Design, synthesis, and evaluation of aza-peptide Michael acceptors as selective and potent inhibitors of caspases-2, -3, -6, -7, -8, -9, and -10

Article abstract of DOI:10.1021/jm0601405

Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3, -6, -7, -8, -9, and -10. The second-order rate constants are in the order of 10⁶ M^-1 s^-1. The aza-peptide Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CH=CH-CON(CH₂-1-Naphth)₂ is the most potent compound and it inhibits caspase-3 with a k₂ value of 5620000 M^-1 s^-1. The inhibitor 18t is 13700, 190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10, respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains. High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of the inhibitors with respect to the P1′ moiety.

Full text of DOI:10.1021/jm0601405

5728
J. Med. Chem. 2006, 49, 5728-5749
Design, Synthesis, and Evaluation of Aza-Peptide Michael Acceptors as Selective and Potent
Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
O¨ zlem Dogˇan Ekici,† Zhao Zhao Li,† Amy J. Campbell,† Karen Ellis James,† Juliana L. Asgian,† Jowita Mikolajczyk,‡
Guy S. Salvesen,‡ Rajkumar Ganesan,§ Stjepan Jelakovic,§ Markus G. Gru¨tter,§ and James C. Powers*,†
School of Chemistry and Biochemistry and the Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology,
Atlanta, Georgia 30332-0400, Program in Apoptosis and Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road,
La Jolla, California 92037, and Department of Biochemistry, UniVersity of Zurich, 8057-Zurich, Switzerland
ReceiVed February 8, 2006
Aza-peptide Michael acceptors are a novel class of inhibitors that are potent and specific for caspases-2, -3,
-6, -7, -8, -9, and -10. The second-order rate constants are in the order of 106 M-1 s-1. The aza-peptide
Michael acceptor inhibitor 18t (Cbz-Asp-Glu-Val-AAsp-trans-CHdCH-CON(CH2-1-Naphth)2 is the most
potent compound and it inhibits caspase-3 with a k2 value of 5620000 M-1 s-1. The inhibitor 18t is 13700,
190, 6.4, 594, 37500, and 173-fold more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and -10,
respectively. Aza-peptide Michael acceptors designed with caspase specific sequences are selective and do
not show any cross reactivity with clan CA cysteine proteases such as papain, cathepsin B, and calpains.
High-resolution crystal structures of caspase-3 and caspase-8 in complex with aza-peptide Michael acceptor
inhibitors demonstrate the nucleophilic attack on C2 and provide insight into the selectivity and potency of
the inhibitors with respect to the P1′ moiety.
Introduction
diseases such as stroke, Alzheimer’s disease, Huntington’s
disease, Parkinson’s disease, amyotropic lateral sclerosis (ALS),
multiple sclerosis (MS), and spinal muscular atrophy.4 Caspases
are recognized as novel therapeutic targets for central nervous
diseases in which cell death occurs mainly by an apoptosis
mechanism.5-8 Hence, potent and specific inhibitors of caspases
could lead to the development of potential drugs, which can
modulate the apoptotic process.
A number of different classes of inhibitors have been
developed for cysteine proteases including reversible transition
state inhibitors and a variety of irreversible inhibitors.9 Relatively
few classes have been applied to clan CD cysteine proteases,
particulary caspases. Several substrate based caspase inhibitors
have been extensively used to address the roles of caspases in
cellular systems over the past few years.10 These include
reversible inhibitors such as peptide aldehydes (Ac-YVAD-
CHO, Ac-DEVD-CHO, Ac-WEHD-CHO) and irreversible
inhibitors such as fluoro- or chloromethyl ketones (Z-VAD-
FMK, Z-YVAD-FMK/CMK, Z-DEVD-FMK/CMK). A few of
these inhibitors have even entered preclinical studies with animal
models of human diseases. However, the major disadvantage
of these inhibitors is their lack of selectivity. Several of these
caspase inhibitors have been recently shown to efficiently
inactivate other cysteine proteases, such as cathepsins B, L, and
S.11 Cathepsin B inhibition by Ac-YVAD-CMK rescued neu-
ronal cells against glucose/oxygen deprivation-induced apop-
Cysteine proteases, also known as thiol proteases have been
identified in the biological systems of bacteria, protozoa, fungi,
plant viruses, and mammals. Cysteine proteases acylate the pep-
tide bond via a nucleophilic thiol and are involved in numerous
physiologically important processes. Cysteine proteases have
been grouped into evolutionary families and clans by Rawlings
and Barrett.1,2 The majority of structurally relevant cysteine
proteases belong to the well-known clan CA, which includes
papain, cathepsins, and calpains. Several important cysteine
proteases such as caspases, legumain, gingipain, clostripain, and
separase belong to a new but smaller clan of cysteine proteases,
which is termed clan CD. The tertiary structure of clan CD
proteases shows a unique R/â fold unlike any other protein,
with the catalytic residues in the order His, Cys in the sequence.
Clan CD cysteine proteases differ from the clan CA proteases
also in their substrate specificity requirements. The substrate
specificity of clan CD proteases is determined by the S1 pocket,
whereas the S2 pocket is the major determinant of substrate
specificity with the clan CA cysteine proteases.
Caspases, also known as cysteinyl aspartate-specific pro-
teases, are found in worms to flies to humans. Currently, >15
members are identified, with 11 members in humans.3 One
distinctive feature of this family of enzymes is their near absolute
specificity for Asp residue at P1. The only other known
mammalian protease with the same specificity requirement is
the lymphocyte serine protease granzyme B, which acts as a
caspase activator. Caspases have been the focus of both
industrial and academic research since their discovery. Over
the past decade, the exact roles and functions of individual
caspases in cellular systems have been intensely investigated.
Some caspases are important mediators of inflammation,
whereas others are involved in apoptosis (programmed cell
death). Excessive neuronal apoptosis leads to a variety of
a Abbreviations: AA, amino acid residue; AAsn, aza-asparagine; AAsp,
aza-aspartic acid; Ac, acetyl; AcOH, acetic acid; AFC, 7-amino-4-trifluoro-
methylcoumarin; AMC, 7-amino-4-methylcoumarin; Boc, tert-butoxycar-
bonyl; Bzl, benzyl, CH2Ph; Cbz, benzyloxycarbonyl; CDCl3, deuterated
chloroform; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-
sulfonate; CMK, chloromethyl ketone; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; DMSO-d6, dimethyl sulfoxide-d6 deuterated;
DTT, dithiothreitol; E-64c, N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl-
3-methylbutanamide; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; EDTA, ethylenediaminetetraacetic acid; EtOAc, ethyl acetate;
EtOH, ethanol; FMK, fluoromethyl ketone; HEPES, N-2-hydroxyethylpip-
erazine-N′-2-ethanesulfonic acid; HOBt, N-hydroxybenzotriazole; iBCF,
isobutyl chloroformate; iBu, isobutyl; MeOH, methanol; NMM, N-meth-
ylmorpholine; OBzl, benzyloxy; Orn, ornithine; PIPES, 1,4-piperazinedi-
ethanesulfonic acid; pNA, p-nitroanilide; tBu, tert-butyl; TFA, trifluoroacetic
acid.
* Corresponding author. Tel: (404) 894-4038, Fax: (404) 894-2295.
E-mail: james.powers@chemistry.gatech.edu.
† Georgia Institute of Technology.
‡ The Burnham Institute.
§ University of Zurich.
10.1021/jm0601405 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/23/2006

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5729
CD cysteine proteases.23 The aza-peptide Michael acceptor
structure resembles the substrate peptide sequence (1, Figure
1), where the carbonyl group of the double bond moiety replaces
the carbonyl group of the scissile bond. Replacement of the
R-carbon of the P1 amino acid with a nitrogen results in the
formation of the aza-peptide, which allows the ready synthesis
of a variety of derivatives. In the fumarate derivative 3, the
Michael acceptor is on the N-terminus of the amino acid residue,
while in our aza-peptide design, the warhead is at the C-terminal
end of the inhibitor. On the other hand, the design of vinyl
sulfones and R,â-unsaturated carbonyl derivatives was based
on the optimal peptide sequence of the target enzyme, where
the carbonyl of the scissile bond was replaced by the double
bond moiety.
Figure 1. Aza-peptide Michael acceptor design.
tosis, suggesting that other proteases may be involved in the
processes previously designated only to caspases. Furthermore,
several lysosomal cysteine proteases were shown to be involved
in various apoptosis models, although the mechanisms are not
yet clear.12,13 Thus, there is a considerable need for potent and
selective caspase inhibitors to elucidate the roles of individual
caspases in apoptosis and as potential therapeutics.
In an effort to obtain greater potency and selectivity, we
elaborated our aza-peptide Michael acceptor design in the P′
portion of the inhibitor. Although considerable effort has been
devoted to the optimization of interactions of caspase inhibitors
at the S1-S4 sites,24,25 interactions of substrates or inhibitors
at the S1’ site in the past were analyzed in only very few
studies.26,27 Recently, a few peptidomimetic compounds binding
to the groove located near the S1′ site of caspase-3 were
identified and characterized structurally.26 We were particularly
interested in designing inhibitors for the key executioner
caspases-3, -6, and -7 and initiator caspases-2, -8, -9, and -10.
Here we also report crystal structures of caspase-3 and caspase-8
in complex with aza-peptide Michael acceptors with different
P1′ substitutions to elucidate the structural determinants for the
preference and discrimination at this site. The analysis of the
crystal structures of the caspase:Michael acceptor inhibitor
complexes also provide insights into the difference in reactivity
of cis (18g) and trans (18h) isomers.
A variety of other inhibitors with electrophilic warheads have
been reported as irreversible inhibitors effective for clan CA
cysteine proteases.9 One of the first Michael acceptors described
in the literature is the fumarate derivative of E-64c (3, Figure
1).14 This inhibitor contains an R,â-unsaturated carbonyl moiety
and was found to be an irreversible inhibitor of cathepsin B,
cathepsin H, and cathepsin L. Vinyl sulfones and R,â-unsatur-
ated carbonyl derivatives (4, Figure 1) have been developed as
highly potent inhibitors for exopeptidases such as DPPI and
many clan CA cysteine endopeptidases including papain,
cathepsins B, L, S, and K, calpains, and cruzain.15,16 Michael
acceptor inhibitors have great potential for use as drugs. One
Michael acceptor CRA-3316 developed by Celera as a cruzain
inhibitor to treat Chagas’s disease is also in clinical trials.17
Another Michael acceptor AG7088 (Ruprintrivir) has entered
phase II clinical studies with humans as a potential nasally
delivered antirhinoviral agent.18-21 Recently, Michael acceptor
inhibitors based on the AG7088 structure drew considerable
attention in the treatment of SARS (Severe Acute Respiratory
Syndrome) due to their potential as antiviral agents against the
SARS coronavirus.22
Chemistry. We synthesized a variety of aza-peptide Michael
acceptors with P1 Asp residue as potential inhibitors for
caspases. We previously reported the general method to prepare
aza-peptide Michael acceptors in our preliminary communica-
tion.23 Aza-peptide Michael acceptors for caspases were syn-
thesized by coupling the appropriate substituted peptidyl
hydrazides with P1 Asp side chain with the desired fumarate
derivative.
A variety of fumarate esters and fumarate mono- and diamides
have been synthesized in order to couple to the substituted
peptidyl hydrazides (Figure 2). The monobenzyl fumarate 7 was
Our laboratory has recently reported aza-peptide Michael
acceptor inhibitors (2, Figure 1) that are highly specific for clan
Figure 2. Synthesis of fumarate and maleate derivatives. Reagents used: (i) NMM, EDC, BzlOH, DMF; (ii) KOH, EtOH, r.t.; (iii) NMM, iBCF,
CH2Cl2; (iv) EtOH, r.t.

5730 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
Figure 3. Synthesis of Aza-Asp Michael acceptors. Reagents used: (i) BrCH2COO-tBu, NMM, DMF; (ii) EDC, HOBt, DMF or NMM, iBCF,
DMF; (iii) TFA, CH2Cl2.
obtained by the addition of 1 eq of benzyl alcohol to monoethyl
fumarate 5 using EDC and NMM as coupling reagents followed
by the hydrolysis of the ethyl ester 6 in methanol by aqueous
NaOH. Mono- and disubstituted amide derivatives 10 and 11
were obtained by coupling the desired primary or secondary
amine to monoethyl fumarate 5 and subsequent hydrolysis of
the ethyl ester 8 or 9 with 1.2 eq of KOH in ethanol. Monoethyl
maleate 13 was synthesized by the alcoholysis reaction of maleic
anhydride 12 with ethanol.
Peptidyl hydrazides 14 were obtained by reacting blocked
mono-, di-, or tripeptidyl methyl esters with excess hydrazine
in methanol. The aza-Asp side chain was introduced by
alkylation of the peptidyl hydrazide with tert-butyl bromoacetate
to form the substituted peptidyl hydrazide 15.
The final aza-peptide Michael acceptors (16-21 (a-u),
Figure 3), were synthesized primarily by the EDC/HOBt
coupling method with the peptide sequences Cbz-Val-NH-
NH-CH2COO-tBu, Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-NH-
NH-CH2COO-tBu, Cbz-Val-Glu(O-tBu)-Val-NH-NH-CH2-
COO-tBu, Cbz-Ile-Glu(O-tBu)-Thr-NH-NH-CH2COO-tBu,
and Cbz-Leu-Glu(O-tBu)-Thr-NH-NH-CH2COO-tBu using an
excess of the desired fumarate derivative. To simplify the
purification process and maximize the yield, some aza-peptide
Michael acceptors were synthesized by the mixed anhydride
method using the peptide sequences Cbz-Glu(O-tBu)-Val-NH-
NH-CH2COO-tBu, Cbz-Ile-Glu(O-tBu)-Thr-NH-NH-CH2-
COO-tBu, and Cbz-Leu-Glu(O-tBu)-Thr-NH-NH-CH2COO-
tBu. In the final step of the aza-peptide Michael acceptor
synthesis, the tert-butyl protecting groups on the aza-Asp, Glu,
and Asp residues were deprotected using TFA in CH2Cl2 at 0
°C.
caspase-3 and caspase-8, respectively, as determined by posi-
tional scanning of a synthetic combinatorial library.29,30 The
IETD sequence for caspases-6 and -8 is the cleavage sequence
of a natural caspase-8 substrate, the caspase-3 proenzyme.28 The
VEVD sequence was chosen as the optimal substrate for
caspase-6 in accordance with the branched amino acid prefer-
ence of this enzyme. The EVD sequence was selected to obtain
a truncated caspase-3 inhibitor. Similarly, the VD sequence was
chosen to represent a general inhibitor for all the caspases
(caspases-2, -3, -6, -7, -8, -9, and -10).
The aza-peptide Michael acceptors with an aza-Asp residue
at P1 position and designed with the appropriate target sequences
were tested against various caspases for kinetic activity. The
aza-peptide Michael acceptors exhibit excellent inhibitory
potency against caspases-2, -3, -6, -7, -8, -9, and -10 (Table 1).
The second-order inhibition rates are as high as 106 M-1 s-1
.
Generally, the DEVD sequence is the most reactive with
caspases-2, -3, and -7. The IETD and LETD sequences are more
reactive with the caspases-6, -8, and -9. The VEVD sequence
is equally potent as IETD for caspases-6 and -8. Interestingly,
the DEVD sequence is the best sequence for caspase-10 although
this enzyme was originally included into group II along with
caspases-6,-8, and -9. Also the most potent caspase-8 inhibitor
has the DEVD sequence. Its favorable interaction with caspase-8
has been demonstgrated in a crystal structure.31 Similarly, the
most potent caspase-9 inhibitor is the one with the EVD
tripeptide sequence. This result seems to be an exception to the
requirement of the tetrapeptide sequence of caspases for efficient
substrate cleavage.
Aza-peptide Michael acceptors are easily extended at their
P′ site. To gain more insight about the potency and selectivity
of the aza-peptide Michael acceptor inhibitors, we modified the
substituents on the fumarate moiety extending into the P′ site.
We utilized various groups such as simple alkyl, alkenyl, and
aryl groups, as well as halogens, esters, and amides in the hopes
of obtaining selectivity in the P′ site among the various caspases.
Results and Discussion
The optimal peptide sequences of the target caspases were
utilized in the design of the aza-peptide Michael acceptors. The
optimal sequences were derived either from natural caspase
cleavage sites or from peptide mapping of caspases with libraries
of AMC substrates.28 On the basis of their S4 subsite prefer-
ences, Thornberry and co-workers divided the caspases into three
groups: group I caspases (1, 4, and 5) prefer aromatic amino
acid residues, group II caspases (6, 8, 9, and 10) have a
preference for branched apolar residues, and group III caspases
(2, 3, and 7) prefer an aspartic acid residue at their P4 positions.
The DEVD and LETD sequences are optimal sequences for
Caspase-2. In contrast to the other caspases, caspase-2
requires a P5 residue for efficient substrate cleavage. The
optimal sequence of caspase-2 is VDVAD, but the caspase-3
specific DEVD sequence still worked best for caspase-2.
Because of the P5 requirement, the second-order rates of
inhibition with caspase-2 remained in the range of 102 M-1 s-1
,
except for the inhibitors 18g and 18h. The most potent inhibitor
of caspase-2 was the cis isomer 18g with a k2 value of 26200

