent constraint as implemented in Gold 3.2
Cbz
software [16] through a covalent bond between the vinyl b-carbon
Fmoc
Fmoc
e
and the sulphur atom of the Cys285 thiol group of the enzyme. Each
pose was ranked according to its ASP scoring function [17] result.
The top ranking solutions were visually inspected and for each
ligand the highest scoring pose was chosen as the binding pose. The
aspartyl side chain at P1 is involved in a series of polar interactions
with the side chains of Arg179, Gln283 and Arg341 residues, i.e.
similarly to the aldehyde Ac-DEVD-CHO inhibitor (Fig. 3). But quite
surprisingly, all ligands 3aed and 6aeh present the Cbz and Fmoc
groups occupying partially the S2 groove. In particular, the Fmoc-
28.8 ꢁ 3.5
Ac-DEVD-CHO
1.6 ꢁ 0.2 nM
ND ¼ Not done.
N-Fmoc dipeptidyl vinyl sulfones 6g and 6h are ca 3- and 6-fold
more active than their N-Fmoc-Asp vinyl sulfone 3c and 3d
counterparts, respectively.
3. The order of inhibitory activity against caspase-3 depends on
the nature of the P2 residue and varies in the order
Ile > Val > Ala.
4. Except for compound 6d, all the dipeptidyl methyl vinyl
sulfones present better activity against caspase-3 when
compared to the related phenyl vinyl sulfones.
protected compounds 6geh present additional
pep stacking
interactions with the benzyl ring of Phe381H (Fig. 3). Moreover, the
N-terminal amino acid residue of 6aeh, presents the corresponding
side chain pointing towards Arg341 and Ser339, rather than
towards the S2 groove, as observed for Ac-DEVD-CHO. This
modeling study indicates that the second residue is interacting
with the S3 pocket rather than with the S2 site, which might
explain, at least in part, the moderate activity displayed by vinyl
sulfones 6aeh.
The inhibitory activity of compound 6h towards caspase-3 was
also determined using the progress curve method [12]. The pseudo-
first-order rate constants, kobs, varied linearly with inhibitor
concentration, yielding a second-order rate constant of inactiva-
tion, kinact/Ki, of 1.51 Mꢀ1 sꢀ1 for 6h. Also, the addition of excess
substrate 30 min after 6h addition did not result in an increase in
activity (Fig. 2), indicating that vinyl sulfone 6h acts irreversibly.
The value of kinact/Ki obtained for 6h is ca 100e5000 times
smaller than those reported for other dipeptidyl Michael acceptors
containing vinyl amide or ester scaffolds. For example, the aza-
peptides Cbz-Val-AAsp-trans-CH]CHeCONHCH2Ph and Cbz-Val-
AAsp-trans-CH]CHeCO2Et inhibit caspase-3 with second-order
3. Conclusion
In conclusion, vinyl sulfones containing Asp at P1 were shown to
be only moderate irreversible inhibitors of caspase-3, with IC50
values in the
mM range. Dipeptidyl vinyl sulfones displayed
improved activity over their counterparts containing Asp as the
rate constants of 185 and 8000 Mꢀ1 ꢀ1, respectively [13]. This
s
result is quite surprising as vinyl sulfones are ca. 2e10 times more
Fig. 2. Progress curves from competition experiments with 6h. Standard caspase-3
Fig. 3. Caspase-3 binding groove displaying the co-crystallized Ac-DEVD-CHO and
inhibitors 3d (red), 6d (magenta) and 6h (blue). Polar hydrogens are displayed for all
molecules in white. The orange and blue region identifies the S2 subsite, the yellow
marks the location of the Arg341, the green marks the location of Cys285 and the cyan
marks the location of Ser339. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
enzyme assays were run in the presence or absence of 6h (300
was determined as a function of assay time. Substrate was added at 10
substrate (100 M) was added 30 min after the assay was initiated, in order to
m
M), and the activity
m
M, an excess of
m
determine whether the inhibition was reversible. The figure shown is a representative
example of two experiments.