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5731

5732 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
M-1 s-1. 18g was 10-fold more reactive than the trans isomer
18h (k2 ) 2640 M-1 s-1). This result was interesting since for
the other caspases (3, 6, 7, 8, 9, and 10) 18g was less potent
when compared to its trans isomer 18h. The order of reactivity
of the inhibitors with the DEVD sequence according to the
substituent Y is as follows: COOEt (cis) > COOEt (trans) >
CON(CH3)CH2Ph > CON(CH2-1-Naphth)2 > CON-tetrahyd-
roquinoline > CON(CH3)CH2CH2Ph > COPh > CONHCH2-
Ph > CONHCH2-4-F-Ph > CONHCH2CH2Ph > CON(CH2-
Ph)2. It seems that the esters are the best substitutents and the
disubstituted amides are more preferred over the monosubsti-
tuted amides.
Caspases-6 and -8. The IETD sequence works best with
caspase-6, whereas the LETD sequence is ideal for caspase-8.
Caspase-8 prefers leucine > valine > aspartic acid at its P4
position. This preference offers some room for obtaining
selectivity of caspase-8 over caspase-6, where caspase-6 prefers
valine > threonine > leucine at the same position (P4).27 The
best caspase-6 inhibitor is 20k (Cbz-Ile-Glu-Thr-AAsp-CHd
CH-CONPh) with a k2 value of 99200 M-1 s-1. Unfortunately,
this inhibitor also potently inactivates caspase-8 (k2 ) 245000
M-1 s-1). The order of reactivity with differing Y groups for
caspase-6 within the IETD sequence is as follows: CONHPh
> CO2Et (trans) > CON(CH2Ph)2 > CON(CH3)CH2Ph > CO2-
CH2Ph > CONHCH2CH2Ph > CONHCH2Ph. There is no
distinct preference for any substitution pattern at the caspase-6
S′ subsite. The monosubstituted amide derivative 20k is the most
preferred, but the disubstituted amide derivatives 20o, 20p, and
20r are also potent inhibitors. The other caspase-6 specific
sequence (VEVD) also yielded very potent inhibition. The
inhibitor 19h (Cbz-Val-Glu-Val-AAsp-CHdCH-COOEt) in-
hibits caspase-6 with a k2 value of 83600 M-1 s-1. However,
the VEVD sequence seems to be less selective. Not only does
it inhibits caspase-8 potently (k2 ) 175800 M-1 s-1), it also
Caspase-3. Considerably high rates were obtained for the
inhibitors with the caspase-3 specific DEVD sequence against
caspase-3. The most potent caspase-3 inhibitor was 18t (Cbz-
Asp-Glu-Val-AAsp-trans-CHdCH-CON(CH2-1-Naphth)2) with
a second-order inhibition rate of 5620000 M-1 s-1. With
caspase-3, the order of reactivity of the inhibitors with various
substituents on the double bond moiety is CON(CH2-1-Naphth)2
> CON(CH2Ph)2 > CON(CH3)CH2Ph > CON-tetrahydro-
quinoline > CO2Et (trans) > CONHCH2-4-F-Ph > CONHCH2-
CH2Ph > CONHCH2Ph > CO2CH2Ph > CON(CH3)CH2CH2Ph
> CO2Et (cis) > COPh. Generally, the bulky aromatic groups
were preferred in the prime site of this enzyme. The disubstituted
amide derivatives worked better than the monosubstituted ones
and the ester derivatives. Replacement of the amide or the ester
group by a carbonyl group caused a decrease in potency by
10-fold. The ketone 18f (Cbz-Asp-Glu-Val-AAsp-trans-CHd
CH-COPh) inhibits caspase-3 more slowly with a k2 value of
122000 M-1 s-1. The ester 18g, the inhibitor with the cis
configuration of the double bond moiety is a potent inhibitor
of caspase-3 (k2 ) 1060000 M-1 s-1). However, the trans ethyl
ester derivative 18h is twice as potent against caspase-3 (k2 )
2130000 M-1 s-1). Considerable decrease in potency was
observed with the EVD sequence based inhibitor 17h (k2 )
38600 M-1 s-1) with caspase-3, which emphasizes the impor-
tance of an acidic P4 residue for increased potency. In addition,
our caspase-3 inhibitors are equally if not more potent than
chloromethyl ketone inhibitors such as Cbz-DEVD-CMK which
inhibits caspase-3 with a k2 of 41200 M-1 s-1
.
Although the LETD sequence is determined to be the best
one for caspase-8, other caspase-specific sequences are also
preferred by this enzyme. The most potent caspase-8 inhibitor
is the one that is actually designed for caspase-3 with the DEVD
sequence. Hence, 18h (Cbz-Asp-Glu-Val-AAsp-trans-CHd
CH-COOEt) has a second-order rate constant of 272960 M-1
s-1 with caspase-8. Also, the inhibitor 20k with the IETD
sequence is a very potent caspase-8 inhibitor (k2 ) 245000 M-1
s-1). The same Y group (CONHPh) when attached to a LETD
sequence (21k) makes a less potent inhibitor with caspase-8
(k2 ) 176000 M-1 s-1). For the inhibitors with the LETD
sequence the order of reactivity with caspase-8 with differing
Y groups is as follows: COOEt (trans) > CON-tetrahydro-
quinoline > CON(CH3)CH2-1-Naphth > CONHPh > CON-
HCH2-4-F-Ph > CON(CH3)CH2Ph > CON(CH2Ph)2 > CONH-
CH2CH2Ph > COOCH2Ph > CON(CH3)CH2CH2Ph > CONH-
CH2Ph. The inhibitor 21h (Cbz-Leu-Glu-Thr-AAsp-trans-CHd
CH-COOEt) is the best one among this series with a k2 value
of 237000 M-1 s-1. The tetrahydroquinoline derivative 21u
(Cbz-Leu-Glu-Thr-AAsp-trans-CHdCH-CON-tetrahydroquin-
oline) is less potent than the ethyl ester derivative 21h, but it is
more selective over caspase-6. Interestingly, one of the most
potent caspase-8 inhibitors is the tripeptide derivative 17h (Cbz-
Glu-Val-AAsp-trans-CHdCH-COOEt) with a k2 value of
244000 M-1 s-1. In general, higher rates were obtained with
caspase-8 where Y is a small sized group such as ethyl and
when Y is not extending deeply into the S′ subsite.
has a k2/KI value of 1000000 M-1 s-1 11
. More importantly, the
aza-peptide Michael acceptors are much more selective (see the
Cross-Reactivity Section).
Caspase-7. The DEVD sequence worked also best with
caspase-7. The second-order rate constants were in the range
of 105 M-1 s-1. Cbz-Asp-Glu-Val-AAsp-trans-CHdCH-CON-
(CH2-1-Naphth)2 (18t) is the most potent inhibitor with a
second-order rate constant of 875000 M-1 s-1. The order of
reactivity with the substituted Y (Figure 3) group among the
DEVD series is as follows: CON(CH2-1-Naphth)2 > CON-
(CH2Ph)2 > CONHCH2-4-F-Ph > CON(CH3)CH2Ph > CONH-
CH2CH2Ph > CONHCH2Ph > CO2Et (trans) > CON(CH3)CH2-
CH2Ph > CO2Et (cis) > CON-tetrahydroquinoline > CO2CH2Ph
> COPh. Similar to caspase-3, bulky aromatic groups such as
benzyl or naphthyl groups which extend deep into the pocket
are preferred at the caspase-7 S′ subsite. There is no distinct
preference for disubstituted amide group preference as in the
case of caspase-3. However, amide substituents still work
generally better than the esters. The fluorophenyl derivative 18m
(Cbz-Asp-Glu-Val-AAsp-trans-CHdCH-CONHCH2-4-F-Ph)
is still the best monosubstituted amide inhibitor for both
caspase-3 and caspase-7. There is also a decrease in potency
with the tripeptide inhibitor 17h (k2 ) 52400 M-1 s-1) indicating
that the P4 amino acid is an important factor in potency with
caspase-7.
Caspase-9. The rates of inhibition of caspase-9 by the aza-
peptide Michael acceptors are not as high as the other members
of the caspase family we have tested. However, the LETD is
still the best sequence with rates in the range of 10-103 M-1
s-1. The best inhibitors are 21s and 21o with k2 values of 5030
M-1 s-1 and 4320 M-1 s-1, respectively. However, the most
potent caspase-9 inhibitor is the tripeptide derivative 17h (Cbz-
Glu-Val-AAsp-trans-CHdCH-COOEt) with a k2 value of
32900 M-1 s-1. Surprisingly, a considerable decrease in potency
is observed with the same Y group (ethyl) when attached to a
tetrapeptide sequence. The inhibitor 21h (Cbz-Leu-Glu-Thr-
AAsp-trans-CHdCH-COOEt) inhibits caspase-9 very weakly
(k2 ) 37 M-1 s-1).

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5733
Table 2. Cross Reactivity of Caspase Inhibitors with Clan CA and Other Clan CD Enzymesa
k2 (M-1 s-1
)
Inhibitor
Cathepsin B
Papain
Calpain
Legumain
Clostripain
Gingipain K
18h
18i
18l
21i
Cbz-Asp-Glu-Val-AAsp-CHdCH-COOEt
Cbz-Asp-Glu-Val-AAsp-CHdCH-COOCH2Ph
Cbz-Asp-Glu-Val-AAsp-CHdCH-CONHCH2Ph
Cbz-Leu-Glu-Thr-AAsp-CHdCH-COOCH2Ph
NI
NI
NI
<10
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
ND
ND
NI
ND
ND
<10
ND
ND
NI
a NI ) No inhibition, ND ) not determined. Enzymes: human liver cathepsin B, papaya latex papain, calpain I from porcine erythrocytes, S. mansoni
legumain, clostripain from Clostridium histolyticum, gingipain K from Porphyromonas gingiValis.
Caspase-10. The best inhibitors of caspase-10 have the
caspase-3 optimal sequence DEVD. The rates of inhibition are
in the range of 103-104 M-1 s-1 and the best inhibitor is Cbz-
Asp-Glu-Val-AAsp-trans-CHdCH-COOEt (18h, k2 ) 49900
M-1 s-1). The order of reactivity with differing Y groups with
the DEVD sequence is as follows: COOEt (trans) > CON-
HCH2-4-F-Ph > CON(CH2-1-Naphth)2 > CONHCH2CH2Ph >
CON(CH3)CH2Ph > COOCH2Ph > CONHCH2Ph > COOEt
(cis) > CON(CH3)CH2CH2Ph > CON-tetrahydroquinoline >
CON(CH2Ph)2. The tripeptide inhibitor 17h works equally well
as some of the tetrapeptide inhibitors with caspase-10 (k2 )
8940 M-1 s-1). Once again, this result indicates that with group
II caspases (6, 8, 9, 10) the absence of the P4 residue is not as
crucial as for group III caspases (2, 3, 7) for potency and
selectivity.
Caspase Selectivity. In general, the most potent inhibitors
of a target caspase were the ones that were designed with its
ideal substrate sequence, although caspases-8, -9, and -10 were
exceptions in some cases. Interestingly, the most specific
inhibitors while still very potent were not the most potent ones.
The most specific inhibitor for caspase-3 was Cbz-Asp-Glu-
Val-AAsp-trans-CHdCH-CON(CH2Ph)2 (18r, k2 ) 3000000
M-1 s-1), which is 27300, 588, 8.4, 348, 6670, and 476-fold
more selective for caspase-3 over caspases-2, -6, -7, -8, -9, and
-10, respectively. The most specific caspase-6 inhibitor was Cbz-
Ile-Glu-Thr-AAsp-trans-CHdCH-COOEt (20h, k2 ) 88,700
M-1 s-1). It is 296, 13.2, 167, 1.6, and 12.9-fold more selective
for caspase-6 over caspases-2, -3, -7, -8, and -10, respectively.
The most selective caspase-8 inhibitor was Cbz-Leu-Glu-Thr-
AAsp-trans-CHdCH-COOEt (21h, k2 ) 237000 M-1 s-1),
which is 42.6, 12.6, and 6400-fold more selective for caspase-8
over caspases-3, -6, and -9, respectively.
The selectivity of the aza-peptide Michael acceptor inhibitors
with the DEVD sequence for caspase-3 over caspase-7 is 6.9-
17-fold. Selectivity between caspase-3 and caspase-7 was hard
to achieve, since the DEVD sequence is ideal for both enzymes.
The selectivity of caspase-3 over caspase-2 is as high as 105
fold, because of the P5 requirement of caspase-2. The selectivity
for caspase-3 over caspase-6 is usually around 500-fold.
However, when one obtained selectivity over caspase-6, it was
difficult to obtain selectivity also for caspase-8. The range of
selectivity for caspase-3 over caspase-8 was only up to 37-fold,
except in the case of the inhibitor 18r. It has been previously
shown that the prime site of caspase-8 differs from that of the
other caspases by the presence of a helix-turn-helix insertion
ranging from residues 245-253, which is unique to caspase-
8.32 Clearly, we have taken advantage of this structural
difference by designing an inhibitor with a dibenzyl amide
substituent on the double bond moiety, which resulted in optimal
selectivity. Generally, caspase-10 behaved similarly to caspase-
8; however, it yielded less potent inhibitors. Last, the rates of
inhibition of caspase-9 were quite low when compared to the
other caspases (3, 6, 7, 8, and 10).
VD sequence in the hopes of obtaining a potent and broad
spectrum inhibitor for caspases. Due to enhanced aqueous
solubility, it has been previously shown that the fluoromethyl
ketone inhibitor Z-VD-fmk was more active than the tri- and
tetrapeptide based inhibitors such as Z-VAD-fmk in certain cell
culture models of apoptosis.33 Unfortunately, our dipeptide
inhibitor Cbz-Val-AAsp-trans-CHdCH-COOEt (16h) weakly
inactivated caspase-3 (k2 ) 8000 M-1 s-1), caspase-7 (k2 )
2680 M-1 s-1), caspase-9 (k2 ) 5260 M-1 s-1) and caspase-10
(k2 ) 389 M-1 s-1). It did not show any inhibition of caspases-
2, -6, or -8. The two amide derivatives 16j and 16l showed
little potency toward caspase-3 (k2 values are 184 M-1 s-1 and
185 M-1 s-1, respectively), caspase-7 (k2 values are 55 M-1
s-1 and 60 M-1 s-1, respectively), caspase-9 (k2 values are 32
M-1 s-1 and 20 M-1 s-1, respectively), but no activity against
caspases-2, -6, -8 or -10. The dipeptidyl aza-peptide Michael
acceptors with simple alkyl, alkenyl or halogen substituents
remained unreactive toward all the caspases (2, 3, 6, 7, 8, 9,
and 10). The decreased potency of the dipeptide derivatives
toward caspases is in accordance with the previous observation
of the requirement of at least tripeptides for effective binding
and recognition.
Cross Reactivity with Clan CA and Other Clan CD
Proteases. The aza-peptide Michael acceptors designed for
caspases show little to no inhibition of the clan CA cysteine
proteases papain, cathepsin B, and calpain, where the specificity
is determined primarily by the S2 subsite. The inhibitors 18h,
18i, 18l, and 21i showed essentially no inhibition against papain,
cathepsin B, and calpain after an incubation period of 1 h (Table
2).
We also tested the cross reactivity of our inhibitors with other
members of clan CD proteases such as legumain, clostripain,
and gingipain K. Legumain has a strict requirement for Asn at
P1, whereas clostripain and gingipain K prefer Orn and Lys at
the same position. Aza-peptide Michael acceptors designed for
caspases did not show any activity against those enzymes after
being incubated for 1 h. Likewise, the aza-peptide Michael
acceptor inhibitors with legumain, clostripain, and gingipain K
specific sequences did not show any cross reactivity with clan
CA enzymes or with each other.23 Clearly, the aza-peptide
Michael acceptor design is highly specific for clan CD enzymes.
Stability Studies. We tested the stability of the aza-peptide
Michael acceptors toward simple thiol nucleophiles such as thiol
DTT contained in the caspase assay buffer. A representative
group of inhibitors (2.5-4 mM) were treated with 10 mM DTT
in the caspase assay buffer with a pH of 7.2 at 25 °C. The
reactivity of the Michael acceptor double bond was monitored
by UV spectrum at 250 nm for a period of 15 h. The half-lives
were obtained from first-order rate plots of ln(At/A0) versus time.
We observed that the ester derivative 18h (Cbz-Asp-Glu-
Val-AAsp-CHdCH-COOEt) had a half-life of 10 min. The
amide substituted Michael acceptors appeared to be less reactive
toward DTT under the same conditions. The monosubstituted
amide derivative 18l (Cbz-Asp-Glu-Val-AAsp-CHdCH-
CONHCH2Ph) had a half-life of 58 min, where the disubstituted
In addition to the tri- and tetrapeptidyl aza-peptide Michael
acceptors we also made several dipeptidyl derivatives with the

5734 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
times faster than ethyl acrylamide with second-order rate
constants of 0.66 M-1 s-1 and 7.8 × 10-3 M-1 s-1, respectively.
Our results of the reactivity of the ester and amide substituted
inhibitors indicate a strong correlation with the observed
reactivity of acrylates and acrylamides with glutathione.
Mechanism of Inhibition. In general, the inactivation of
cysteine proteases by Michael acceptor inhibitors proceeds
through the nucleophilic attack of the active site cysteine thiol
group on the C3-carbon of the Michael acceptor double bond.
The mechanism of inhibition of caspases by aza-peptide Michael
acceptors could involve irreversible thioalkylation of the active
site Cys by the two pathways shown in Figure 4. Theoretically,
if the substituent on the double bond is an acyl derivative such
as ester or amide, the formation of both thioether adducts 22
and 23 is possible upon a nucleophilic attack taking place either
at the C-2 or C-3 carbon on the double bond moiety. If the
substituent is a simple alkyl or halogen, only the C-3 carbon is
the possible site for the Michael addition reaction.
Figure 4. Proposed mechanism of inhibition of caspases by aza-peptide
Michael acceptors.
amide derivative 18o (Cbz-Asp-Glu-Val-AAsp-CHdCH-CON-
(CH3)CH2Ph) was twice as long stable with a half-life of 116
min. The dienyl derivative 16b (Cbz-Val-AAsp-CHdCH-CHd
CH-CH3) was essentially stable over the period of 15 h of
monitoring.
Aza-peptide Michael acceptors designed for other clan CD
proteases such as legumain, gingipain, and clostripain specific
sequences were also tested for stability against DTT.23 The half-
life of the legumain inhibitor Cbz-Ala-Ala-AAsn-CHdCH-
COOEt in the legumain assay buffer (pH ) 5.8) with excess
DTT was 19 min. When tested at a higher pH (6.8) the half-
life of the same inhibitor was shortened to 3.7 min.
It should be noted that the pH of the assay buffer has a
considerable effect on the stability of the inhibitors against DTT.
The DTT thiol has a pKa value of 9.2. The lower the pH, the
lower is the concentration of the deprotonated thiol group of
DTT. Hence, a nucleophilic attack on the Michael acceptor
double bond is more favored at higher pH values.
We also anticipate that the nature of the P′ substituent on the
double bond contributes significantly to the overall stability of
the inhibitor. In general, the order of stability of our inhibitors
is as follows: alkyl > amide > ester. It has been previously
demonstrated by Freidig and co-workers that simple acrylates
are more reactive than simple acrylamides to thiol nucleophiles
such as glutathione.34 Ethyl acrylate reacts with glutathione 85
On the basis of the results of the NMR study and the stability
studies of our inhibitors with DTT, we previously hypothesized
that a nucleophilic attack of the active site thiol group at the
C-2 would be more favorable.23 However, to determine con-
clusively the point of attack, crystal structures of five caspase-
double bond complexes were determined.
X-ray Structure Analysis of Caspase-Inhibitor Com-
plexes. We have determined four crystal structures of caspase-
3-inhibitor complexes and the structure of one caspase-8-
inhibitor complex (Tables 3 and 4). The Michael acceptors used
for the structural studies with caspase-3 are the two stereoiso-
mers (cis and trans) of the ethyl ester derivative (18g and 18h),
a benzyl ester derivative (18i), and a N-methyl-N-benzyl amide
derivative (18o). We have determined the structure of caspase-8
complexed with 21t which is a tetrahydroisoquinoline derivative.
These inhibitor complexes are unique since they have inhibitor
moieties which interact with S′ subsites of the enzyme unlike
most caspase-inhibitor complex structures published to date.
The overall structures of the polypeptide chain in the
complexes are quite similar to the other known crystal structures
of the caspase family.35-38 The superposition of the CR atoms
Table 3. X-ray Data Collection Statistics of the Caspase:Michael Acceptor Complex Crystals
Caspase-3:
Cbz-DEVaD-
CHdCH-CO2Et
(trans) 18h
Caspase-3:
Cbz-DEVaD-
CHdCH-CO2Et
(cis) 18g
Caspase-3:
Cbz-DEVaD-
CHdCH-CO2Bzl
18i
Caspase-3:
Cbz-DEVaD-
Caspase-8:
Cbz-LETaD-
CHdCH-CON(CH3)Bzl
CHdCH-CO-tetrahydroquinoline
18o
21t
Resolution range (Å)
(last shell)
Unique reflections
Redundancy
20-1.76
20-1.86
20-1.93
20-2.45
10-1.95
(1.86-1.76)
25781 (3441)
4.3 (4.0)
(1.96-1.86)
22830 (2606)
5.0 (4.7)
(2.03-1.93)
19537 (2531)
3.3 (3.1)
(2.58-2.45)
10094 (1402)
5.8 (5.4)
(2.05-1.95)
21841 (1561)
12 (13)
R
merge (%)
5.4 (20.8)
95.3 (87.8)
19.1 (6.6)
7.6 (43.9)
97.8 (84.1)
17.3 (4.5)
7.8 (26.2)
94.3 (85)
12.8 (4.2)
7.9 (24.3)
99.2 (95.8)
19.6 (7.0)
5.2 (9.0)
98.5 (99.5)
36.4 (24.7)
Completeness (%)
Average I/σ
Table 4. Refinement Statistics of Caspase:Michael Acceptor Crystal Structures
Caspase-3:
Cbz-DEVaD-
CHdCH-CO2Et
(trans) 18h
Caspase-3:
Cbz-DEVaD-
CHdCH-CO2Et
(cis) 18g
Caspase-3:
Cbz-DEVaD-
CHdCH-CO2Bzl
18i
Caspase-3:
Cbz-DEVaD-
Caspase-8:
Cbz-LETaD-
CHdCH-CON(CH3) Bzl
CHdCH-CO-tetrahydroquinoline
18o
21t
Resolution (Å)
Rcryst
Rfree
20-1.76
15.9
18.4
20-1.86
18.4
21.5
20-1.93
16.1
19.7
20-2.45
16.8
21.0
10-1.95
16.8
20.1
Number of Atoms
Protein
Ligand
Water
1996
49
419
1996
49
303
1996
54
363
1996
55
303
1912
56
411
Average B-Factors
Protein (Å2)
11.94
18.98
33.61
2C1E
15.47
30.7
31.8
13.4
29.5
33.3
2C2M
19.73
42.5
27.0
17.3
23.1
37.0
2C2Z
Ligand (Å2)
Water (Å2)
PDB accession code
2C2K
2C2O

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5735
Figure 5. Stereoview of the atomic model of (a) caspase-3 in complex with the trans Cbz-Asp-Glu-Val-AAsp-CHdCH-CO2Et (18h) and (b)
caspase-8 in complex with Cbz-Leu-Glu-Thr-AAsp-CHdCH-CO-tetrahydroisoquinoline (21t) overlaided with an electron density map contoured
at 1.0 s. Potential hyrdogen bond interaction are drawn in brown dashed lines. The residues involved in interaction with the inhibitor and catalytic
dyad Cys163/His121 are highlighted. (c) Stereoview of the active site residues of caspase-3 in complex with the trans ethyl ester 18h (blue)
overlaided on top of structure of caspase-3:peptidyl aldehyde Ac-DEVD-CHO complex (green pdb code: 1PAU). The atoms of the inhibitor (18h)
are highlighted in yellow. The catalytic His121 of 18h complex is rotated approximately 20° about its Chi2 angle, which enables it to form a
hydrogen bond with P1-Asp and P1′ carbonyl groups.
of the subunits result in a root-mean-square deviation of 0.2 Å
for caspase-8 and 0.35 Å for caspase-3-inhibitor complexes.
This indicates that large scale conformational transitions do not
occur upon binding of the P′ portion of the inhibitor structure;
however, conformational changes do occur in parts of the
enzyme directly interacting with the inhibitor to improve the
fit. The interactions of the P1-P4 moieties of the Michael
acceptor inhibitors bind to the substrate binding cleft of the
caspase active site similar to the structures previously observed
in peptidyl aldehyde or peptidyl halogen methyl ketone inhibitor
complexes35-38 and closely mimick substrate binding from P1
to P4.
The high resolution crystal structures clearly demonstrate that
the nucleophilic attack takes place at the C-2 carbon more
precisely on the re face. This results in a covalent thioether
bond between the C-2 carbon and the sulfur atom of the active
site Cys residue. As a result of the nucleophilic attack, the
hybridization of both the C-2 and C-3 carbons change from sp2
to sp3 yielding a tetrahedral C-3 carbon and a chiral tetrahedral
C-2 carbon with the R-configuration (Figure 5a,b).
Caspase-3-Inhibitor Complexes. As observed in the crystal
structures of caspases complexed with halomethyl ketones,26,37
the P1 azapeptide carbonyl points toward the “oxyanion hole”
and it is stabilized by the amide protons of Gly122 and Cys163.
A unique feature of all the crystal structures of caspase-3
Michael acceptor complexes is the rotation of the catalytic
His121 about Chi2 by approximately 20 deg. In this new
orientation the imidazole ring is ideally placed to form a
hydrogen bond to the carboxylate of P1-Asp (Figure 5c). The
distance of this hydrogen bond ranges from 3.3 to 3.6 Å in our
complexes. This interaction between the catalytic His121 and
P1-Asp is not observed in any other caspase crystal structure
reported so far. Apart from this interaction, His121 also forms
a hydrogen bond to the azapeptide P1′ carbonyl. The interactions

5736 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
the P1′ moiety is a N-methyl-benzyl amide derivative. The
phenyl ring points toward the one side of the S1′ pocket and
stabilized by hydrophobic contacts with Thr166 and Tyr204
while the N-methyl group is stabilized by Phe128 (Figure 6b).
This is most reactive caspase-3 inhibitor of those studied
crystallographically. In the case of the benzyl derivative 18i,
the electron density for the phenyl ring is not well defined as
indicated by high B values. In the ethyl ester derivative formed
from the cis and trans isomers 18h and 18g, the ethyl group
interacts with the side chains of Phe128 and His121. To avoid
steric hindrance and in tandem to maintain the hydrophobic
contact with the ethyl group of the inhibitor, the side chain of
Met61 of the 179-loop adopts a different conformation. This
conformation is quite different from that observed in all other
known caspase-3 structures. As expected, the atoms of the
products formed from the two stereoisomers (18g and 18h) of
the ethyl ester overlay well on superposition. Additionally, in
both structures, the C-2 carbon adopts a R-configuration
indicating the attack is on the re face of the inhibitor (Figure
6a).
Caspase-8. Unlike in caspase-3:Michael acceptor inhibitor
complexes, in the caspase-8 complex the P1′ carbonyl is
stabilized by the side chain of P2-Thr instead of the catalytic
His317. The P4-Leu is stabilized in the manner similar to the
P4-Ile as exemplified in the crystal structure of caspase-8
complex with the peptidyl aldehyde Ac-IETD-CHO.36 The
phenyl ring of the capping residue Cbz in 21t stacks favorably
on top of Pro415, subsequently the puckering of this Pro is
different from the one which is observed in other caspase-8-
inhibitor complex structures.36,38,39
Figure 6. Surface representation of the active site of caspase-3 with
the active site and prime site residues are highlighted. (a) Superposition
of atoms of the ethyl ester inhibitor Cbz-Asp-Glu-Val-AAsp-CHdCH-
COOEt (18g, cis isomer in yellow) and the ethyl ester 18h (trans isomer
in green). (b) Superposition of atoms of the trans benzyl ester inhibitor
Cbz-Asp-Glu-Val-AAsp-CHdCH-COOCH2Ph 18i (in yellow) and the
N-methylbenzyl amide derivative Cbz-Asp-Glu-Val-AAsp-CHdCH-
CON(CH3)CH2Ph (18o (in green). The inhibitor 18i is able to form a
hydrogen bond with the catalytic His121 while 18o is not involved in
such an interaction.
The S1′ site of caspase-8 is different from all other caspases
due to the helix-turn-helix insertion in the 179-loop.38 We have
determined the structure of caspase-8 in complex with 21t. This
compound is a tetrahydroisoquinoline derivative in which the
tetrahydroisoquinoline occupies the hydrophobic S1′ pocket
defined by the residues Leu254, Ile257, and Tyr324 (Figure
7). As a consequence, this bulky hydrophobic moiety displaces
the 179-loop by about 1.0 Å when compared to other caspase-
8-inhibitor complexes. More importantly, significant changes
in the side chain conformation of Arg258 are observed. Arg258
recognizes P3-Glu, and its backbone carbonyl is critical for
maintaining a hydrogen bond with Nꢀ1 of the catalytic His317.
In the complex with 21t the side chain of Arg258 is pointing
toward Ser175 and in addition is involved in a hydrogen bond
with the backbone carbonyl of Ser175. Such conformational
of the P2 and P3 residues are similar to those reported in other
caspase-3 structures.35, 37
The P1′ site in caspase-3 is predominantly hydrophobic, and
it is defined by four loop regions. The 179-loop (between strand
â1 and helix R2), the 240-loop (between strand â3 and helix
R3), and the C-terminal loop belong to the alpha subunit,
whereas the fourth loop (between strand â4 and helix R3) is
from the â subunit. The approximate solvent accessible volume
of the prime site cavity is 900 Å3. In the Michael acceptor 18o,
Figure 7. Superposition of the crystal structure of complex of caspase-8 with the inhibitor Cbz-Leu-Glu-Thr-AAsp-CHdCH-CON-
tetrahydroquinoline (21t in blue) with the crystal structure of caspase-8:peptidyl aldehyde Ac-IETD-CHO (in green, pdb code 1QTN). Large scale
movement of the 179-loop apart from the different conformation of Arg 258 are depicted. The hydrogen bond between the P1′ carbonyl and the
side chain of P2-Thr is shown as a brown dashed line.

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5737
changes might cost binding energy, which in turn is compen-
sated by the formation of favorable hydrogen bonds or by
hydrophobic interactions. Such differences are particularly
observed in caspase-8, as it has a unique helix-turn-helix
insertion loop at that site.38
prefers smaller P′ substituents such as the ethyl group. Currently,
we are continuing to refine our design on the P′ site, where we
would like exploit the further possibilities for some polar
interactions in the prime site. By introducing substituents on
the double bond with a variety of different electron-withdrawing
characters, we are also hoping to improve the stability of aza-
peptide Michael acceptors toward thiol nucleophiles such as the
thiol DTT.
Aza-peptide Michael acceptors’ potency and selectivity for
caspases make them great candidates for potential use as probes
in cellular function studies and as drugs. Due to the strict
requirement of the negatively charged Asp residue at P1 and
the peptide chain itself, the hydrophobic character of our
inhibitors is significantly low. By introducing nonpeptide
moieties to replace the peptide backbone and shielding the side
chain carboxylic acid group with certain isosteres, one can
increase the hydrophobicity and hence the bioavailability of our
caspase inhibitors.
Stereochemistry. In our design of the aza-peptide Michael
acceptors the double bond moiety has two different substitutents.
One is the peptide chain and the other is the differing Y group,
which consists of various groups such as alkyl, alkenyl, aryl,
halogen, ester, or amide. Therefore, the double bond in aza-
peptide Michael acceptors has two isomers: the cis isomer and
the trans isomer. We focused our attention of making mainly
fumarate analogues. However, we made one inhibitor with the
cis configuration on the double bond moiety (18g). This inhibitor
turned out to be the most potent inhibitor of caspase-2 (k2 )
26200 M-1 s-1), when compared to the fumarate analogues.
18g was also quite potent with the other caspases as well (Table
1). It was less potent than the trans isomer 18h, but overall it
was more reactive than some of the fumarate analogues. The
weaker inhibition by the cis isomer (18g) compared to the trans
isomer (18h) could be due to an inherent difference in chemical
reactivity of the two isomers and/or to weaker binding of the
cis isomer to the enzyme. In an attempt to search for some
structural reasons for this behavior, we have cocrystallized both
the isomers with caspase-3. The crystal structure of the cis and
trans isomer complexes were determined at 1.76 Å and 1.86 Å
resolution, respectively. In the cis complex the B-factor values
of the inhibitor in general and P1′ atoms in particular are higher
than the trans complex, indicating higher mobility and/or lower
occupancy in the active site. Consequently, the difference in
electron density for the bound inhibitor in the active sites of
the caspase-3 cis complex is weaker than for the trans isomer.
Nonetheless the electron density maps were good enough to fit
the inhibitor model into the active site. The superposition of
the cis and the trans complexes reveals that the bound covalent
adducts overlap well with similar side chain rearrangements in
the two complex structures. From structural considerations alone
one would argue that both compounds bind with equal potency
which is confirmed by kinetic data (1060000 M-1 s-1 for the
cis and 2130000 M-1 s-1 for the trans isomer). But, with larger
P1′ substituents, there is likely to be a more significant difference
between the cis and trans isomers.
Experimental Section
Materials and Methods. Amino acid methyl esters were
purchased from Bachem Bioscience Inc., King of Prussia, PA.
Tripeptides were synthesized using standard coupling procedures
1
such as the mixed anhydride method. The H NMR spectra were
obtained using a Varian Mercury Vx 400 MHz spectrometer.
Electrospray ionization (ESI), fast-atom-bombardment (FAB) and
high-resolution mass spectrometry were performed using Micromass
Quattro LC and VG Analytical 70-SE instruments. Elemental
analysis was performed by Atlantic Microlab Inc., Norcross, GA.
Peptidyl Hydrazides. Anhydrous hydrazine (10 equiv) was
added to a stirred solution of a peptidyl methyl ester (1 equiv) in
MeOH and the reaction mixture was stirred vigorously for 16 h at
room temperature. Excess hydrazine and solvent were removed
under vacuum and the residue was washed with ether several times
to give the peptidyl hydrazide as a white solid. Compounds were
purified further on silica gel column using the eluent 2:18:5 MeOH:
CH2Cl2:EtOAc when needed. MS and 1H NMR (CDCl3 or DMSO-
d6) were consistent with the proposed structures.
Cbz-Val-NH-NH2, white solid, yield 92%; Cbz-Glu(O-tBu)-
Val-NH-NH2, white solid, yield 47-53%; Cbz-Asp(O-tBu)-Glu-
(O-tBu)-Val-NH-NH2, white solid, yield 43%; Cbz-Leu-Glu(O-
tBu)-Thr-NH-NH2, white solid, yield 92%; Cbz-Ile-Glu(O-tBu)-
Thr-NH-NH2, white solid, yield 91%; Cbz-Val-Glu(O-tBu)-Val-
NH-NH2, white solid, yield 75%.
Peptidyl-AA2-NH-NH-CH2COOtBu. General Procedure.
To a stirred solution of a peptidyl hydrazide (1 equiv) in DMF,
NMM (1.1 equiv) and neat tert-butyl bromoacetate (1.1 equiv) were
added dropwise at -15 °C and the mixture was allowed to react
for 16 h at room temperature. The solvent was removed under
vacuum and the residue was dried in vacuo. Purification on a silica
gel column with the proper eluent gave the product with yields of
18-68%. MS and 1H NMR (CDCl3 or DMSO-d6) were consistent
with the proposed structures.
Conclusion. Aza-peptide Michael acceptors with the Asp
residue at P1 are a novel class of inhibitors for caspases-2, -3,
-6, -7, -8, -9, and -10. The inhibitors have excellent inhibitory
potency and selectivity for clan CD cysteine proteases, where
the second-order rate constants are as high as 5620000 M-1
s-1. Aza-peptide Michael acceptors designed for caspases have
been tested with a variety of other cysteine proteases for cross
reactivity. They do not show cross reactivity with the clan CA
proteases such as papain, cathepsin B, and calpain. Aza-peptide
Michael acceptors designed for caspases show little to no cross
reactivity with legumain which has a specifity for Asn at P1.
They do not react with other clan CD proteases gingipain K
and clostripain with specificities Lys and Orn at P1, respectively.
Cbz-Val-NH-NH-CH2COOtBu was purified by column chro-
matography on silica gel using 1:20:4.2 MeOH:CH2Cl2:EtOAc as
1
the eluent; white solid, yield 64%. H NMR (DMSO-d6): 0.90 (t,
6H, Val), 1.40 (s, 9H, tBu), 1.86 (m, 1H, Val), 3.37 (d, 2H, NHCH2-
COOH), 3.72 (t, 1H, R-H), 4.99 (s, 2H, Cbz), 5.13 (d, 1H, NH),
7.30 (s, 5H, Ph), 9.38 (d, 1H, NH).
Cbz-Glu(O-tBu)-Val-NH-NH-CH2COOtBu was purified by
column chromatography on silica gel using 2:18:5 MeOH:CH2Cl2:
EtOAc as the eluent; white solid, yield 78%. MS (ESI) m/z 565.3
Analysis of our crystal structures reveals that the nucleophilic
attack takes place on the C2-carbon of the double bond; thus,
additional substitution might be tolerated in the C3-carbon. In
an effort to obtain greater selectivity among caspases-2, -3, -6,
-7, -8, -9, and -10, we modified the P′ substituent on the double
bond for each appropriate caspase sequence. Our results indicate
that caspases-3 and -7 can tolerate bulky and aromatic residues
in the prime site. Caspases-6 and -10 can accommodate a variety
of different substituents at the same position, whereas caspase-8
1
[(M + 1)+]. H NMR (CDCl3): 0.95 (t, 6H, Val), 1.49 (s, 18H,
tBu), 1.85-2.20 (m, 3H, Val and Glu), 2.21 (m, 2H, Glu), 3.45-
3.70 (m, 3H, NHCH2 and NHCH2), 4.25-4.30 (m, 2H, R-H), 5.05
(m, 2H, Cbz), 5.85 (d, 1H, NH), 7.05 (d, 1H, NH), 7.20-7.40 (m,
5H, Ph), 8.00 (m, 1H, NH).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-NH-NH-CH2COOtBu was
purified by column chromatography on silica gel using 2:18:5