A.S. Newton et al. / European Journal of Medicinal Chemistry 45 (2010) 3858e3863
3861
single amino acid residue, with this effect being particularly
noticeably for the Fmoc-protected compounds. Importantly, methyl
sulfones are preferred over phenyl sulfones, which agree with
Goode’s findings that phenyl ketone is less potent than aliphatic
ketone caspase-3 inhibitors [18]. The most active compound, Fmoc-
128.53, 128.26, 127.79, 67.65, 47.99, 37.91. HRMS-EI: m/z calc
19H19NO6S (Mþ) 389.0933, found 389.0930.
C
4.1.2.2. CbzAspVSMe (3b). Obtained in 90% yield. m.p. 106e107 ꢂC.
IR (cmꢀ1) 3327, 1784, 1695, 1525. 1H NMR (400 Mhz, MeOD)
7.36
d
VD-VSMe, 6h, presented an IC50 value of 29
m
M. We extended these
(m, 5H), 6.89 (dd, 1H, J ¼ 15.1 Hz, J ¼ 4.8 Hz, CH]CHSO2Me), 6.67
(dd, 1H, J ¼ 15.1 Hz, J ¼ 1.59 Hz, CH]CHSO2Me), 5.10 (s, 2H),
4.79e4.75 (m, 1H), 2.94 (s, 3H), 2.68 (m, 2H). 13C NMR (100 MHz,
enzyme-inhibition studies by testing the ability of the most active
vinyl sulfones 3 and 6 to block another downstream enzyme critical
in the execution of apoptosis, caspase-7. Vinyl sulfones 3 and 6 were
inactive for the tested concentrations. These results indicate that
our vinyl sulfones are selective for caspase-3.
MeOD)
d 173.40, 157.90, 147.30, 138.09, 131.48, 129.50, 129.06,
128.87, 67.76, 49.74, 42.70, 38.87. HRMS-EI: m/z calc C14H17NO6S
(Mþ) 327.0777, found 327.0783.
The docking studies indicate that the Asp vinyl sulfone core may
be suitable for optimal enzyme binding. Docking studies suggest
that there is space to improve the caspase-3 inhibitory activity by
modifying the N-terminal protecting group and introducing
a dipeptide sequence that can improve interaction with S3 subsite.
Further efforts aimed at improving compound potency are under
way and will be reported in due course.
4.1.2.3. FmocAspVSPh (3c). Obtained in 100% yield. m.p. 97e98 ꢂC.
IR (cmꢀ1) 3409, 1716, 1641, 1532. 1H NMR (400 MHz, DMSO-d6)
d
12.44 (sl, 1H), 7.88 (d, 2H, J ¼ 7.5 Hz), 7.84 (d, 2H, J ¼ 7.3 Hz),
7.74e7.62 (m, 6H), 7.41 (t, 2H, J ¼ 7.3 Hz), 7.29 (t, 2H, J ¼ 7.3 Hz), 6.92
(dd, 1H, J ¼ 15.4 Hz, J ¼ 4.6 Hz, CH]CHSO2Ph), 6.65 (d, 1H,
J ¼ 15.4 Hz, CH]CHSO2Ph), 4.59 (sl, 1H), 4.34e4.26 (m, 2H), 4.19 (t,
1H, J ¼ 6.7 Hz), 2.65 (dd, 1H, J ¼ 16.1 Hz, J ¼ 5.3 Hz), 2.51 (dd, 1H,
J ¼ 16.1Hz, J ¼ 5.3 Hz). 13C NMR (100 MHZ, DMSO-d6)
d 171.35,
4. Experimental protocols
155.36,146.22,143.89,143.69,140.75,133.76,130.04,129.66,127.68,
127.21, 127.09, 125.17, 120.17, 65.51, 48.24, 46.67, 37. HRMS-EI: m/z
calc C26H23NO6S (Mþ) 477.1246, found 477.1244.