5738 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
MeOH:CH2Cl2:EtOAc as the eluent, and then rechromatographed
using 1:9:5 MeOH:CH2Cl2:EtOAc as the eluent; white solid, yield
54%. 1H NMR (DMSO-d6): 0.81 (d, 6H, Val), 1.35-1.41 (t, 27H,
tBu), 1.71 (m, 1H, Val), 1.84 (m, 2H, Glu), 2.17 (m, 2H, Glu),
2.44 (dd, 1H, Asp), 2.49 (dd, 1H, Asp), 4.02 (t, 1H, R-H), 4.26-
4.35 (dm, 2H, R-H), 5.02 (q, 2H, Cbz), 7.32 (s, 5H, Ph), 7.61 (d,
1H, NH), 7.79 (d, 1H, NH), 7.91 (d, 1H, NH), 9.43 (d, 1H, NH).
Cbz-Ile-Glu(O-tBu)-Thr-NH-NH-CH2COOtBu was purified
by column chromatography on silica gel using 1:9 MeOH:CH2Cl2
as the eluent; white solid, yield 26%. MS (ESI) m/z 680 [(M +
1)+].1H NMR (DMSO-d6): 0.7-0.9 (t, 6H, Ile CH3), 0.9-1.0 (d,
3H, Thr CH3), 1-1.2 (m, 2H, Ile CH2), 1.3-1.5 (s, 18H, tBu),
1.6-1.8 (m, 2H, Ile CH and Glu CH2), 1.8-1.9 (m, 1H, Glu CH2),
2.1-2.3 (m, 2H, Glu CH2), 3.4 (d, 2H, NCH2), 3.9 (m, 2H, R-H),
4.1 (m, 1H, R-H), 4.35 (m, 1H, Thr CH-OH), 4.8 (d, 1H, NH),
5.03 (s, 2H, Cbz), 5.05 (d, 1H, NH), 7.3-7.4 (m, 5H, phenyl), 7.7
(d, 1H, NH), 8.05 (d, 1H, NH), 9.2 (s, 1H, NH).
1H, CHdCHCON), 7.14-7.31 (m, 2H, Ph), 7.32 (t, 2H, Ph), 7.66
(d, 2H, Ph), 10.47 (s, 1H, NH).
trans-3-Benzylcarbamoylacrylic Acid (HOOC-CHdCH-
CONHCH2Ph). To a stirred solution of benzylamine (1 equiv),
HOBt (1.1 equiv), and monoethyl fumarate (1.1 equiv) in DMF
was added EDC (1.1 equiv) and the mixture was allowed to react
for 16 h at room temperature. The DMF was removed under vacuum
and the residue was treated with EtOAc. The organic layer was
then washed with 2% citric acid, saturated NaHCO3, and brine.
The organic layer was dried over Na2SO4, filtered, and concentrated.
Column chromatography on silica gel with 1:1 EtOAc:hexanes as
the eluent gave the trans-3-benzylcarbamoylacrylic acid ethyl ester
as a white solid with a 63% yield. Hydrolysis of the ethyl ester
with NaOH (1.1 equiv) followed by acidic workup gave the trans-
1
3-benzylcarbamoylacrylic acid as a white solid in 65% yield. H
NMR (DMSO-d6): 4.37 (d, 2H, CH2Ph), 6.55 (d, 1H, CHdCH-
CONH), 6.93 (d, 1H, CHdCH-COOH), 7.29 (m, 5H, Ph), 8.96 (t,
1H, NH). In general, carbamoylacrylic acids were obtained in 60-
79% yield.
Cbz-Leu-Glu(O-tBu)-Thr-NH-NH-CH2COOtBu was puri-
fied by column chromatography on silica gel using 2:18:5 MeOH:
1
trans-3-(4-Fluorobenzylcarbamoyl)acrylic Acid (HOOC-
CHdCH-CONHCH2-4-F-Ph). trans-3-(4-Fluorobenzylcarbam-
oyl)acrylic acid ethyl ester was obtained by mixed anhydride
coupling of equimolar amounts of monoethyl fumarate and 4-fluo-
robenzylamine to give a pink solid (66% yield). EtOOC-CHd
CH-CONHCH2-4-F-Ph was hydrolyzed in MeOH using NaOH
(1M aqueous, 1.1 equiv) under standard deblocking conditions to
CH2Cl2:EtOAc as the eluent, white solid, yield 68%. H NMR
(DMSO-d6): 0.84 (t, 6H, Leu), 0.98 (d, 3H, Thr), 1.37-1.44 (m,
20H, tBu and Leu), 1.62-1.73 (dm, 2H, Glu), 1.88 (m, 1H, Leu),
2.22 (m, 2H, Glu), 3.94 (m, 1H, R-H), 4.03-4.13 (dm, 2H, R-H),
4.37 (m, 1H, Thr), 4.99 (s, 2H, Cbz), 6.65 7.33 (m, 5H, Ph), 7.42
(d, 1H, NH), 7.63 (d, 1H, NH), 8.03 (d, 1H, NH), 9.26 (s, 1H,
NH).
1
give a clear, colorless syrup (45% yield). H NMR (DMSO-d6):
Cbz-Val-Glu(O-tBu)-Val-NH-NH-CH2COOtBu was purified
by column chromatography on silica gel using 1:9:5 MeOH:CH2-
Cl2:EtOAc as the eluent, and then rechromatographed using 1:9:5
4.34-4.35 (d, 2H, N-CH2-Ph), 6.52-6.55 (d, 1H, J ) 15.2 Hz,
CHdCHCON), 6.93-6.94 (d, 1H, J ) 15.6 Hz, CHdCHCON),
7.14-7.16 (t, 2H, Ph), 7.27-7.31 (t, 2H, Ph), 8.99 (t, 1H, NH).
trans-3-Phenethylcarbamoylacrylic Acid (HOOC-CHdCH-
CONHCH2CH2Ph). Monoethyl fumarate (1 equiv) was dissolved
in dry THF and cooled to -15 °C. NMM (1 equiv) and IBCF were
added dropwise and the mixture was allowed to react for 20 min.
Phenethylamine was added to the above mixture at -15 °C. The
mixture was stirred for 1 h at -15 °C and was allowed to react for
16 h at room temperature. The solvent was evaporated and the
residue was treated with EtOAc and H2O. The organic layer was
washed with 1 M HCl, saturated NaHCO3, and brine, dried over
Na2SO4, filtered, and concentrated to give trans-3-phenethylcar-
bamoylacrylic acid ethyl ester. Hydrolysis of the ethyl ester with
NaOH (1.1 equiv) followed by acidic workup gave the trans-3-
phenethylcarbamoylacrylic acid as a yellow solid in 79% yield. 1H
NMR (DMSO-d6): 2.75 (t, 2H, NHCH2CH2Ph), 3.38 (q, 2H,
NHCH2CH2Ph), 6.55 (d, 1H, CHdCH-CONH), 6.98 (d, 1H, CHd
CH-COOH), 7.20 (m, 5H, Ph), 8.59 (t, 1H, NH).
1
MeOH:CH2Cl2:EtOAc as the eluent; white solid, yield 18%. H
NMR (DMSO-d6): 0.78-0.85 (dd, 12H, Val), 1.40 (d, 18H, tBu),
1.71 (m, 1H, Val), 1.83 (m, 2H, Glu), 1.91 (m, 1h, Val), 2.17 (m,
2H, Glu), 3.86 (t, 1H, R-H), 4.04 (t, 1H, R-H), 4.32 (m, 1H, R-H),
5.01 (s, 2H, Cbz), 5.12 (m, 1H, NH), 7.33 (s, 5H, Ph), 7.76 (d, 1H,
NH), 8.00 (d, 1H, NH), 9.43 (d, 1H, NH).
Monoethyl Maleate (HOOC-CHdCH-COOEt). Monoethyl
maleate was obtained as a colorless oil from the reaction of maleic
anhydride with absolute ethanol at room temperature for 18 h (yield
) 86%).40 1H NMR (DMSO-d6): 1.19 (t, 3H, OCH2CH3), 4.11 (q,
2H, OCH2CH3), 6.33 (s, 2H, CHdCH).
trans-3-Benzyloxycarbonylacrylic Acid (HOOC-CHdCH-
COOCH2Ph). Equimolar amounts of fumaric acid and benzyl
alcohol were dissolved in anhydrous DMF. NMM (1 equiv) was
added at 0 °C followed by EDC after 15 min. The reaction was
stirred overnight at room temperature. DMF was evaporated and
the crude residue was redissolved in EtOAc. The product was
extracted with saturated aqueous NaHCO3. The aqueous layer was
then acidified with 1N HCl to pH 2. The product was extracted
with EtOAc, and the organic layer was washed with water and dried
(MgSO4). The solvent was evaporated and the crude residue was
subjected to column chromatography (MeOH/CH2Cl2) to give a
white powder (51% yield). 1H NMR (DMSO-d6): 5.21 (s, 2H, CHd
CH-COOCH2Ph), 6.73 (s, 2H, CH)CH-COOCH2Ph), 7.29-7.43
(m, 5H, Ph). MS (ESI) m/z 207 [(M + 1)+].
trans-3-Benzylmethylcarbamoylacrylic Acid (HOOC-CHd
CH-CON(CH3)CH2Ph). trans-3-Benzylmethylcarbamoylacrylic
acid ethyl ester was obtained by mixed anhydride coupling of
equimolar amounts of monoethyl fumarate and N-methylbenzyl-
amine to give a clear, pink syrup (76% yield). EtOOC-CHdCH-
CON(CH3)CH2Ph was hydrolyzed in MeOH using NaOH (1M
aqueous, 1.1 equiv) under standard deblocking conditions to give
1
a clear, colorless syrup (77% yield). H NMR (CDCl3): 3.03 (s,
3H, N-CH3), 4.62-4.67 (d, 2H, N-CH2-Ph), 6.83-6.87 (d, 1H,
J ) 16 Hz, CHdCHCON), 7.15-7.17 (d, 1H, J ) 8 Hz, CHd
CHCON), 7.25-7.50 (m, 5H, Ph).
trans-3-Butylcarbamoylacrylic Acid (HOOC-CHdCH-
CONH-nBu). This compound was obtained by EDC/HOBt cou-
pling in CHCl3 followed by standard NaOH hydrolysis (white solid,
trans-3-(Methylphenethylcarbamoyl)acrylic Acid (HOOC-
CHdCH-CON(CH3)CH2CH2Ph). trans-3-(Methylphenethylcar-
bamoyl)acrylic acid ethyl ester was obtained by mixed anhydride
coupling of equimolar amounts of monoethyl fumarate and N-
methylphenethylamine to give a clear colorless syrup (64% yield).
EtOOC-CHdCH-CON(CH3)CH2CH2Ph was hydrolyzed in MeOH
using NaOH (1M aqueous, 1.1 equiv) under standard deblocking
1
yield ) 60%). H NMR (DMSO-d6): 0.86 (t, 3H, NHCH2CH2-
CH2CH3), 1.28 (m, 2H, NHCH2CH2CH2CH3), 1.41 (m, 2H,
NHCH2CH2CH2CH3), 3.12 (m, 2H, NHCH2CH2CH2CH3), 6.49 (d,
1H, CHdCH-CONH), 6.87 (d, 1H, CHdCH-COOH), 8.43 (t, 1H,
NH).
trans-3-Phenylcarbamoylacrylic Acid (HOOC-CHdCH-
CONHPh). trans-3-Phenylcarbamoylacrylic acid ethyl ester was
obtained by mixed anhydride coupling of equimolar amounts of
monoethyl fumarate and aniline to give a white solid (59% yield).
EtOOC-CHdCH-CONHPh was hydrolyzed in MeOH using
NaOH (1M aqueous, 1.1 equiv) under standard deblocking condi-
tions to give a white solid (81% yield). 1H NMR (DMSO-d6):
6.61-6.65 (d, 1H, J ) 15.2 Hz, CHdCHCON), 7.02-7.14 (m,
1
conditions to give a clear, colorless syrup (81% yield). H NMR
(DMSO-d6): 2.82 (s, 3H, N-CH3), 3.54 (t, 2H, N-CH2-CH2-
Ph), 3.61 (t, 2H, N-CH2-CH2-Ph), 6.43-6.47 (d, 1H, J ) 15.2
Hz, CHdCHCON), 6.98-6.7.02 (d, 1H, J ) 15.6 Hz, CHd
CHCON), 7.14-7.31 (m, 5H, Ph).
trans-3-Dibenzylcarbamoylacrylic Acid (HOOC-CHdCH-
CON(CH2Ph)2). trans-3-Dibenzylcarbamoylacrylic acid ethyl ester

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5739
was obtained by mixed anhydride coupling of equimolar amounts
of monoethyl fumarate and dibenzylamine to give a clear, pink
syrup (87% yield). EtOOC-CHdCH-CON(CH2Ph)2 was hydro-
lyzed in MeOH using NaOH (1M aqueous, 1.1 equiv) under
standard deblocking conditions to give a clear, colorless syrup which
chromatography on silica gel with 0.5:49.5:50 MeOH:CH2Cl2:
EtOAc as the eluent; white solid, yield 8%. MS (ESI) m/z 474.3
[(M + 1)+]. 1H NMR (DMSO-d6): 0.83-0.89 (dd, 6H, Val), 1.39
(s, 9H, tBu), 1.75 (d, 3H, CH3), 1.92 (m, 1H, Val), 3.83 (t, 1H,
R-H), 5.02 (q, 2H, Cbz), 6.14 (m, 2H, db), 6.40 (m, 1H, db), 7.09
(m, 1H, db), 7.33 (s, 5H, Ph), 7.58 (d, 1H, NH), 10.77 (s, 1H,
NH-N).
Cbz-Val-AAsp(O-tBu)-CHdCH-CH2CH2Ph was obtained by
the EDC/HOBt coupling method, and purified by column chroma-
tography on silica gel with 0.5:49.5:50 MeOH:CH2Cl2:EtOAc as
the eluent; white solid, yield 53%. MS (ESI) m/z 538.3 [(M + 1)+].
Cbz-Val-AAsp(O-tBu)-cis-CHdCH-Cl was obtained by the
EDC/HOBt coupling method, and purified by column chromatog-
raphy on silica gel with 0.5:49.5:50 MeOH:CH2Cl2:EtOAc as the
eluent; white solid, yield 25%. MS (ESI) m/z 468.4 [(M + 1)+].
1H NMR (DMSO-d6): 0.89 (d, 6H, Val), 1.43 (s, 9H, tBu), 1.95
(m, 1H, Val), 3.84 (t, 1H, R-H), 5.04 (s, 2H, Cbz), 6.59 (s, 1H,
db), 6.81 (d, 1H, db), 7.35 (s, 5H, Ph), 7.56 (d, 1H, NH), 10.81 (s,
1H, NH-N).
1
was recrystallized overnight from hexane/EtOAc (30% yield). H
NMR (DMSO-d6): 4.57 (s, 2H, N-CH2-Ph), 4.65 (s, 2H, N-CH2-
Ph), 6.59-6.63 (d, 1H, J ) 15.2 Hz, CHdCHCON), 7.15-7.17
(d, 1H, J ) 8 Hz, CHdCHCON), 7.25-7.50 (m, 10H, 2 ? Ph).
trans-3-(3,4-Dihydro-2H-quinolin-1-ylcarbonyl)acrylic Acid
(HOOC-CHdCH-CO-tetrahydroquinoline). trans-3-(3,4-Di-
hydro-2H-quinolin-1-ylcarbonyl)acrylic acid ethyl ester was ob-
tained by mixed anhydride coupling of equimolar amounts of
monoethyl fumarate and 1,2,3,4-tetrahydroquinoline to give a brown
syrup (83% yield). EtOOC-CHdCH-CO-tetrahydroquinoline was
hydrolyzed in MeOH using NaOH (1M aqueous, 1.1 equiv) under
standard deblocking conditions to give a clear syrup, which was
recrystallized using cold EtOAc to give a yellow powder (68%
yield).
trans-3-(Methyl-1-naphthylmethylcarbamoyl)acrylic Acid
(HOOC-CHdCH-CON(CH3)CH2-1-Naphth). trans-3-(Methyl-
1-naphthylmethylcarbamoyl)acrylic acid ethyl ester was obtained
by mixed anhydride coupling of equimolar amounts of monoethyl
fumarate and N-methyl-1-naphthylmethylamine hydrochloride to
give a clear, colorless syrup (99% yield). EtOOC-CHdCH-CON-
(CH3)CH2-1-Naphth was hydrolyzed in MeOH using NaOH (1M
aqueous, 1.1 equiv) under standard deblocking conditions to give
a white solid after recrystallization from cold EtOAc (13% yield).
1H NMR (DMSO-d6): 3.01 (s, 3H, CH3), 5.01 (s, 2H, CH2), 6.61-
6.65 (d, 1H, J ) 15.2 Hz, CHdCHCON), 7.17-7.21 (d, 1H, CHd
CHCON), 7.37-7.60 (m, 4H, naphthyl), 7.85-8.01 (m, 3H,
naphthyl).
Cbz-Val-AAsp(O-tBu)-CHdCH-Ph-4-Cl was obtained by the
EDC/HOBt coupling method, and purified by column chromatog-
raphy on silica gel with 0.5:49.5:50 MeOH:CH2Cl2:EtOAc as the
eluent; white solid, yield 62%. MS (ESI) m/z 544.3 [(M + 1)+].
1H NMR (DMSO-d6): 0.86-0.93 (dd, 6H, Val), 1.41 (s, 9H, tBu),
1.98 (m, 1H, Val), 3.80 (t, 1H, R-H), 5.03 (q, 2H, Cbz), 7.30 (s,
5H, Ph), 7.40 (d, 1H, db), 7.52 (d, 1H, db), 7.76 (m, 4H, Ph-Cl),
10.95 (s, 1H, NH-N).
Cbz-Val-AAsp(O-tBu)-CHdCH-CONH-nBu was obtained by
the EDC/HOBt coupling method, and purified by column chroma-
tography on silica gel with 1:9:10 MeOH:CH2Cl2:EtOAc as the
eluent; white solid, yield 53%. MS (ESI) m/z 533.2 [(M + 1)+].
1H NMR (DMSO-d6): 0.83-0.87 (m, 9H, Val and NHCH2CH2-
CH2CH3), 1.27 (m, 2H, NHCH2CH2CH2CH3), 1.41 (s, 11H, tBu
and NHCH2CH2CH2CH3), 1.96 (m, 1H, Val), 3.12 (dd, 2H, NHCH2-
CH2CH2CH3), 3.90 (t, 1H, R-H), 5.03 (q, 2H, Cbz), 6.91 (d, 1H,
db), 7.08 (d, 1H, db), 7.28-7.34 (m, 5H, Ph), 7.47 (d, 1H, NH),
8.37 (t, 1H, NHCH2CH2CH2CH3), 10.93 (s, 1H, NH-N).
trans-3-Di-1-Naphthylcarbamoylacrylic Acid (HOOC-CHd
CH-CON(CH2-1-Naphth)2). trans-3-Di-1-Naphthylcarbamoyl-
acrylic acid ethyl ester was obtained by mixed anhydride coupling
of equimolar amounts of monoethyl fumarate and di-1-naphthyl-
amine(Ref.) to give a clear, yellow syrup (71% yield). EtOOC-
CHdCH-CON(CH2-1-Naph)2 was hydrolyzed in EtOH using
NaOH (1M aqueous, 1.5 equiv) under standard deblocking condi-
Cbz-Val-AAsp(O-tBu)-CHdCH-CONHCH2Ph was obtained
by the EDC/HOBt coupling method, and purified by column
chromatography on silica gel with 1:49:50 MeOH:CH2Cl2:EtOAc
as the eluent; white solid, yield 72%. MS (ESI) m/z 567.4 [(M +
1
tions to give a light tan solid. (52% yield). H NMR (DMSO-d6):
5.21 (s, 4H, N-CH2-1-Naph), 6.61-6.62 (d, 1H, J ) 1.2 Hz, CHd
CHCON), 6.69-6.72 (d, 1H, J ) 14 Hz, CHdCHCON), 7.17-
8.11 (m, 14H, N(CH2-1-Naph)2).
1
1)+]. H NMR (DMSO-d6): 0.85 (d, 6H, Val), 1.40 (s, 9H, tBu),
1.96 (m, 1H, Val), 3.90 (t, 1H, R-H), 4.33 (d, 2H, NHCH2Ph), 5.00
(s, 2H, Cbz), 6.97 (d, 1H, db), 7.14-7.32 (m, 11H, Ph and db),
7.49 (d, 1H, NH), 8.90 (t, 1H, NHCH2Ph), 10.92 (s, 1H, NH-N).
Cbz-Val-AAsp(O-tBu)-CHdCH-COOEt. EDC/HOBt Coup-
ling Method. To a stirred solution of Cbz-Val-NH-NH-CH2-
COOtBu (0.34 mM, 1 equiv), HOBt (0.37 mM, 1.1 equiv), and
monoethyl fumarate (0.37 mM, 1.1 equiv) in DMF was added EDC
(0.37 mM, 1.1 equiv) and the mixture was allowed to react for 16
h at room temperature (2 eq of EDC/HOBt were used with the
tetrapeptides). The DMF was removed under vacuum and the
residue was treated with 10 mL EtOAc. The organic layer was then
washed with 2 × 10 mL 2% citric acid, 1 × 15 mL saturated
NaHCO3, and 2 × 10 mL brine. The organic layer was dried over
Na2SO4, filtered, and concentrated. Column chromatography on
silica gel with 1:49:50 MeOH:CH2Cl2:EtOAc as the eluent gave
Mixed Anhydride Coupling Method. Coupling of some of the
bulky peptides, such as Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-NH-
NH-CH2COO-tBu and Cbz-Leu-Glu(O-tBu)-Thr-NH-NH-CH2-
COO-tBu, with the monoethyl fumarate, was accomplished using
the mixed anhydride coupling method. To a solution of the
monoethyl fumarate (5 equiv) in DMF at 0 °C was added
N-methylmorpholine (NMM, 5 equiv) followed by isobutyl chlo-
roformate (IBCF, 5 equiv). After the reaction mixture was allowed
to stir for 30 min, the substituted hydrazide (1 equiv) dissolved in
DMF, was added to the mixture. After 10 min the ice bath was
removed and the reaction mixture was stirred for 16 h at room
temperature. The DMF was evaporated and the residue was washed
and purified using the same procedure as described above for the
EDC/HOBt coupling method. MS and 1H NMR (DMSO-d6, CDCl3
or acetone-d6) were consistent with the proposed structures.
1
the product as a white solid with 63% yield. MS and H NMR
(DMSO-d6) were consistent with the proposed structure. MS (ESI)
m/z 506.3 [(M + 1)+]. 1H NMR (CDCl3): 0.97-1.02 (dd, 6H, Val),
1.28 (t, 3H, OCH2CH3), 1.46 (s, 9H, tBu), 2.18 (m, 1H, Val), 3.98
(t, 1H, R-H), 4.21 (q, 2H, OCH2CH3), 5.11 (s, 2H, Cbz), 5.21 (d,
1H, NH), 6.86 (d, 1H, db), 7.33 (s, 6H, Ph and db), 8.49 (s, 1H,
NH-N).
Cbz-Val-AAsp(O-tBu)-CHdCH-CH3 was obtained by the
EDC/HOBt coupling method, and purified by column chromatog-
raphy on silica gel with 0.5:49.5:50 MeOH:CH2Cl2:EtOAc as the
eluent; white solid, yield 53%. MS (ESI) m/z 448.4 [(M + 1)+].
1H NMR (DMSO-d6): 0.85-0.89 (dd, 6H, Val), 1.39 (s, 9H, tBu),
1.94 (m, 1H, Val), 3.83 (t, 1H, R-H), 5.02 (q, 2H, Cbz), 6.41 (m,
1H, db), 6.72 (m, 1H, db), 7.32 (s, 5H, Ph), 7.58 (d, 1H, NH).
Cbz-Val-AAsp(O-tBu)-CHdCH-CHdCH-CH3 was ob-
tained by the EDC/HOBt coupling method, and purified by column
Cbz-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-COOEt was ob-
tained using the mixed anhydride method and was purified using
column chromatography on silica gel using 1:9:10 MeOH:CH2Cl2:
1
EtOAc as the eluent, white solid, yield 49%. H NMR (DMSO-
d6): 0.90 (m, 6H, Val), 1.20 (t, 3H, OCH2CH3), 1.41 (m, 18H,
tBu), 1.60-2.00 (m, 3H, Val, Glu), 2.21 (m, 2H, Glu), (m, 4H,
NCH2COOH, and OCH2CH3), 4.20-4.40 (m, 2H, R-H), 5.05 (m,
2H, Cbz), 7.10 (-CO-CHdCH-COOEt), 7.20-7.40 (m, 5H, Ph),
8.00 (m, 2H, NH). ESI (M+1) Calcd. for C34H51N4O11: 691.35
Observed m/z 691.30.

5740 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
COPh was obtained by the EDC/HOBt coupling method, and
purified by column chromatography on silica gel with 1:499:500
MeOH:CH2Cl2:EtOAc as the eluent; white solid, yield 40%. MS
graphed using 1:1 EtOAc:CH2Cl2 as the eluent; white solid, yield
29%. MS (ESI) m/z 937.5 [(M + 1)+]. 1H NMR (DMSO-d6): 0.85
(t, 6H, Val), 1.35-1.41 (m, 27H, tBu), 1.72 (m, 1H, Glu), 1.86
(m, 1H, Glu), 2.00 (m, 1H, Val), 2.17 (m, 2H, Glu), 2.43 (dd, 1H,
Asp), 2.61-2.64 (dd, 1H, Asp), 2.73 (t, 2H, NHCH2CH2Ph), 3.35
(q, 2H, NHCH2CH2Ph), 4.17 (t, 1H, R-H), 4.29-4.35 (m, 2H, R-H),
5.02 (q, 2H, Cbz), 6.89 (d, 1H, db), 7.07 (d, 1H, db), 7.17-7.31
(m, 10H, Ph), 7.59 (d,1H, NH), 7.94 (d, 1H, NH), 8.51 (t, 1H,
NHCH2CH2Ph), 10.95 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CON(CH3) CH2Ph was obtained by the EDC/HOBt coupling
method, and purified by column chromatography on silica gel with
1:9:5 MeOH:CH2Cl2:EtOAc as the eluent, and then rechromato-
graphed using 1:1 EtOAc:hexanes as the eluent; white solid, yield
34%. MS (ESI) m/z 937.5 [(M + 1)+]. 1H NMR (DMSO-d6): 0.86
(t, 6H, Val), 1.34-1.41 (m, 27H, tBu), 1.65 (m, 1H, Glu), 1.88
(m, 1H, Glu), 1.97 (m, 1H, Val), 2.17 (m, 2H, Glu), 2.43 (dd, 1H,
Asp), 2.59 (dd, 1H, Asp), 2.88-2.98 (d, 3H, N(CH3)CH2Ph), 4.16
(t, 1H, R-H), 4.29-4.34 (dm, 2H, R-H), 4.54 (q, 1H, N(CH3)CH2-
Ph), 4.67 (s, 1H, N(CH3)CH2Ph), 5.02 (q, 2H, Cbz), 7.14 (d, 1H,
db), 7.20 (d, 1H, db), 7.28-7.37 (m, 10H, Ph), 7.56 (d,1H, NH),
7.94 (t, 1H, NH), 10.97 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CON(CH3)CH2CH2Ph was obtained by the EDC/HOBt coupling
method, and purified by column chromatography on silica gel with
1:19:20 MeOH:CH2Cl2:EtOAc as the eluent, and then rechromato-
graphed using 1:1 EtOAc:CH2Cl2 as the eluent; white solid, yield
45%. MS (ESI) m/z 951.5 [(M + 1)+]. 1H NMR (acetone-d6): 0.86
(t, 6H, Val), 1.41-1.48 (m, 27H, tBu), 1.75 (m, 1H, Glu), 1.89
(m, 1H, Glu), 2.01 (m, 1H, Val), 2.21 (m, 2H, Glu), 2.46 (dd, 1H,
Asp), 2.62 (dd, 1H, Asp), 2.80 (t, 2H, N(CH3)CH2CH2Ph), 2.85-
2.98 (d, 3H, N(CH3)CH2CH2Ph), 3.73 (m, 2H, N(CH3)CH2CH2-
Ph), 4.18 (t, 1H, R-H), 4.32 (dm, 2H, R-H), 5.00 (q, 2H, Cbz),
6.88 (t, 1H, NH), 7.17-7.34 (m, 12H, Cbz-Ph, N(CH3)CH2CH2Ph,
and CHdCH), 7.59 (d, 1H, NH), 7.85 (d, 1H, NH), 7.97 (d, 1H,
NH), 9.89 (d, 1H, NH-N).
1
(ESI) m/z 894.4 [(M + 1)+]. H NMR (DMSO-d6): 0.82 (t, 6H,
Val), 1.34-1.42 (m, 27H, tBu), 1.70 (m, 1H, Glu), 1.85 (m, 1H,
Glu), 1.96 (m, 1H, Val), 2.15 (m, 2H, Glu), 2.44 (dd, 1H, Asp),
2.58-2.60 (dd, 1H, Asp), 4.14 (t, 1H, R-H), 4.26-4.33 (dm, 2H,
R-H), 5.02 (q, 2H, Cbz), 7.23 (d, 1H, db), 7.31 (s, 5H, Cbz), 7.51
(m, 3H, Ph), 7.66 (t,1H, NH), 7.80 (d, 1H, db), 7.99 (d, 2H, Ph),
11.02 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-cis-CHdCH-
COOEt was obtained using the EDC/HOBt coupling method from
monoethyl maleate, purified by column chromatography on silica
gel with 1:19:20 MeOH:CH2Cl2:EtOAc as the eluent; white solid,
1
yield 53%. MS (ESI) m/z 862.5 [(M + 1)+]. H NMR (DMSO-
d6): 0.85 (d, 6H, Val), 1.18 (t, 3H, OCH2CH3), 1.33-1.39 (m, 27H,
tBu), 1.74 (m, 1H, Glu), 1.86 (m, 1H, Glu), 1.95 (m, 1H, Val),
2.18 (m, 2H, Glu), 2.45 (dd, 1H, Asp), 2.61 (dd, 1H, Asp), 4.09 (t,
1H, R-H), 4.16 (q, 2H, OCH2CH3), 4.33 (m, 2H, R-H), 5.01 (q,
2H, Cbz), 6.61 (d, 1H, db), 7.25 (d, 1H, db), 7.33 (s, 5H, Ph), 7.62
(d, 1H, NH), 7.93 (t, 2H, NH), 11.05 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-trans-CHd
CH-COOEt was obtained using the EDC/HOBt coupling method
from monoethyl fumarate, purified by column chromatography on
silica gel with 50:45:5 CH2Cl2:EtOAc:MeOH as the eluent; white
1
solid, yield 68%. H NMR (CDCl3): 1.00 (d, 6H, Val CH3), 1.29
(t, 3H, OEt), 1.45 (s, 27H, t-Bu), 1.90 (m, 1H, Val CH), 2.10 (d of
m, 2H, Glu CH2), 2.40 (d of m, 2H, Glu CH2), 2.80 (d of m, 2H,
Asp CH2), 3.20 (s, 2H, AAsp CH2), 4.19 (q, 3H, OEt and R-H),
4.30 (b, 1H, R-H), 4.45 (b, 1H, R-H), 5.12 (q, 2H, Cbz), 6.05 (d,
1H, NH), 6.81 and 7.20 (d of d, 2H, CHdCH), 7.33 (m, 6H, Ph
and NH), 7.90 (d, 1H, NH), 9.00 (s, 1H, NH).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
COOCH2Ph was obtained by the EDC/HOBt coupling method,
and purified by column chromatography on silica gel with 1:499:
500 MeOH:CH2Cl2:EtOAc as the eluent; white solid, yield 49%.
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CON(CH2Ph)2 was obtained by the EDC/HOBt coupling method,
and purified by column chromatography on silica gel with 2:1
EtOAc:hexane as the eluent, and then rechromatographed using 1:1
EtOAc:hexanes as the eluent; white solid, yield 62%. MS (ESI)
1
MS (ESI) m/z 924.5 [(M + 1)+]. H NMR (DMSO-d6): 0.80 (t,
6H, Val), 1.35-1.41 (m, 27H, tBu), 1.75 (m, 1H, Glu), 1.84 (m,
1H, Glu), 1.94 (m, 1H, Val), 2.17 (m, 2H, Glu), 2.45 (dd, 1H, Asp),
2.60 (dd, 1H, Asp), 4.13 (t, 1H, R-H), 4.28-4.35 (dm, 2H, R-H),
5.02 (q, 2H, Cbz), 5.19 (s, 2H, Bzl), 6.70 (d, 1H, db), 7.25-7.37
(m, 11H, Ph and db), 7.57 (d,1H, NH), 7.92 (d, 1H, NH), 7.98 (d,
1H, NH), 11.02 (s, 1H, NH-N).
1
m/z 1013.4 [(M + 1)+]. H NMR (DMSO-d6): 0.87 (t, 6H, Val),
1.35-1.39 (m, 27H, tBu), 1.73 (m, 1H, Glu), 1.87 (m, 1H, Glu),
2.02 (m, 1H, Val), 2.23 (m, 2H, Glu), 2.48 (dd, 1H, Asp), 2.65
(dd, 1H, Asp), 4.19 (t, 1H, R-H), 4.33 (m, 2H, R-H), 4.55 (d, 2H,
N(CH2Ph)2), 4.63 (s, 2H, N(CH2Ph)2), 5.00 (q, 2H, Cbz), 7.14 (d,
1H, db), 7.21-7.34 (m, 16H, db, Cbz-Ph, and N(CH2Ph)2), 7.60
(d, 1H, NH), 7.91-7.98 (dd, 2H, NH), 11.00 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CONHCH2Ph was obtained by the EDC/HOBt coupling method,
and purified by column chromatography on silica gel with 1:9:10
MeOH:CH2Cl2:EtOAc as the eluent; white solid, yield 49%. MS
1
(ESI) m/z 923.5 [(M + 1)+]. H NMR (DMSO-d6): 0.85 (t, 6H,
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CON(CH2-1-Naphth)2 was obtained by the EDC/HOBt coupling
method, and purified by column chromatography on silica gel with
2:1 EtOAc:hexane as the eluent; white solid, yield 17%. 1H NMR
(acetone-d6): 1.04 (d, 6H, Val), 1.35-1.43 (m, 27H, tBu), 1.97
(m, 2H, Glu), 2.17-2.38 (m, Glu, Val, Asp), 2.71 (dd, 1H, Asp),
4.30 (t, 1H, R-H), 4.51 (m, 2H, R-H), 5.10 (dd, 2H, N(CH2-1-
Naph)2), 5.24 (s, 2H, N(CH2-1-Naph)2), 5.33 (s, 2H, Cbz), 6.85
(d, 1H, db), 7.29-7.95 (m, 16H, db, Cbz-Ph, and N(CH2-1-Naph)2),
8.18 (d, 1H, NH), 9.81 (s, 1H, NH-N).
Val), 1.35-1.41 (m, 27H, tBu), 1.72 (m, 1H, Glu), 1.84 (m, 1H,
Glu), 1.96 (m, 1H, Val), 2.17 (m, 2H, Glu), 2.44 (dd, 1H, Asp),
2.60 (dd, 1H, Asp), 4.17 (t, 1H, R-H), 4.28 (m, 2H, R-H), 4.36 (d,
2H, NHCH2Ph), 5.02 (q, 2H, Cbz), 6.95 (d, 1H, db), 7.12 (d, 1H,
db), 7.22-7.31 (m, 10H, Ph), 7.57 (d, 1H, NH), 7.94 (d, 1H, NH),
8.92 (t, 1H, NHCH2Ph), 10.96 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CONHCH2-4-F-Ph was obtained by the EDC/HOBt coupling
method, and purified by column chromatography on silica gel with
1:19:20 MeOH:CH2Cl2:EtOAc as the eluent; white solid, yield 67%.
1
MS (ESI) m/z 941.4 [(M + 1)+]. H NMR (DMSO-d6): 0.86 (t,
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CO-tetrahydroquinoline was obtained by the EDC/HOBt coupling
method, and purified by column chromatography on silica gel with
1:2 CH2Cl2:EtOAc as the eluent; yellow solid, yield 54%. MS (ESI)
6H, Val), 1.34-1.41 (m, 27H, tBu), 1.75 (m, 1H, Glu), 1.88 (m,
1H, Glu), 2.01 (m, 1H, Val), 2.20 (m, 2H, Glu), 2.48 (dd, 1H, Asp),
2.65 (dd, 1H, Asp), 4.18 (t, 1H, R-H), 4.34 (m, 4H, R-H and
NHCH2Ph-4-F), 5.01 (q, 2H, Cbz), 6.93 (d, 1H, db), 7.12 (d, 2H,
NHCH2Ph-4-F), 7.25-7.32 (m, 8H, Cbz-Ph, NHCH2Ph-4-F, and
db), 7.59 (d, 1H, NH), 7.90-7.99 (dd, 2H, NH), 8.94 (t, 1H,
NHCH2Ph-4-F), 10.98 (s, 1H, NH-N).
1
m/z 949.4 [(M + 1)+]. H NMR (DMSO-d6): 0.89 (t, 6H, Val),
1.35-1.42 (m, 27H, tBu), 1.75 (m, 1H, Glu), 1.87 (m, 3H, Glu
and NCH2CH2CH2), 2.02 (m, 1H, Val), 2.20 (m, 2H, Glu), 2.48
(dd, 1H, Asp), 2.65 (dd, 1H, Asp), 2.70 (t, 2H, NCH2CH2CH2),
3.73 (t, 2H, NCH2CH2CH2), 4.19 (t, 1H, R-H), 4.33 (m, 2H, R-H),
5.01 (q, 2H, Cbz), 6.95 (m, 1H, db), 7.04 (d, 1H, db), 7.14-7.33
(m, 9H, Cbz-Ph, and quinoline), 7.60 (d, 1H, NH), 7.96 (dd, 2H,
NH), 11.02 (s, 1H, NH-N).
Cbz-Asp(O-tBu)-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-
CONHCH2CH2Ph was obtained by the EDC/HOBt coupling
method, and purified by column chromatography on silica gel with
1:9:5 MeOH:CH2Cl2:EtOAc as the eluent, and then rechromato-