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 spectra were collected on
a Nicolet Impact 400 FTIR infrared spectrophotometer. High reso-
lution 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 chromatography 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
4.1.2.4. FmocAspVSMe(3d). Obtained in 88% yield. m.p. 127e128 ꢂC.
IR (cmꢀ1) 3327, 1729, 1689, 1539. 1H NMR (400 MHz, DMSO-d6)
d
12.46 (sl,1H), 7.90 (d, 2H, J ¼ 7.3 Hz), 7.73e7.69 (m, 3H), 7.42 (t, 2H,
J ¼ 7.3 Hz), 7.34 (t, 2H, J ¼ 7.3 Hz), 6.74 (dd, 1H, J ¼ 14.9 Hz,
J ¼ 4.5 Hz, CH]CHSO2Me), 6.65 (d, 1H, J ¼ 16.1 Hz, CH]CHSO2Me),
4.59 (sl, 1H), 4.33 (d, 2H, J ¼ 7.0 Hz), 4.23 (t, 1H, J ¼ 8.0 Hz), 3.00 (s,
3H), 2.64 (dd, 1H, J ¼ 16.0 Hz, J ¼ 6.0 Hz), 2.52 (dd, 1H, J ¼ 16.0 Hz,
J ¼ 6.0 Hz). 13C NMR (100 MHz, DMSO-d6)
d 171.42, 155.40, 145.20,
143.90, 143.78, 140.78, 130.30, 127.71, 127.14, 125.23, 120.21, 65.61,
48.16, 46.72, 42.21, 36.99. Anal. Cald for C21H21NO6S: C, 60.71; H,
5.09; N, 3.37; S 7.72. Found: C, 60.36; H, 5.39; N, 3.36; S 7.37.
4.1.2.5. CbzAlaAspVSPh(6a). Obtained in 95% yield. m.p.102e103 ꢂC.
chemical shifts are expressed in
d
(ppm) referenced to the solvent
IR (cmꢀ1) 3340,1723,1675,1525.1H NMR (400 MHz, MeOD)
d 7.89 (d,
used and the proton coupling constants (J) in hertz.
2H, J ¼ 7.7 Hz), 7.65e7.53 (m, 3H), 7.33(m, 5H), 6.99 (dd,1H, J ¼ 15 Hz,
J ¼ 3.6 Hz, CH]CHSO2Me), 6.89 (d, 1H, J ¼ 15 Hz, CH]CHSO2Me),
5.09e4.95 (m, 3H), 4.09e4.03 (m, 1H), 2.74 (dd, 1H, J ¼ 16.9 Hz,
J ¼ 5.9 Hz), 2.61 (dd,1H, J ¼ 16.9 Hz, J ¼ 8.1 Hz),1.28 (d, 3H, J ¼ 7.2 Hz).
4.1.1. General procedure for the preparation of vinyl sulfones 2aed
and 5aeh
To a suspension of NaH 60% (1.8 mmol, 1.1 equiv) in THF (5 ml),
at 0 ꢂC, was added 1 eq. of the appropriate sulfone. The resulting
solution was stirred at temperature room for 30 min. A solution of 1
eq. of the aldehyde 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 and used directly for
the next step.
13C NMR (100 MHz, MeOD)
d 175.54, 173.45, 173.30, 158.19, 146.13,
141.71, 138.08, 134.75, 134.69, 132.54, 132.36, 130.54, 130.47, 129.47,
129.07,129.00,128.91,128.65, 67.83, 67.70, 52.60, 52.13, 38.37, 28.25,
17.77. HRMS-EI: m/z calc C22H24N2O7S (Mþ) 460.1304, found
460.1307.
4.1.2.6. CbzValAspVSPh (6b). Obtained in 99% yield. m.p.104e105 ꢂC.
IR (cmꢀ1) 3052,1716,1682,1511.1H NMR (400 MHz, MeOD)
d 7.88 (d,
2H, J ¼ 7.4 Hz), 7.67 (t,1H, J ¼ 7.4 Hz), 7.58 (t, 2H, J ¼ 7.4 Hz), 7.36 (m,
5H), 6.97 (dd, 1H, J ¼ 15.2 Hz, J ¼ 4.5 Hz, CH]CHSO2Ph), 6.70 (d, 1H,
J ¼ 14,9 Hz, CH]CHSO2Me), 5.10e4.98 (m, 3H), 3.85 (d, 1H,
J ¼ 6.5 Hz), 2.72e2.70 (m, 2H), 2.06e1.97 (m, 1H), 0.89 (m, 6H). 13C
4.1.2. General procedure for the preparation of vinyl sulfones 3aed
and 6aeh
Vinyl sulfones were 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.