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5741
Cbz-Val-Glu(O-tBu)-Val-AAsp(O-tBu)-CHdCH-COOEt was
obtained by the EDC/HOBt coupling method, and purified by
column chromatography on silica gel with 1:9:5 MeOH:CH2Cl2:
EtOAc as the eluent; white solid, yield 71%. MS (ESI) m/z 790.3
[(M + 1)+]. 1H NMR (DMSO-d6): 0.81-0.87 (dd, 12H, Val), 1.22
(t, 3H, OCH2CH3), 1.24-1.41 (d, 18H, tBu), 1.72 (m, 1H, Glu),
1.83 (m, 1H, Glu), 1.96 (m, 1H, Val), 2.19 (m, 2H, Glu), 3.86 (t,
1H, R-H), 4.17 (m, 3H, R-H and OCH2CH3), 4.32 (m, 1H, R-H),
5.01 (s, 2H, Cbz), 6.64 (d, 1H, db), 7.20 (d, 1H, db), 7.30-7.33
(m, 5H, Ph), 7.97 (t, 2H, NH), 11.02 (s, 1H, NH-N).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-COOEt was
synthesized using the mixed anhydride coupling method and
purified by column chromatography using 4:1 (1:9 MeOH:CH2-
Cl2):EtOAc as an eluent, and then rechromatographed using 4:1
(1:19 MeOH:CH2Cl2):EtOAc as the eluent; white solid, yield 36%.
MS (ESI) m/z 806 [(M + 1)+].
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-COOCH2-
Ph was obtained using the EDC/HOBt coupling method, purified
by column chromatography on silica gel with 50:45:5 CH2Cl2:
EtOAc:MeOH as the eluent; white solid, yield 37%. 1H NMR
(CDCl3): 0.91 and 0.94 (m, 6H, Ile CH3), 1.20 (m, 5H, Ile CH2
and Thr CH3), 1.45 (s, 19H, t-Bu and Ile CH), 2.05 and 2.20 (d of
m, 2H, Glu CH2), 2.35 and 2.50 (d of m, 2H, Glu CH2), 3.21 (s,
2H, AAsp CH2), 4.12 (m, 2H, R-H), 4.30 (m, 1H, R-H), 4.44 (m,
1H, Thr CH), 5.10 (m, 4H, Cbz and CH2Ph), 5.40 (d, 1H, NH),
6.85 and 7.20 (d of d, 2H, CHdCH), 7.20-7.40 (m, 10H, Ph),
7.50 (d, 1H, NH), 8.00 (d, 1H, NH), 9.40 (s, 1H, NH).
1H, Thr CH), 4.65 (s, 2H, CH2Ph), 5.08 (q, 2H, Cbz), 6.63 (d, 1H,
NH), 7.20-7.40 (m, 13H, CHdCH, Ph and NH), 7.52 (d, 1H, NH),
7.84 (d, 1H, NH), 9.75 (s, 1H, NH).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH3)-
CH2CH2Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 71%. 1H NMR
(CDCl3): 0.90 (m, 3H, Ile CH3), 0.96 (m, 3H, Ile CH3), 1.20 (m,
3H, Thr CH3), 1.45 (s, 18H, t-Bu), 1.80-1.90 (m, 3H, Ile CH and
CH2), 2.10 (m, 2H, Glu CH2), 2.40 (m, 2H, Glu CH2), 2.85 (m,
2H, CH2Ph), 2.97 (d, 3H, NCH3), 3.30 (m, 2H, AAsp CH2), 3.54
(m, 2H, NCH2), 4.20 (m, 2H, R-H), 4.41 (m, 2H, R-H and Thr
CH), 5.11 (q, 2H, Cbz), 5.60 (d, 1H, NH), 7.10-7.40 (m, 13H,
CHdCH, Ph and NH), 7.85 (b, 1H, NH), 9.50 (d, 1H, NH).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH-
2Ph)2 was obtained using the EDC/HOBt coupling method, purified
by column chromatography on silica gel with 50:45:5 CH2Cl2:
EtOAc:MeOH as the eluent; white solid, yield 46%. 1H NMR
(acetone-d6): 0.89 and 0.99 (m, 6H, Ile CH3), 1.21 (m, 3H, Thr
CH3), 1.45 (s, 18H, t-Bu), 1.60 (m, 3H, Ile CH and CH2), 1.95 and
2.10 (d of m, 2H, Glu CH2), 2.39 (m, 2H, Glu CH2), 2.84 (s, 2H,
AAsp CH2), 4.15 (t, 1H, R-H), 4.35 (m, 1H, R-H), 4.45 (m, 2H,
R-H and Thr CH), 4.67 (m, 4H, CH2Ph), 5.09 (q, 2H, Cbz), 5.60
(d, 1H, NH), 7.20-7.40 (m, 17H, CHdCH and Ph), 7.55 (d, 1H,
NH), 7.85 (d, 1H, NH), 9.75 (s, 1H, NH).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-COOEt was
synthesized using the mixed anhydride coupling method and
purified by column chromatography using 4:1 (1:19 MeOH:CH2-
Cl2):EtOAc as the eluent; white solid, yield 35%. MS (ESI) m/z
806 [(M + 1)+]. 1H NMR (DMSO-d6): 0.85 (t, 6H, Leu CH3), 1.1
(d, 3H, Thr CH3), 1.2-1.3 (t, 3H, OCH2CH3), 1.3-1.5 (d, 20H,
tBu and Leu CH2), 1.6 (m, 1H, CH Leu), 1.7 (m, 1H, Glu CH2),
1.9 (m, 1H, Glu CH2), 2.2 (m, 2H, Glu CH2), 3.9-4.1 (m, 4H,
NCH2COOH and R-H), 4.1-4.2 (m, 3H, OCH2CH3 and CH-OH),
4.3 (m, 2H, R-H), 5.0 (s, 2H, Cbz), 6.6 (d, 1H, CHdCH), 7.20-
7.40 (m, 6H, Ph and CHdCH), 7.50 (d, 1H, NH), 7.75 (d, 1H,
NH), 8.0 (d, 1H, NH).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CONHPh
was obtained using the EDC/HOBt coupling method, purified by
column chromatography on silica gel with 50:45:5 CH2Cl2:EtOAc:
1
MeOH as the eluent; white solid, yield 67%. H NMR (acetone-
d6): 0.89 and 0.99 (m, 6H, Ile CH3), 1.24 (m, 5H, Thr CH3 and Ile
CH2), 1.45 (s, 18H, t-Bu), 1.60 (m, 1H, Ile CH), 1.95 and 2.15 (d
of m, 2H, Glu CH2), 2.39 (m, 2H, Glu CH2), 2.86 (s, 2H, AAsp
CH2), 4.16 (m, 1H, R-H), 4.35 (m, 1H, R-H), 4.45 (m, 2H, R-H
and Thr CH), 5.10 (q, 2H, Cbz), 6.70 (d, 1H, NH), 6.80 and 7.20
(d of d, 2H, CHdCH), 7.08 (m, 2H, Ph), 7.35 (m, 6H, Ph), 7.52
(m, 1H, NH), 7.78 (t, 2H, Ph), 7.90 (b, 1H, NH), 9.60 (s, 1H, NH),
9.70 (s, 1H, NH).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CONHC-
H2Ph was obtained using the EDC/HOBt coupling method, purified
by column chromatography on silica gel with 50:45:5 CH2Cl2:
EtOAc:MeOH as the eluent; white solid, yield 30%. 1H NMR
(CDCl3): 0.92 and 0.95 (m, 6H, Ile CH3), 1.19 (m, 5H, Ile CH2
and Thr CH3), 1.45 (s, 18H, t-Bu), 1.80 (m, 1H, Ile CH), 1.90 (m,
2H, Glu CH2), 2.30 and 2.40 (d of m, 2H, Glu CH2), 3.20 (s, 2H,
AAsp CH2), 4.12 (m, 1H, R-H), 4.38 (m, 3H, R-H, Thr CH and
CH2Ph), 4.52 (m, 1H, CH2Ph), 5.10 (s, 2H, Cbz), 5.53 (d, 1H, NH),
6.85 (d, 1H, CHdCH), 7.15-7.40 (m, 11H, CHdCH and Ph), 7.70
(s, 1H, NH), 8.05 (b, 1H, NH), 8.35 (b, 1H, NH), 9.45 (s, 1H,
NH).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CONHC-
H2CH2Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 77%. 1H NMR
(CDCl3): 0.92 and 0.98 (m, 6H, Ile CH3), 1.19 (m, 5H, Ile CH2
and Thr CH3), 1.45 (s, 18H, t-Bu), 1.80 (m, 1H, Ile CH), 1.95 and
2.10 (d of m, 2H, Glu CH2), 2.35 and 2.50 (d of m, 2H, Glu CH2),
2.80 (m, 2H, CH2Ph), 3.20 (s, 2H, AAsp CH2), 3.52 (m, 2H, NCH2),
4.15 (m, 2H, R-H), 4.25 (m, 1H, R-H), 4.40 (m, 1H, Thr CH),
5.11 (s, 2H, Cbz), 5.45 (d, 1H, NH), 6.85 (d, 1H, CHdCH), 7.15-
7.40 (m, 12H, CHdCH, Ph and NH), 7.70 (b, 1H, NH), 8.05 (b,
1H, NH), 9.40 (s, 1H, NH).
Cbz-Ile-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH3)-
CH2Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 77%. 1H NMR
(acetone-d6): 0.90 and 0.99 (m, 6H, Ile CH3), 1.21 (m, 3H, Thr
CH3), 1.45 (s, 18H, t-Bu), 1.60 (m, 3H, Ile CH and CH2), 1.96 (m,
2H, Glu CH2), 2.38 (m, 2H, Glu CH2), 2.84 (s, 2H, AAsp CH2),
2.95 and 3.08 (d, 3H, NCH3), 4.15-4.30 (m, 3H, R-H), 4.42 (m,
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-COOCH-
2Ph was obtained by the EDC/HOBt coupling method, and purified
by column chromatography on silica gel with 1:499:500 MeOH:
CH2Cl2:EtOAc as the eluent; white solid, yield 52%. MS (ESI)
1
m/z 868.4 [(M + 1)+]. H NMR (DMSO-d6): 0.83 (t, 6H, Leu),
1.02 (d, 3H, Thr), 1.36-1.40 (t, 20H, tBu and Leu), 1.60 (m, 1H,
Glu), 1.72 (m, 1H, Glu), 1.88 (m, 1H, Leu), 2.21 (m, 2H, Glu),
3.92 (m, 1H, R-H), 4.03 (m, 1H, R-H), 4.17 (m, 1H, R-H), 4.34
(m, 1H, Thr), 4.99 (s, 2H, Cbz), 5.19 (s, 2H, Bzl), 6.67-6.71 (d,
1H, db), 7.32-7.38 (m, 10H, Ph), 7.40 (d, 1H, db), 7.76 (d, 1H,
NH), 8.04 (d, 1H, NH), 10.80 (s, 1H, NH-N).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCHCONHPh was
obtained using the EDC/HOBt coupling method, purified by column
chromatography on silica gel with 50:45:5 CH2Cl2:EtOAc:MeOH
as the eluent; white solid, yield 80%. 1H NMR (acetone-d6): 0.93
(d, 6H, Leu CH3), 1.22 (m, 3H, Thr CH3), 1.44 (s, 18H, t-Bu),
1.67 (t, 2H, Leu CH2), 1.78 (m, 1H, Leu CH), 1.95 and 2.15 (d of
m, 2H, Glu CH2), 2.38 (m, 2H, Glu CH2), 2.87 (s, 2H, AAsp CH2),
4.30-4.35 (m, 3H, R-H), 4.42 (m, 1H, Thr CH), 5.09 (q, 2H, Cbz),
6.75 (d, 1H, NH), 6.80 and 7.20 (d of d, 2H, CHdCH), 7.08 (m,
2H, Ph), 7.35 (m, 6H, Ph), 7.52 (m, 1H, NH), 7.78 (t, 2H, Ph),
7.88 (b, 1H, NH), 9.63 (s, 1H, NH), 9.72 (s, 1H, NH).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCHCONHC-
H2Ph was obtained using the EDC/HOBt coupling method, purified
by column chromatography on silica gel with 50:45:5 CH2Cl2:
EtOAc:MeOH as the eluent; white solid, yield 38%. 1H NMR
(CDCl3): 0.94 (d, 6H, Leu CH3), 1.20 (m, 3H, Thr CH3), 1.45 (s,
18H, t-Bu), 1.64 (m, 3H, Leu CH and CH2), 2.00 (m, 2H, Glu
CH2), 2.34 and 2.42 (d of m, 2H, Glu CH2), 3.20 (s, 2H, AAsp
CH2), 4.10 (m, 1H, R-H), 4.21 (m, 2H, R-H), 4.40-4.50 (m, 3H,
Thr CH and CH2Ph), 5.05 (q, 2H, Cbz), 5.42 (d, 1H, NH), 6.85 (d,
1H, CHdCH), 7.15-7.40 (m, 11H, CHdCH and, Ph), 7.55 (b,
1H, NH), 7.70 (b, 1H, NH), 8.20 (b, 1H, NH), 9.40 (s, 1H, NH).

5742 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCHCONHCH2-
4-F-Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 46%. 1H NMR
(acetone-d6): 0.92 (d, 6H, Leu CH3), 1.21 (d, 3H, Thr CH3), 1.45
(s, 18H, t-Bu), 1.66 (t, 2H, Leu CH2), 1.76 (m, 1H, Leu CH), 1.95
and 2.10 (d of m, 2H, Glu CH2), 2.36 (m, 2H, Glu CH2), 3.20 (s,
2H, AAsp CH2), 4.29 (m, 3H, R-H), 4.40 (m, 1H, Thr CH), 4.50
(d, 2H, CH2Ph), 5.10 (q, 2H, Cbz), 6.76 (d, 1H, NH), 6.91 (d, 1H,
CHdCH), 7.07 (d. 2H, CHdCH and Ph), 7.25-7.40 (m, 8H, Ph),
7.50 (b, 1H, NH), 7.95 (b, 1H, NH), 8.23 (b, 1H, NH), 9.70 (s,
1H, NH).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCHCONHC-
H2CH2Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 55%. 1H NMR
(CDCl3): 0.94 (d, 6H, Leu CH3), 1.20 (m, 3H, Thr CH3), 1.45 (s,
18H, t-Bu), 1.65 (m, 3H, Leu CH and CH2), 2.05 (m, 2H, Glu
CH2), 2.40 (m, 2H, Glu CH2), 2.78 (m, 2H, CH2Ph), 3.20 (s, 2H,
AAsp CH2), 3.50 (m, 2H, NCH2), 4.24 (m, 3H, R-H), 4.41 (m,
1H, Thr CH), 5.05 (s, 2H, Cbz), 5.62 (d, 1H, NH), 6.80 and 7.10
(d of d, 2H, CHdCH), 7.15-7.40 (m, 12H, Ph and NH), 8.00 (b,
1H, NH), 9.45 (s, 1H, NH).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH3)-
CH2Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 77%. 1H NMR
(acetone-d6): 0.94 (d, 6H, Leu CH3), 1.20 (m, 3H, Thr CH3), 1.45
(s, 18H, t-Bu), 1.65 (m, 2H, Leu CH2), 1.80 (m, 1H, Leu CH),
1.95-2.10 (d of m, 2H, Glu CH2), 2.38 (d of m, 2H, Glu CH2),
2.85 (s, 2H, AAsp CH2), 2.95 and 3.08 (d, 3H,NCH3), 4.30 (m,
3H, R-H), 4.42 (m, 1H, Thr CH), 4.65 (s, 2H, CH2Ph), 5.08 (q,
2H, Cbz), 6.75 (d, 1H, NH), 7.20-7.40 (m, 12H, CHdCH and
Ph), 7.44 (d, 1H, NH), 7.90 (d, 1H, NH), 9.75 (s, 1H, NH).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH3)-
CH2CH2Ph was obtained using the EDC/HOBt coupling method,
purified by column chromatography on silica gel with 50:45:5 CH2-
Cl2:EtOAc:MeOH as the eluent; white solid, yield 76%. 1H NMR
(CDCl3): 0.94 (m, 6H, Leu CH3), 1.21 (m, 3H, Thr CH3), 1.45 (s,
18H, t-Bu), 1.55 (m, 2H, Leu CH2), 1.70 (m, 1H, Leu CH), 2.13
(m, 2H, Glu CH2), 2.43 (m, 2H, Glu CH2), 2.81 (m, 2H, CH2Ph),
2.88 and 2.92 (d, 3H, NCH3), 3.56 (m, 4H, NCH2 and AAsp CH2),
4.30 (m, 2H, R-H), 4.43 (m, 2H, R-H and Thr CH), 5.10 (q, 2H,
Cbz), 5.55 (m, 1H, NH), 7.10-7.40 (m, 12H, CHdCH and Ph),
7.45 (d, 1H, NH), 7.95 (b, 1H, NH), 9.42 (d, 1H, NH).
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH2-
Ph)2 was obtained using the EDC/HOBt coupling method, purified
by column chromatography on silica gel with 50:45:5 CH2Cl2:
EtOAc:MeOH as the eluent; white solid, yield 85%. 1H NMR
(acetone-d6): 0.93 (d, 6H, Leu CH3), 1.21 (m, 3H, Thr CH3), 1.45
(s, 18H, t-Bu), 1.67 (m, 2H, Leu CH2), 1.80 (m, 1H, Leu CH),
1.95 and 2.10 (d of m, 2H, Glu CH2), 2.39 (m, 2H, Glu CH2), 2.88
(s, 2H, AAsp CH2), 4.25-4.35 (m, 3H, R-H), 4.43 (m, 1H, Thr
CH), 4.66 (m, 4H, CH2Ph), 5.09 (q, 2H, Cbz), 6.75 (d, 1H, NH),
7.20-7.40 (m, 17H, CHdCH and Ph), 7.55 (d, 1H, NH), 7.90 (b,
1H, NH), 9.75 (s, 1H, NH).
45:5 CH2Cl2:EtOAc:MeOH as the eluent; white solid, yield 66%.
1H NMR (CDCl3): 0.95 (m, 6H, Leu CH3), 1.22 (m, 3H, Thr CH3),
1.45 (s, 18H, t-Bu), 1.55 (m, 2H, Leu CH2), 1.70 (m, 1H, Leu CH),
2.15 (m, 2H, Glu CH2), 2.45 (m, 2H, Glu CH2), 2.92 and 2.99 (d,
3H, NCH3), 3.30 (m, 2H, AAsp CH2), 4.31 (m, 2H, R-H), 4.47
(m, 2H, R-H and Thr CH), 5.09 (m, 4H, CH2-naphth and Cbz),
5.50 (b, 1H, NH), 7.10 (d, 1H, NH), 7.20-7.40 (m, 10H, CHdCH
and Ph), 7.52 (m, 2H, naphth), 7.79 (d, 1H, NH), 7.90 (m, 2H,
naphth), 9.45 (d, 1H, NH).
Deblocking of the t-Butyl Protecting Group in the Aza-Asp
Peptide Inhibitors. The aza-peptide inhibitors were treated with
1:1 TFA: CH2Cl2 at 0 °C for 1 h and at room temperature for 2 h.
TFA and CH2Cl2 were removed under vacuum and the final
products were recrystallized from ether/hexanes mostly as white
solids.
N2-(N-Benzyloxycarbonylvalyl)-N1-trans-(but-2-enoyl)-N1-car-
boxymethylhydrazine (Cbz-Val-AAsp-CHdCH-CH3, 16a).
HRMS (ESI) Calcd. for C19H25N3O6; 391.1822; Observed m/z:
391.1797. N2-(N-Benzyloxycarbonylvalyl)-N1-carboxymethyl-N1-
trans-(hexa-2,4-dienoyl)hydrazine (Cbz-Val-AAsp-CHdCH-
1
CHdCH-CH3, 16b). H NMR (DMSO-d6): 0.90 (d, 6H, Val),
1.78 (d, 3H, CH3), 1.97 (m, 1H, Val), 3.87 (t, 1H, R-H), 5.05 (q,
2H, Cbz), 6.14 (m, 2H, db), 6.38 (m, 1H, db), 7.09 (dd, 2H, db),
7.33 (m, 5H, Ph), 7.59 (d, 1H, NH), 10.81 (s, 1H, NH-N). HRMS
(ESI) Calcd. for C21H27N3O6: 417.1978; Observed m/z: 417.1974.
Anal. (C21H27N3O6‚1.43H2O‚0.05TFA) C, H, N.
N2-(N-Benzyloxycarbonylvalyl)-N1-carboxymethyl-N1-trans-
(3-phenethylacryloyl)hydrazine (Cbz-Val-AAsp-CHdCH-CH2-
CH2Ph, 16c). HRMS (ESI) Calcd. for C26H31N3O6: 482.2291;
Observed m/z: 482.2301. Anal. (C26H31N3O6‚0.84H2O) C, H, N.
N2-(N-Benzyloxycarbonylvalyl)-N1-carboxymethyl-N1-cis-(3-
chloroacryloyl)hydrazine (Cbz-Val-AAsp-cis-CHdCH-Cl, 16d).
1H NMR (DMSO-d6): 0.86 (d, 6H, Val), 1.93 (m, 1H, Val), 3.84
(t, 1H, R-H), 5.01 (s, 2H, Cbz), 6.57 (s, 1H, db), 6.76 (d, 1H, db),
7.29-7.34 (m, 5H, Ph), 7.53 (d, 1H, NH), 10.80 (s, 1H, NH-N).
HRMS (ESI) Calcd. for C18H22N3O6Cl: 411.1275; Observed m/z:
411.1195. Anal. (C18H22N3O6Cl‚0.04H2O) C, H, N.
N2-(N-Benzyloxycarbonylvalyl)-N1-carboxymethyl-N1-trans-
(3-(4-chlorophenyl) acryloyl)hydrazine (Cbz-Val-AAsp-CHd
1
CH-Ph-4-Cl, 16e). H NMR (DMSO-d6): 0.86-0.95 (dd, 6H,
Val), 1.94 (m, 1H, Val), 3.84 (t, 1H, R-H), 5.03 (q, 2H, Cbz), 7.27-
7.32 (m, 5H, Ph), 7.42 (d, 1H, db), 7.55 (d, 1H, db), 7.77 (m, 4H,
Ph-Cl), 10.97 (s, 1H, NH-N). HRMS (ESI) Calcd. for C24H26N3O6-
Cl: 488.1588; Observed m/z: 488.1563. Anal. (C24H26N3O6Cl‚
0.29H2O) C, H, N.
N2-(N-Benzyloxycarbonylvalyl)-N1-carboxymethyl-N1-trans-
(3-ethoxycarbonylacryloyl)hydrazine (Cbz-Val-AAsp-CHdCH-
1
COOEt, 16h). H NMR (DMSO-d6): 0.87 (d, 6H, Val), 1.15-
1.23 (m, 3H, OCH2CH3), 1.98 (m, 1H, Val), 3.89 (t, 1H, R-H),
4.13 (q, 2H, OCH2CH3), 5.03 (s, 2H, Cbz), 6.65 (d, 1H, db), 7.28-
7.34 (m, 6H, db and Ph), 7.54 (d, 1H, NH), 11.00 (s, 1H, NH).
HRMS (FAB) Calcd. for C21H27N3O8: 449.1798; Observed m/z:
449.1876. Anal. (C21H27N3O8‚0.29H2O) C, H, N.
N2-(N-Benzyloxycarbonylvalyl)-N1-trans-(3-butylcarbamoy-
lacryloyl)-N1-carboxymethylhydrazine (Cbz-Val-AAsp-CHd
CH-CONH-nBu, 16j). 1H NMR (DMSO-d6): 0.83-0.87 (m, 9H,
Val and NHCH2CH2CH2CH3), 1.27 (m, 2H, NHCH2CH2CH2CH3),
1.37-1.41 (m, 2H, NHCH2CH2CH2CH3), 1.97 (m, 1H, Val), 3.12
(dd, 2H, NHCH2CH2CH2CH3), 3.93 (t, 1H, R-H), 5.03 (q, 2H, Cbz),
6.91 (d, 1H, db), 7.08 (d, 1H, db), 7.28-7.34 (m, 5H, Ph), 7.47 (d,
1H, NH), 8.37 (t, 1H, NHCH2CH2CH2CH3), 10.93 (s, 1H, NH-
N). HRMS (ESI) Calcd. for C23H32N4O7: 477.2349; Observed
m/z: 477.2357. Anal. (C23H32N4O7‚0.46H2O) C, H, N.
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CO-tet-
rahydroquinoline was obtained using the EDC/HOBt coupling
method, purified by column chromatography on silica gel with 50:
45:5 CH2Cl2:EtOAc:MeOH as the eluent; white solid, yield 85%.
1H NMR (CDCl3): 0.94 (m, 6H, Leu CH3), 1.21 (m, 3H, Thr CH3),
1.45 (s, 18H, t-Bu), 1.60 (m, 2H, Leu CH2), 1.70 (m, 1H, Leu CH),
1.90 (m, 2H, quinoline CH2), 2.10 (m, 2H, Glu CH2), 2.45 (m, 2H,
Glu CH2), 2.67 (t, 2H, quinoline CH2), 3.40 (m, 2H, AAsp CH2),
3.90 (m, 2H, NCH2), 4.30 (m, 2H, R-H), 4.45 (m, 2H, R-H and
Thr CH), 5.10 (q, 2H, Cbz), 5.55 (b, 1H, NH), 6.90 (b, 1H, NH),
7.10-7.40 (m, 10H, CHdCH and Ph), 7.50 (d, 1H, Ph), 7.95 (d,
1H, NH), 9.40 (d, 1H, NH).
N1-trans-(3-Benzylcarbamoylacryloyl)-N2-(N-benzyloxycarbo-
nylvalyl)-N1-carboxymethylhydrazine (Cbz-Val-AAsp-CHd
CH-CONHCH2Ph, 16l). 1H NMR (DMSO-d6): 0.85 (d, 6H, Val),
1.97 (m, 1H, Val), 3.96 (t, 1H, R-H), 4.35 (d, 2H, NHCH2Ph), 5.01
(s, 2H, Cbz), 6.97 (d, 1H, db), 7.15 (d, 1H, db), 7.19-7.32 (m,
10H, Ph), 7.47 (d, 1H, NH), 8.91 (t, 1H, NHCH2Ph), 10.94 (s, 1H,
NH-N). HRMS (ESI) Calcd. for C26H30N4O7: 510.2193; Observed
m/z: 510.2208. Anal. (C26H30N4O7‚0.56H2O) C, H, N.
Cbz-Leu-Glu(O-tBu)-Thr-AAsp(O-tBu)-CHdCH-CON(CH3)-
CH2-1-Naphth was obtained using the EDC/HOBt coupling
method, purified by column chromatography on silica gel with 50:

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5743
N2-(N-Benzyloxycarbonylglutamylvalyl)-N1-carboxymethyl-
N1-trans-(3-ethoxycarbonylacryloyl) hydrazine (Cbz-Glu-Val-
AAsp-CHdCH-COOEt, 17h). H NMR (DMSO-d6): 0.84 (m,
6H, Val), 1.20-1.21 (t, 3H, OCH2CH3), 1.70-2.10 (m, 3H, Val,
Glu), 2.21 (m, 2H, Glu), 2.40 (CHdCH-COOEt) 4.05-4.22 (m,
4H, NCH2COOH and OCH2CH3), 4.50-4.60 (m, 2H, R-H), 5.05
(m, 2H, Cbz), 7.12 (CO-CHdCH-COOEt) 7.20-7.40 (m, 5H, Ph),
7.60 (1H, NH), 7.85 (m, 2H, NH), 11.00 (m, COOH). HRMS (FAB,
M+1)) Calcd. for C26H35N4O11: 579.2302; Observed m/z 579.2342.
Anal. (C26H34N4O11) C, H, N.
and db), 7.59 (d, 1H, NH), 7.90-7.99 (dd, 2H, NH), 8.94 (t, 1H,
NHCH2Ph-4-F), 10.98 (s, 1H, NH-N). HRMS (ESI) Calcd. for
C35H41N6O13F: 772.2788; Observed m/z: 772.2747. Anal. (C35H41-
N6O13F‚2.08H2O) C, H, N.
1
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
methyl-N1-trans-(3-phenethylcarbamoylacryloyl)hydrazine (Cbz-
Asp-Glu-Val-AAsp-CHdCH-CONHCH2CH2Ph, 18n). 1H NMR
(DMSO-d6): 0.87 (t, 6H, Val), 1.75 (m, 1H, Glu), 1.89 (m, 1H,
Glu), 2.01 (m, 1H, Val), 2.21 (m, 2H, Glu), 2.46 (dd, 1H, Asp),
2.62 (dd, 1H, Asp), 2.73 (t, 2H, NHCH2CH2Ph), 3.73 (d, 2H,
NHCH2CH2Ph), 4.18 (t, 1H, R-H), 4.32 (m, 2H, R-H), 5.01 (s, 2H,
Cbz), 6.89 (d, 1H, db), 7.08 (d, 1H, db), 7.16-7.32 (m, 10H, Ph),
7.59 (d, 1H, NH), 7.89 (d, 1H, NH), 7.98 (d, 1H, NH), 8.52 (t, 1H,
NHCH2CH2Ph), 10.97 (s, 1H, NH-N). HRMS (ESI) Calcd. for
C36H44N6O13: 768.3044; Observed m/z: 768.2949. Anal. (C36H44-
N6O13‚1.22H2O) C, H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-trans-(3-
benzoylacryloyl)-N1-carboxymethylhydrazine (Cbz-Asp-Glu-
Val-AAsp-CHdCH-COPh, 18f). 1H NMR (DMSO-d6): 0.82 (d,
6H, Val), 1.73 (m, 1H, Glu), 1.85 (m, 1H, Glu), 1.98 (m, 1H, Val),
2.20 (m, 2H, Glu), 2.48 (dd, 1H, Asp), 2.62 (dd, 1H, Asp), 4.16 (t,
1H, R-H), 4.33 (m, 2H, R-H), 5.01 (s, 2H, Cbz), 7.24 (d, 1H, db),
7.32 (s, 5H, Ph), 7.54 (t, 3H, Ph), 7.67 (t, 1H, NH), 7.79 (d, 1H,
NH), 7.93 (t, 1H, NH), 7.97 (d, 2H, Ph), 11.06 (s, 1H, NH-N).
HRMS (ESI) Calcd. for C34H39N5O13: 725.2623; Observed m/z:
725.2543. Anal. (C34H39N5O13‚H2O‚0.61TFA) C, H, N.
N1-trans-(3-Benzylmethylcarbamoylacryloyl)-N2-(N-benzyl-
oxycarbonylaspartylglutamylvalyl)-N1-carboxymethylhydra-
zine (Cbz-Asp-Glu-Val-AAsp-CHdCH-CON(CH3)CH2Ph, 18o).
1H NMR (DMSO-d6): 0.85 (d, 6H, Val), 1.73 (m, 1H, Glu), 1.87
(m, 1H, Glu), 1.97 (m, 1H, Val), 2.20 (m, 2H, Glu), 2.48 (dd, 1H,
Asp), 2.63 (dd, 1H, Asp), 2.88-2.98 (d, 3H, N(CH3)CH2Ph), 4.17
(t, 1H, R-H), 4.32 (m, 2H, R-H), 4.47-4.58 (q, 1H, N(CH3)CH2-
Ph), 4.66 (s, 1H, N(CH3)CH2Ph), 5.00 (s, 2H, Cbz), 7.14 (d, 1H,
db), 7.20 (d, 1H, db), 7.28-7.36 (m, 10H, Ph), 7.57 (d, 1H, NH),
7.91-7.95 (m, 2H, NH), 10.99 (s, 1H, NH-N). HRMS (ESI) Calcd.
for C36H44N6O13: 768.3045; Observed m/z: 768.3039. Anal.
(C36H44N6O13‚EtOAc) C, H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
lmethyl-N1-cis-(3-ethoxycarbonylacryloyl)hydrazine (Cbz-Asp-
1
Glu-Val-AAsp-cis-CHdCH-COOEt, 18g). H NMR (DMSO-
d6): 0.85 (d, 6H, Val), 1.18 (t, 3H, OCH2CH3), 1.74 (m, 1H, Glu),
1.86 (m, 1H, Glu), 1.95 (m, 1H, Val), 2.18 (m, 2H, Glu), 2.45 (dd,
1H, Asp), 2.61 (dd, 1H, Asp), 4.09 (t, 1H, R-H), 4.16 (q, 2H, OCH2-
CH3), 4.33 (m, 2H, R-H), 5.01 (s, 2H, Cbz), 6.61 (d, 1H, db), 7.25
(d, 1H, db), 7.33 (s, 5H, Ph), 7.62 (d, 1H, NH), 7.93 (t, 2H, NH),
11.05 (s, 1H, NH-N). HRMS (ESI) Calcd. for C30H39N5O14:
693.2566; Observed m/z: 693.2516. Anal. (C30H39N5O14‚1H2O) C,
H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
methyl-N1-trans-(3-phenethylmethylcarbamoylacryloyl)hydra-
zine (Cbz-Asp-Glu-Val-AAsp-CHdCH-CON(CH3)CH2-CH2Ph,
18p). 1H NMR (DMSO-d6): 0.86 (t, 6H, Val), 1.75 (m, 1H, Glu),
1.89 (m, 1H, Glu), 2.01 (m, 1H, Val), 2.21 (m, 2H, Glu), 2.46 (dd,
1H, Asp), 2.62 (dd, 1H, Asp), 2.80 (t, 2H, N(CH3)CH2CH2Ph),
2.85-2.98 (d, 3H, N(CH3)CH2CH2Ph), 3.73 (d, 2H, N(CH3)CH2-
CH2Ph), 4.18 (t, 1H, R-H), 4.32 (m, 2H, R-H), 5.00 (s, 2H, Cbz),
6.94 (d, 1H, db), 7.05 (dd, 1H, db), 7.15-7.32 (m, 10H, Ph), 7.59
(d, 1H, NH), 7.91 (d, 1H, NH), 7.97 (d, 1H, NH), 10.93 (d, 1H,
NH-N). HRMS (ESI) Calcd. for C37H46N6O13: 783.3196; Ob-
served m/z: 783.3182. Anal. (C37H46N6O13‚1.09H2O‚0.24TFA) C,
H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylthreonyl)-N1-car-
boxylmethyl-N1-trans-(3-ethoxycarbonylacryloyl)hydrazine (Cbz-
Asp-Glu-Val-AAsp-CHdCH-COOEt, 18h). 1H NMR (DMSO-
d6): 0.86 (d, 6H, Val CH3), 1.21 (t, 3H, OEt), 1.75 and 1.98 (d of
m, 2H, Glu CH2), 1.85 (m, 1H, Val CH), 2.18 (m, 2H, Glu CH2),
2.47 and 2.60 (d of m, 2H, Asp CH2), 3.32 (s, 2H, AAsp CH2),
4.15 (q, 2H, OEt), 4.31 (m, 3H, R-H), 5.00 (s, 2H, Z), 6.58 (d, 1H,
CHdCH), 7.20-7.35 (m, 7H, CHdCH, Ph and NH), 7.58 (d, 1H,
NH), 7.97 (m, 2H, NH), 10.95 (b, 3H, COOH). HRMS (FAB,
M+1) calcd. for C30H40N5O14: 694.2572; Observed m/z 694.2599.
Anal. (C30H39N5O14) C, H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
methyl-N1-trans-(3-dibenzylcarbamoylacryloyl)hydrazine (Cbz-
Asp-Glu-Val-AAsp-CHdCH-CON(CH2Ph)2, 18r). 1H NMR
(DMSO-d6): 0.87 (t, 6H, Val), 1.73 (m, 1H, Glu), 1.87 (m, 1H,
Glu), 2.02 (m, 1H, Val), 2.23 (m, 2H, Glu), 2.48 (dd, 1H, Asp),
2.65 (dd, 1H, Asp), 4.19 (t, 1H, R-H), 4.33 (m, 2H, R-H), 4.55 (d,
2H, N(CH2Ph)2), 4.63 (s, 2H, N(CH2Ph)2), 5.00 (s, 2H, Cbz), 7.14
(d, 1H, db), 7.21-7.34 (m, 16H, db, Cbz-Ph, and N(CH2Ph)2), 7.60
(d, 1H, NH), 7.91-7.98 (dd, 2H, NH), 11.00 (s, 1H, NH-N).
HRMS (ESI) Calcd. for C42H48N6O13: 844.3352; Observed m/z:
844.3210. Anal. (C42H48N6O13‚0.53H2O‚0.40TFA) C, H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
methyl-N1-trans-(3-di-1-naphthylcarbamoylacryloyl)hydraz-
ine (Cbz-Asp-Glu-Val-AAsp-CHdCH-CON(CH2-1-Naphth)2,
18t). 1H NMR (acetone-d6): 1.03 (d, 6H, Val), 2.04-2.48 (m, 5H,
Glu, Val, Asp), 2.88 (dd, 1H, Asp), 2.99 (dd, 1H, Asp), 4.46 (m,
1H, R-H), 4.57 (m, 2H, R-H), 5.09 (d, 2H, N(CH2-1-Naph)2), 5.29-
5.38 (m, 4H, N(CH2-1-Naph)2), Cbz), 6.89 (d, J ) 7.2 Hz, 1H,
db), 7.30-7.99 (m, 21H, db, NH, Cbz-Ph, and N(CH2-1-Naph)2),
8.15 (d, 1H, NH), 9.87 (s, 1H, NH-N). HRMS (ESI) Calcd. for
C50H53N6O13: 945.3572; Observed m/z: 945.3665.
N1-trans-(3-Benzyloxycarbonylacryloyl)-N2-(N-benzyloxycar-
bonylaspartylglutamylvalyl)-N1-carboxymethylhydrazine (Cbz-
Asp-Glu-Val-AAsp-CHdCH-COOCH2Ph, 18i). 1H NMR (DM-
SO-d6): 0.81 (d, 6H, Val), 1.74 (m, 1H, Glu), 1.86 (m, 1H, Glu),
1.95 (m, 1H, Val), 2.18 (m, 2H, Glu), 2.45 (dd, 1H, Asp), 2.61
(dd, 1H, Asp), 4.13 (t, 1H, R-H), 4.29 (m, 2H, R-H), 4.99 (s, 2H,
Cbz), 5.18 (s, 2H, Bzl), 6.69 (d, 1H, db), 7.25-7.37 (m, 11H, Ph,
and db), 7.60 (d, 1H, NH), 7.93 (t, 2H, NH), 11.02 (s, 1H, NH-N).
HRMS (ESI) Calcd. for C35H41N5O14: 755.2728; Observed m/z:
755.2631. Anal. (C35H41N5O14‚0.59H2O) C, H, N.
N1-trans-(3-Benzylcarbamoylacryloyl)-N2-(N-benzyloxycarbo-
nylaspartylglutamylvalyl)-N1-carboxymethylhydrazine (Cbz-
Asp-Glu-Val-AAsp-CHdCH-CONHCH2Ph, 18l). 1H NMR (DM-
SO-d6): 0.85 (t, 6H, Val), 1.75 (m, 1H, Glu), 1.88 (m, 1H, Glu),
2.01 (m, 1H, Val), 2.19 (m, 2H, Glu), 2.48 (dd, 1H, Asp), 2.61
(dd, 1H, Asp), 4.17 (t, 1H, R-H), 4.30 (m, 2H, R-H), 4.35 (d, 2H,
NHCH2Ph), 5.00 (s, 2H, Cbz), 6.96 (d, 1H, db), 7.12 (d, 1H, db),
7.21-7.32 (m, 10H, Ph), 7.58 (d, 1H, NH), 7.90 (d, 1H, NH), 7.96
(d, 1H, NH), 8.92 (t, 1H, NHCH2Ph), 10.97 (s, 1H, NH-N). HRMS
(ESI) Calcd. for C35H42N6O13: 754.2888; Observed m/z: 754.2853.
Anal. (C35H42N6O13‚1.63H2O) C, H, N.
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
methyl-N1-trans-(3-(3,4-dihydro-2H-quinolin-1-ylcarbonyl)acryl-
oyl)hydrazine (Cbz-Asp-Glu-Val-AAsp-CHdCH-CO-tetrahy-
droquinoline, 18u). 1H NMR (DMSO-d6): 0.89 (t, 6H, Val), 1.75
(m, 1H, Glu), 1.87 (m, 3H, Glu and NCH2CH2CH2), 2.02 (m, 1H,
Val), 2.20 (m, 2H, Glu), 2.48 (dd, 1H, Asp), 2.65 (dd, 1H, Asp),
2.70 (t, 2H, NCH2CH2CH2), 3.73 (t, 2H, NCH2CH2CH2), 4.19 (t,
1H, R-H), 4.33 (m, 2H, R-H), 5.01 (s, 2H, Cbz), 6.95 (m, 1H, db),
7.04 (d, 1H, db), 7.14-7.33 (m, 9H, Cbz-Ph, and quinoline), 7.60
N2-(N-Benzyloxycarbonylaspartylglutamylvalyl)-N1-carboxy-
methyl-N1-trans-(3-(4-fluorobenzyl)carbamoylacryloyl)hydra-
zine (Cbz-Asp-Glu-Val-AAsp-CHdCH-CONHCH2-4-F-Ph,
18m). 1H NMR (DMSO-d6): 0.86 (t, 6H, Val), 1.75 (m, 1H, Glu),
1.88 (m, 1H, Glu), 2.01 (m, 1H, Val), 2.20 (m, 2H, Glu), 2.48 (dd,
1H, Asp), 2.65 (dd, 1H, Asp), 4.18 (t, 1H, R-H), 4.34 (m, 4H, R-H
and NHCH2Ph-4-F), 5.01 (s, 2H, Cbz), 6.93 (d, 1H, db), 7.12 (d,
2H, NHCH2Ph-4-F), 7.25-7.32 (m, 8H, Cbz-Ph, NHCH2Ph-4-F,