NMR (100 MHz, CDCl3) d 173.84,173.47,158.73,146.08,141.47,137.90,
134.84, 132.45, 130.58, 129.48, 129.24, 129.07, 128.94, 128.62, 67.89,
62.52, 38.40, 30.71, 19.67, 18.35. HRMS-EI: m/z calc C24H28N2O7S
(Mþ) 488.1617, found 488.1615.
4.1.2.1. CbzAspVSPh (3a). Obtained in 100% yield. m.p. 81e82 ꢂC. IR
(cmꢀ1) 3349, 1721, 1528. 1H NMR (400 Mhz, CDCl3)
d
7.85 (2H, d,
4.1.2.7. CbzIleAspVSPh (6c). Obtained in 100% yield. m.p.150e151 ꢂC.
J ¼ 8.0 Hz), 7.62 (t, 1H, J ¼ 7.6 Hz), 7.52 (t, 2H, J ¼ 7.6 Hz), 7.32 (m,
5H), 6.96 (dd, 1H, J ¼ 15.2 Hz, J ¼ 4.4 Hz, CH]CHSO2Ph), 6.50 (d, 1H,
J ¼ 15.2 Hz, CH]CHSO2Ph), 5.67 (d,1H, J ¼ 9.2 Hz), 5.06 (s, 2H), 4.80
IR (cmꢀ1)3244,1722,1648,1539.1HNMR (400 MHz, MeOD)
d 7.88 (d,
2H, J ¼ 7.1 Hz), 7.67 (t,1H, J ¼ 7.1 Hz), 7.59 (t, 2H, J ¼ 7.1 Hz), 7.34e7.32
(m, 5H), 6.97 (dd, 1H, J ¼ 15.8 Hz, J ¼ 5.5 Hz, CH]CHSO2Ph), 6.69 (d,
1H, J ¼ 15.8 Hz, CH]CHSO2Ph), 5.10e4.96 (m, 3H), 3.90 (d, 1H,
J ¼ 7.1 Hz), 2.75e2.68 (m, 2H), 1.81e1.72 (m, 1H), 1.51e1.41 (m, 1H),
(sl, 1H), 2.75 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 174.23, 156.01,
144.44, 139.45, 135.75,133.93, 131.37, 129.55, 129.40, 128.99, 128.72,

3862
A.S. Newton et al. / European Journal of Medicinal Chemistry 45 (2010) 3858e3863
1.20e1.10 (m,1H), 0.86 (m, 6H). 13C NMR (100 MHz, MeOD)
d
173.85,
amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase-3, resulting
173.31, 158.74, 146.09, 134.80, 132.55, 130.58, 130.50, 129.50, 129.08,
128.99, 128.69, 67.87, 61.59, 47.96, 38.30, 38.06, 25.83, 15.98, 11.63.
HRMS-EI: m/z calc C25H30N2O7S (Mþ) 502.1774, found 502.1782.
in the release of the fluorescent 7-amido-4-methylcoumarin (AMC)
moiety. Briefly, 5
human, recombinant, Calbiochem) were added to 5
inhibitors at various concentrations. The reaction was initiated by
the addition of 190 L of substrate to a final concentration of 20
mL of appropriate dilution of caspase-3 (caspase-3,
mL of the tested
4.1.2.8. CbzAlaAspVSMe (6d). Obtained in 97%yield. m.p.144e145 ꢂC.
m
mM
IR(cmꢀ1)3409,1708,1668,1518.1HNMR(400MHz,DMSO-d6)
d
12.44
in assay buffer (20 mM HEPES, 2 mM 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-CHO, Calbiochem). IC50 values were
determined by non-linear regression analysis. Progress curves, run
in the absence and presence of compound, were used to determine
(sl,1H), 8.27(d,1H, J¼ 8.3Hz),7.54(d,1H, J¼ 6.7Hz), 7.35(m, 5H), 6.77
(s, 2H), 5.02(d, 2H, J¼ 6.4 Hz), 4.81(dd,1H, J¼ 15.6Hz, J ¼ 6.4 Hz), 4.01
(t,1H, J ¼ 7.0 Hz), 2.96 (s, 3H), 2.68 (dd,1H, J ¼ 16.2 Hz, J ¼ 6.0 Hz), 2.49
(dd, 1H, J ¼ 16.2 Hz, J ¼ 6.0 Hz), 1.19 (d, 3H, J ¼ 7.3 Hz). 13C NMR
(100 MHz, DMSO-d6)
d 172.29, 171.50, 155.87, 145.18, 137.01, 130.38,
128.42, 127.88, 127.83, 65.45, 50.21, 46.19, 42.27, 37.50, 17.83. HRMS-
EI: m/z calc C17H22N2O7S (Mþ) 398.1148, found 398.1151.
4.1.2.9. CbzValAspVSMe (6e). Obtained in 99% yield. m.p.157e158 ꢂC.
IR (cmꢀ1) 3052, 1709, 1682, 1648, 1525. 1H NMR (400 MHz, MeOD)
d
7.35 (m, 5H), 6.88 (s, 2H, CH]CHSO2Me), 5.10e5.03 (m, 3H), 3.81 (d,
the nature of inhibition. The reaction was initiated with 10
mM of
1H, J ¼ 7.9 Hz), 2.77 (dd, 1H, J ¼ 16.5 Hz, J ¼ 5.4 Hz), 2.58 (dd, 1H,
substrate when the reaction attain a plateau, after 30 min, an excess
substrate was added to final concentration 100 mM.
J¼ 16.5Hz, J¼ 8.6 Hz), 2.03e1.95 (m,1H), 0.98(d, 3H, J¼ 4 Hz), 0.96(d,
3H, J ¼ 4 Hz). 13C NMR (100 MHz, MeOD)
d 174.29, 173.20, 158.71,
146.62,138.27,131.88,129.50,128.97,128.74, 67.58, 62.81, 48.37, 42.71,
38.47, 31.36, 19.71, 19.21. HRMS-EI: m/z calc C19H26N2O7S (Mþ)
426.1461, found 426.1463.
4.2.2. Caspase-7 assays
Caspase-7 kinetic assays were performed using the same
conditions and the same substrate (Ac-DEVD-AMC, 2 mM stock
solution in DMSO) of caspase-3 assays. The enzyme stock solution
was 200 U/mL (final concentration in the well: 1 U) in the assay
buffer.
4.1.2.10. CbzIleAspVSMe(6f). Obtainedin 99%yield. m.p.125e127 ꢂC.
IR (cmꢀ1) 3327, 3258,1723,1648,1539. E isomer: 1H NMR (400 MHz,
MeOD)
d
7.37e7.32 (m, 5H), 6.87 (dd,1H, J ¼ 15.4 Hz, J ¼ 4.4 Hz, CH]
CHSO2Me), 6.71 (d, 1H, J ¼ 15.4 Hz, CH]CHSO2Me), 5.11e5.09 (m,
2H), 5.00e4.98 (m, 1H), 3.88 (d, 1H, J ¼ 8.9 Hz), 2.88 (s, 3H),
2.75e2.55 (m, 2H), 1.84e1.76 (m, 1H), 1.58e1.49 (m, 1H), 1.23e1.16
4.3. Computational studies
Molecular docking studies were performed, using the GOLD
software [16] (version 3.2) to predict the interactions and binding
modes of all the synthesized inhibitors in the caspase-3 active site,
and to evaluate their relative binding affinities. The 3D structure
coordinates of caspase-3 were obtained after a search in the Protein
Data Bank and electing the structure with code 1PAU with a reso-
lution of 2.5 Å [19]. To prepare the enzyme for the docking studies,
the Ac-DEVD-CHO inhibitor included in the 1PAU structure was
removed, hydrogen atoms were added to caspase-3, crystallo-
graphic waters were removed and the protonation states were
correctly assigned using the Protonate-3D tool within the Molec-
ular Operating Environment (MOE) 2008.10 software package [20].
To evaluate and validate the effectiveness of our model system and
the performance of the docking protocol, a set of studies was first
performed using the co-crystallized inhibitor to reproduce the
experimental binding pose. The best docking pose obtained was
overlaid with the experimental crystal structure showing
a minimal deviation of its atomic coordinates (RMSD ¼ 0.82 Å).
Since the validation study revealed that our model and software
protocol could accurately reproduce the experimentally binding
mode of the Ac-DEVD-CHO inhibitor, a series of synthesized vinyl
sulfones were covalently docked into the caspase-3 binding site
using the ASP fitness function [17].
(m, 1H), 0.95e0.89 (m, 6H). 13C NMR (100 MHz, MeOD)
d 173.97,
173.35, 158.81, 146.62, 138.10, 131.96, 129.52, 128.95, 67.87, 61.68,
47.99, 42.77, 38.05, 37.41, 25.87, 16.05, 11.63. HMRS-EI: m/z calc
C20H28N2O7S (Mþ) 440.1617, found 440.1610.
4.1.2.11. FmocValAspVSPh (6g). Obtained in 91% yield. m.p.
108e109 ꢂC. IR (cmꢀ1). 3314, 1740, 1666, 1516. 1H NMR (400 MHz,
DMSO-d6)
d
12.56 (sl, 1H), 8.33 (d, 1H, J ¼ 8 Hz), 7.93 (d, 2H,
J ¼ 8 Hz), 7.78 (m, 2H), 7.36 (m, 9H), 6.76 (dd, 1H, J ¼ 16 Hz, J ¼ 4 Hz,
CH]CHSO2Ph), 6.68 (d, 1H, J ¼ 16 Hz, CH]CHSO2Ph), 4.84 (sl, 1H),
4.32e4.25 (m, 3H), 3.88 (t, 1H, J ¼ 4 Hz), 2,63 (m, 2H), 2.01 (sl, 1H),
0.88 (d, 6H, J ¼ 8 Hz). 13C NMR (100 MHz, DMSO-d6)
d 172.34,171.76,
160.73, 144.81, 140.94, 139.26, 136.77, 135.44, 133.05, 129.10, 128.65,
128.18, 127.43, 124.24, 122.82, 66.79, 59.11, 50.19, 48.05, 39.76,
30.48, 19.04. HRMS-ESI-TOF: m/z calc C31H32N2O7SNa (Mþ þ Na)
599.1825, found 599.1822.
4.1.2.12. FmocValAspVSMe (6h). Obtained in 93% yield. m.p.
93e94 ꢂC. IR (cmꢀ1). 3291, 1746, 1700, 1660, 1522. 1H NMR
(400 MHz, DMSO-d6)
d
12.53 (sl, 1H), 8.31 (d, 1H, J ¼ 8 Hz), 7.90 (d,
2H, J ¼ 8 Hz), 7.76 (m, 2H), 7.42e7.32 (m, 2H), 6.74 (dd,1H, J ¼ 16 Hz,
J ¼ 4 Hz, CH]CHSO2Me), 6.66 (d, 1H, J ¼ 16 Hz, CH]CHSO2Me),
4.82 (sl, 1H), 4.30e4.22 (m, 3H), 3.85 (t, 1H, J ¼ 4 Hz), 2.96 (s, 3H),
2.61 (m, 2H), 1.99 (sl, 1H), 0.85 (d, 6H, J ¼ 8 Hz). 13C NMR (100 MHz,
Acknowledgments
DMSO-d6)
d 172.54, 171.35, 160.78, 158.55, 144.86, 141.17, 139.26,
128.64, 127.43, 124.24, 122.84, 66.82, 59.11, 50.17, 48.06, 39.74,
37.99, 30.48, 19.04. HRMS-ESI-TOF: m/z calc C26H30N2O7SNa
(Mþ þ Na) 537.1652, found 537.1666.
This work was supported by the Fundação para a Ciência e
Tecnologia (Lisbon, Portugal) by the award of doctoral fellowship to
A.S.N. (SFRH/BD/41276/2007) and post doctoral fellowship to P.M.C.
G. (SFRH/BPD/22631/2005).
4.2. Pharmacology
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hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-7-
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