5744 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
(d, 1H, NH), 7.96 (dd, 2H, NH), 11.02 (s, 1H, NH-N). HRMS
(ESI) Calcd. for C37H44N6O13: 780.3039; Observed m/z: 780.2954.
Anal. (C37H44N6O13‚0.48H2O‚0.47TFA) C, H, N.
COOH). HRMS (FAB, M+1) Calcd. for C37H49N6O12: 769.3408;
Observed m/z 769.3332. Anal. (C37H48N6O12‚2H2O) C, H, N.
N1-trans-(3-Benzylmethylcarbamoylacryloyl)-N2-(N-benzyl-
oxycarbonylisoleucylglutamylthreonyl)-N1-carboxylmethylhy-
drazine (Cbz-Ile-Glu-Thr-AAsp-CHdCH-CON(CH3)CH2Ph,
N2-(N-Benzyloxycarbonylvalylglutamylvalyl)-N1-carboxy-meth-
yl-N1-trans-(3-ethoxycarbonylacryloyl)hydrazine (Cbz-Val-Glu-
Val-AAsp-CHdCH-COOEt, 19h). 1H NMR (DMSO-d6): 0.80-
0.87 (dd, 12H, Val), 1.21 (t, 3H, OCH2CH3), 1.74 (m, 1H, Glu),
1.89 (m, 1H, Glu), 1.94 (m, 1H, Val), 2.20 (m, 2H, Glu), 3.87 (t,
1H, R-H), 4.16 (m, 3H, R-H and OCH2CH3), 4.33 (t, 1H, R-H),
5.01 (s, 2H, Cbz), 6.63 (d, 1H, db), 7.20 (d, 1H, db), 7.33 (m, 5H,
Ph), 7.97 (t, 2H, NH), 11.02 (s, 1H, NH-N). HRMS (ESI) Calcd.
for C31H43N5O12: 677.2986; Observed m/z: 677.2978. Anal.
(C31H43N5O12‚1.41H2O‚0.51hex) C, H, N.
1
20o). H NMR (DMSO-d6): 0.80 (m, 6H, Ile CH3), 1.06 (m, 3H,
Thr CH3), 1.49 (m, 2H, Ile CH2), 1.65 (m, 1H, Ile CH), 1.80 and
1.92 (d of m, 2H, Glu CH2), 2.24 (m, 2H, Glu CH2), 2.88 and 2.99
(d, 3H, NCH3), 3.32 (s, 2H, AAsp CH2), 4.02 (m, 2H, R-H), 4.20
(m, 1H, R-H), 4.40 (m, 1H, Thr CH), 4.55 (m, 2H, CH2Ph), 5.00
(s, 2H, Z), 7.10-7.45 (m, 14H, CHdCH, Ph and NH), 7.80 (m,
1H, NH), 8.05 (m, 1H, NH),10.90 (b, 2H, COOH). HRMS (FAB,
M+1) Calcd. for C37H49N6O12: 769.3408; Observed m/z 769.3394.
Anal. (C37H48N6O12‚2H2O) C, H, N.
N2-(N-Benzyloxycarbonylisoleucylglutamylthreonyl)-N1-car-
boxymethyl-N1-trans-(3-ethoxycarbonylacryloyl)hydrazine (Cbz-
N2-(N-Benzyloxycarbonylisoleucylglutamylthreonyl)-N1-car-
boxylmethyl-N1-trans-(3-methylphenylethylcarbamoylacryloyl)-
hydrazine (Cbz-Ile-Glu-Thr-AAsp-CHdCH-CON(CH3)CH2-
CH2Ph, 20p). 1H NMR (acetone-d6): 0.88 (m, 3H, Ile CH3), 0.97
(m, 3H, Ile CH3), 1.21 (m, 3H, Thr CH3), 1.60 (m, 3H, Ile CH and
CH2), 2.00 (m, 2H, Glu CH2), 2.47 (m, 2H, Glu CH2), 2.88 (m,
4H, CH2Ph and AAsp CH2), 2.98 and 3.10 (d, 3H, CH3), 3.67 (m,
2H, CH2N), 4.13 (m, 1H, R-H), 4.32 (m, 1H, R-H), 4.50 (m, 2H,
R-H and Thr CH), 5.09 (m, 2H, Z), 6.62 (b, 1H, NH), 7.10-7.40
(m, 13H, CHdCH, NH and Ph), 7.65 (b, 1H, NH), 7.90 (b, 1H,
NH). HRMS (FAB) Calcd. for C38H51N6O12: 783.3565; Observed
m/z 783.3569. Anal. (C38H50N6O12‚2H2O) C, H, N.
1
Ile-Glu-Thr-AAsp-CHdCH-COOEt, 20h). H NMR (DMSO-
d6): 0.8 (m, 6H, Ile CH3), 1-1.2 (m, 4H, Thr CH3 and Ile CH2),
1.2-1.3 (t, 3H, OCH2CH3), 1.4 (m, 1H, Ile CH2), 1.6-1.8 (m, 2H,
CH Ile and Glu CH2), 1.8-2.0 (m, 1H, Glu CH2), 2.15-2.35 (m,
2H, Glu CH2), 3.85-4.05 (m, 3H, NCH2COOH and CH-OH), 4.1-
4.3 (m, 3H, OCH2CH3 and R-H), 4.4 (m, 2H, R-H), 5.05 (m, 2H,
Cbz), 6.6-6.65 (d, 1H, CHdCH), 7.20-7.40 (m, 6H, Ph and CHd
CH), 7.80 (m, 1H, NH), 8.1 (m, 1H, NH). HRMS (FAB, M+1)
Calcd. for C31H44N5O13: 694.29356; Observed m/z 694.29979.
Anal. (C31H43N5O13‚H2O) C, H, N.
N1-trans-(3-Benzyloxycarbonylacryloyl)-N2-(N-benzyloxycar-
bonylisoleucylglutamylthreonyl)-N1-carboxylmethylhydrazine
(Cbz-Ile-Glu-Thr-AAsp-CHdCH-COOCH2Ph, 20i). 1H NMR
(DMSO-d6): 0.80 (m, 6H, Ile CH3), 1.02 (m, 3H, Thr CH3), 1.10
(m, 2H, Ile CH2), 1.65 (m, 1H, Ile CH), 1.75 and 1.85 (d of m, 2H,
Glu CH2), 2.25 (m, 2H, Glu CH2), 3.30 (s, 2H, AAsp CH2), 3.90
(m, 2H, R-H), 4.25 (m, 1H, R-H), 4.39 (m, 1H, Thr CH), 5.00 (s,
2H, Z), 5.20 (s, 2H, CH2Ph), 6.70 (d, 1H, NH), 7.20-7.40 (m,
12H, Ph and CHdCH), 7.90 (b, 1H, NH), 8.10 (b, 1H, NH), 8.60
(m, 1H, NH), 10.70 (b, 2H, COOH). HRMS (FAB, M+1) Calcd.
for C36H46N5O13: 756.3092; Observed m/z 756.3037. Anal.
(C36H45N5O13‚2H2O) C, H, N.
N2-(N-Benzyloxycarbonylisoleucylglutamylthreonyl)-N1-car-
boxylmethyl-N1-trans-(3-dibenzylcarbamoylacryloyl)hydraz-
ine (Cbz-Ile-Glu-Thr-AAsp-CHdCH-CON(CH2Ph)2, 20r). 1H
NMR (DMSO-d6): 0.80 (m, 6H, Ile CH3), 1.06 (m, 3H, Thr CH3),
1.50 (m, 2H, Ile CH2), 1.65 (m, 1H, Ile CH), 1.78 and 1.90 (d of
m, 2H, Glu CH2), 2.25 (m, 2H, Glu CH2), 3.32 (s, 2H, AAsp CH2),
4.00 (m, 2H, R-H), 4.25 (m, 1H, R-H), 4.40 (m, 1H, Thr CH),
4.60 (d, 4H, CH2Ph), 5.00 (s, 2H, Z), 7.00-7.35 (m, 13H, CHd
CH, Ph and NH), 7.85 (b, 1H, NH), 8.10 (d, 1H, NH), 8.60 (b, 1H,
NH), 10.75 (b, 2H, COOH). HRMS (FAB, M+1) Calcd. for
C43H53N6O12: 845.3721; Observed m/z 845.3731. Anal. (C43H52-
N6O12‚2H2O) C, H, N.
N2-(N-Benzyloxycarbonylisoleucylglutamylthreonyl)-N1-car-
boxylmethyl-N1-trans-(3-phenylcarbamoylacryloyl)hydrazine
(Cbz-Ile-Glu-Thr-AAsp-CHdCH-CONHPh, 20k). 1H NMR
(DMSO-d6): 0.84 (t, 6H, Ile CH3), 1.06 (m, 3H, Thr CH3), 1.45
(m, 2H, Ile CH2), 1.70 (m, 1H, Ile CH), 1.80 and 1.90 (d of m, 2H,
Glu CH2), 2.25 (m, 2H, Glu CH2), 3.33 (s, 2H, AAsp CH2), 3.88
(t, 1H, R-H), 4.00 (m, 1H, R-H), 4.25 (m, 1H, R-H), 4.39 (m, 1H,
Thr CH), 5.00 (s, 2H, Z), 7.00-7.10 (m, 4H, CHdCH and Ph),
7.30 (m, 7H, Ph and NH), 7.64 (d, 2H, Ph), 7.85 (b, 1H, NH), 8.05
(d, 1H, NH), 8.35 (m, 1H, NH), 8.60 (m, 1H, NH), 10.45 (b, 2H,
COOH). HRMS (FAB, M+1) Calcd. for C35H45N6O12: 741.3095;
Observed m/z 741.3106. Anal. (C35H44N6O12‚2H2O) C, H, N.
N1-trans-(3-Benzylcarbamoylacryloyl)-N2-(N-benzyloxycarbo-
nylisoleucylglutamylthreonyl)-N1-carboxylmethylhydrazine (Cbz-
Ile-Glu-Thr-AAsp-CHdCH-CONHCH2Ph, 20l). 1H NMR (DM-
SO-d6): 0.75-0.87 (m, 6H, Ile CH3), 1.02 (m, 3H, Thr CH3), 1.49
(m, 2H, Ile CH2), 1.65 (m, 1H, Ile CH), 1.74 and 1.85 (d of m, 2H,
Glu CH2), 2.25 (m, 2H, Glu CH2), 3.32 (s, 2H, AAsp CH2), 4.00
(m, 2H, R-H), 4.25 (m, 1H, R-H), 4.40 (m, 3H, Thr CH and CH2-
Ph), 5.00 (s, 2H, Z), 7.00 (d, 1H, CHdCH), 7.40-7.40 (m, 13H,
CHdCH, Ph and NH), 7.90 (b, 1H, NH), 8.05 (b, 1H, NH), 8.90
(b, 1H, NH), 10.60 (b, 2H, COOH). HRMS (FAB, M+1) Calcd.
for C36H47N6O12: 755.3252; Observed m/z 755.3200. Anal.
(C36H46N6O12‚2H2O) C, H, N.
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carboxy-
methyl-N1-trans-(3-ethoxycarbonylacryloyl)hydrazine (Cbz-Leu-
1
Glu-Thr-AAsp-CHdCH-COOEt, 21h). H NMR (DMSO-d6):
0.85 (t, 6H, Leu CH3), 1.05 (d, 3H, Thr CH3), 1.25 (t, 3H,
OCH2CH3), 1.4 (m, 2H, Leu CH2), 1.6 (m, 1H, CH Leu), 1.75 (m,
1H, Glu CH2), 1.9 (m, 1H, Glu CH2), 2.25 (m, 2H, Glu CH2), 3.9-
4.1 (m, 3H, NCH2COOH and R-H), 4.1-4.3 (m, 3H, OCH2CH3
and CH-OH), 4.3-4.4 (m, 2H, R-H), 5.02 (m, 2H, Cbz), 6.6-6.65
(d, 1H, CHdCH), 7.20-7.40 (m, 6H, Ph and CHdCH), 7.50 (d,
1H, NH), 7.78 (m, 1H, NH), 8.05 (m, 1H, NH). HRMS (FAB,
M+1) Calcd. for C31H44N5O13: 694.29356; Observed m/z 694.29963.
Anal. (C31H43N5O13‚H2O) C, H, N.
N1-trans-(3-Benzyloxycarbonylacryloyl)-N2-(N-benzyloxycar-
bonylleucylglutamylthreonyl)-N1-carboxymethylhydrazine (Cbz-
Leu-Glu-Thr-AAsp-CHdCH-COOCH2Ph, 21i). 1H NMR (DM-
SO-d6): 0.84 (t, 6H, Leu), 1.02 (d, 3H, Thr), 1.40 (m, 2H, Leu),
1.60 (m, 1H, Glu), 1.73 (m, 1H, Glu), 1.90 (m, 1H, Leu), 2.22 (m,
2H, Glu), 3.94 (m, 1H, R-H), 4.03 (m, 1H, R-H), 4.20 (m, 1H,
R-H), 4.37 (m, 1H, Thr), 4.99 (s, 2H, Cbz), 5.19 (s, 2H, Bzl), 6.65
(d, 1H, db), 7.32-7.36 (m, 10H, Ph), 7.42 (d, 1H, db), 7.76 (d,
1H, NH), 8.03 (d, 1H, NH), 10.84 (s, 1H, NH-N). HRMS (ESI)
Calcd. for C36H45N5O13: 755.3092; Observed m/z: 755.3150. Anal.
(C36H45N5O13‚0.27H2O‚0.28TFA) C, H, N.
N2-(N-Benzyloxycarbonylisoleucylglutamylthreonyl)-N1-car-
boxylmethyl-N1-trans-(3-phenyethylcarbamoylacryloyl)hydra-
zine (Cbz-Ile-Glu-Thr-AAsp-CHdCH-CONHCH2CH2Ph, 20n).
1H NMR (DMSO-d6): 0.82 (m, 6H, Ile CH3), 1.04 (m, 3H, Thr
CH3), 1.45 (m, 2H, Ile CH2), 1.65 (m, 1H, Ile CH), 1.75 and 1.90
(d of m, 2H, Glu CH2), 2.24 (m, 2H, Glu CH2), 2.73 (t, 2H, CH2-
Ph), 3.32 (m, 4H, AAsp CH2 and NCH2), 3.95 (m, 2H, R-H), 4.22
(m, 1H, R-H), 4.40 (m, 1H, Thr CH), 5.00 (s, 2H, Z), 6.88 (d, 1H,
CHdCH), 7.10-7.45 (m, 13H, CHdCH, Ph and NH), 7.90 (b,
2H, NH), 8.05 (b, 1H, NH), 8.52 (b, 1H, NH), 10.73 (b, 2H,
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-phenylcarbamoylacryloyl)hydrazine (Cbz-
Leu-Glu-Thr-AAsp-CHdCHCONHPh, 21k). 1H NMR (DMSO-
d6): 0.85 (t, 6H, Leu CH3), 1.06 (m, 3H, Thr CH3), 1.49 (m, 2H,
Leu CH2), 1.61 (m, 1H, Leu CH), 1.80 and 1.95 (d of m, 2H, Glu
CH2), 2.26 (m, 2H, Glu CH2), 3.32 (s, 2H, AAsp CH2), 4.03 (m,
2H, R-H), 4.25 (m, 1H, R-H), 4.40 (m, 1H, Thr CH), 5.00 (s, 2H,
Z), 7.00-7.10 (m, 4H, CHdCH and Ph), 7.30 (m, 7H, Ph and NH),
7.64 (d, 2H, Ph), 7.85 (m, 1H, NH), 8.05 (b, 1H, NH), 8.20 (m,
1H, NH), 8.75 (b, 1H, NH), 10.45 (s, 2H, COOH). HRMS (FAB,

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5745
M+1) Calcd. for C35H45N6O12: 741.3095; Observed m/z 741.3064.
Anal. (C35H44N6O12‚2H2O) C, H, N.
NH), 8.65 (m, 1H, NH), 10.75 (b, 2H, COOH). HRMS (FAB,
M+1) Calcd. for C43H53N6O12, 845.3721; Observed m/z 845.3777.
Anal. (C43H52N6O12‚2H2O) C, H, N.
N1-trans-(3-Benzylcarbamoylacryloyl)-N2-(N-benzyloxycarbo-
nylleucylglutamylthreonyl)-N1-carboxylmethylhydrazine (Cbz-
Leu-Glu-Thr-AAsp-CHdCHCONHCH2Ph, 21l). 1H NMR (DM-
SO-d6): 0.85 (t, 6H, Leu CH3), 1.06 (m, 3H, Thr CH3), 1.49 (m,
2H, Leu CH2), 1.61 (m, 1H, Leu CH), 1.80 and 1.90 (d of m, 2H,
Glu CH2), 2.26 (m, 2H, Glu CH2), 3.32 (s, 2H, AAsp CH2), 4.03
(m, 2H, R-H), 4.25 (m, 1H, R-H), 4.45 (m, 3H, Thr CH and CH2-
Ph), 5.00 (s, 2H, Z), 7.00 (d, 1H, CHdCH), 7.20-7.40 (m, 13H,
CHdCH, Ph and NH), 7.80 (b, 1H, NH), 8.05 (d, 1H, NH), 8.90
(d, 1H, NH), 10.65 (b, 2H, COOH). HRMS (FAB, M+1) Calcd.
for C36H47N6O12: 755.3252; Observed m/z 755.3184. Anal.
(C36H46N6O12‚2H2O) C, H, N.
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-(methyl-1-naphthylmethyl)carbamoylacryl-
oyl)hydrazine (Cbz-Leu-Glu-Thr-AAsp-CHdCH-CON(CH3)-
1
CH2-1-Naphth, 21s). H NMR (acetone-d6): 0.92 (m, 6H, Leu
CH3), 1.22 (m, 3H, Thr CH3), 1.65 (m, 2H, Leu CH2), 1.75 (m,
1H, Leu CH), 2.00 (m, 2H, Glu CH2), 2.47 (m, 2H, Glu CH2),
3.08 (d, 4H, AAsp CH2 and NCH3), 4.25 (m, 1H, R-H), 4.35 (m,
2H, R-H), 4.50 (m, 1H, Thr CH), 5.07 (m, 2H, Z), 5.15 (m, 2H,
CH2-naphth), 6.75 (b, 1H, NH), 7.20-7.35 (m, 6H, CHdCH and
Ph), 7.40-7.60 (m, 7H, Ph and NH), 7.90 (m, 2H, naphth), 7.80
(b, 1H, NH), 8.10 (b, 1H, NH). HRMS (FAB, M+1) Calcd. for
C41H51N6O12, 819.3565; Observed m/z 819.3521. Anal. (C41H50N6O12‚
2H2O) C, H, N.
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-(4-fluorobenzyl)carbamoylacryloyl)hydra-
zine (Cbz-Leu-Glu-Thr-AAsp-CHdCHCONHCH2-4-F-Ph, 21m).
1H NMR (DMSO-d6): 0.85 (t, 6H, Leu CH3), 1.04 (m, 3H, Thr
CH3), 1.60 (m, 2H, Leu CH2), 1.75 (m, 1H, Leu CH), 1.90 (m,
2H, Glu CH2), 2.24 (m, 2H, Glu CH2), 3.32 (s, 2H, AAsp CH2),
4.03 (m, 2H, R-H), 4.25 (m, 1H, R-H), 4.40 (m, 3H, Thr CH and
CH2Ph), 5.00 (s, 2H, Z), 6.93 (d, 1H, CHdCH), 7.10-7.40 (m,
10H, CHdCH and Ph), 7.85 (b, 1H, NH), 8.05 (d, 1H, NH), 8.70
(b, 1H, NH), 8.94 (m, 2H, NH), 10.60 (b, 2H, COOH). HRMS
(FAB, M+1) Calcd. for C36H46N6O12F: 773.3153; Observed m/z
773.3061. Anal. (C36H45N6O12F‚2H2O) C, H, N.
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-(3,4-dihydro-2H-quinolin-1-yloxo)acrylo-
yl)hydrazine (Cbz-Leu-Glu-Thr-AAsp-CHdCH-CO-tetrahy-
droquinoline, 21t). 1H NMR (acetone-d6): 0.94 (m, 6H, Leu CH3),
1.22 (m, 3H, Thr CH3), 1.66 (m, 2H, Leu CH2), 1.78 (m, 1H, Leu
CH), 1.98 (m, 2H, quinoline CH2), 2.00 (m, 2H, Glu CH2), 2.46
(m, 2H, Glu CH2), 2.80 (m, 4H, quinoline CH2 and Asp CH2), 3.81
(m, 2H, NCH2), 4.28 (m, 1H, R-H), 4.38 (m, 1H, R-H), 4.50 (m,
2H, R-H and Thr CH), 5.09 (m, 2H, Z), 6.75 (b, 1H, NH), 7.10-
7.40 (m, 10H, CHdCH and Ph), 7.48 (d, 1H, Ph), 7.60 (d, 1H,
NH), 7.90 (d, 1H, NH), 9.00 (s, 1H, NH). HRMS (FAB) Calcd.
for C38H49N6O12: 781.3408; Observed m/z 781.3367. Anal.
(C38H48N6O12‚2H2O) C, H, N.
Enzyme Assays. Caspase-2, -3, -6, -7, -8, -9 and -10. Caspases-
2, -3, -6, -7, -8, -9, and -10 were expressed in E. coli and purified
in Guy Salvesen’s laboratory at the Burnham Institute, La Jolla,
CA, according to the methods previously described by Stennicke
and Salvesen.41 Inhibition rates were determined by the progress
curve method described by Tian and Tsou.42 This method is suitable
for measuring irreversible inhibition rates with fast inhibitors, where
the inhibitor, the substrate, and the enzyme are incubated together
and the rate of substrate hydrolysis is measured continuously. The
rate of substrate hydrolysis in the presence of the inhibitor was
monitored for 20 min. The progress curve of inhibition, where the
product formation approaches an asymptote is described in the
equation
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-phenylethylcarbamoylacryloyl)hydrazine
(Cbz-Leu-Glu-Thr-AAsp-CHdCH-CONHCH2CH2Ph, 21n). 1H
NMR (DMSO-d6): 0.85 (t, 6H, Leu CH3), 1.06 (m, 3H, Thr CH3),
1.45 (m, 2H, Leu CH2), 1.60 (m, 1H, Leu CH), 1.75 and 1.88 (d of
m, 2H, Glu CH2), 2.25 (m, 2H, Glu CH2), 2.75 (t, 2H, CH2Ph),
3.30-3.35 (m, 4H, CH2N and AAsp CH2), 4.00 (m, 2H, R-H), 4.20
(m, 1H, R-H), 4.38 (m, 1H, Thr CH), 5.00 (s, 2H, Z), 6.90-7.15
(d of d, 2H, CHdCH), 7.20-7.40 (m, 12H, Ph and NH), 7.86 (m,
1H, NH), 8.06 (d, 1H, NH), 8.50 (b, 1H, NH), 10.60 (b, 2H,
COOH). HRMS (FAB, M+1) Calcd. for C37H49N6O12: 769.3408;
Observed m/z 769.3201. Anal. (C37H48N6O12‚2H2O) C, H, N.
N1-trans-(3-Benzylmethylcarbamoylacryloyl)-N2-(N-benzyl-
oxycarbonylleucylglutamylthreonyl)-N1-carboxylmethylhydra-
zine (Cbz-Leu-Glu-Thr-AAsp-CHdCH-CON(CH3)CH2Ph, 21o).
1H NMR (DMSO-d6): 0.83 (t, 6H, Leu CH3), 1.06 (m, 3H, Thr
CH3), 1.49 (m, 2H, Leu CH2), 1.65 (m, 1H, Leu CH), 1.80 and
1.92 (d of m, 2H, Glu CH2), 2.24 (m, 2H, Glu CH2), 2.88 and 2.99
(d, 2H, CH3), 3.32 (s, 2H, AAsp CH2), 4.02 (m, 2H, R-H), 4.20
(m, 1H, R-H), 4.40 (m, 1H, Thr CH), 4.55 (m, 2H, CH2Ph), 5.00
(s, 2H, Z), 7.10-7.45 (m, 14H, CHdCH, Ph and NH), 7.80 (m,
1H, NH), 8.05 (m, 1H, NH),10.85 (b, 2H, COOH). HRMS (FAB,
M+1) Calcd. for C37H49N6O12: 769.3408; Observed m/z 769.3320.
Anal. (C37H48N6O12‚2H2O) C, H, N.
ln([P∞] - [P]) ) ln[P∞] - A[I]t
where [P] and [P∞] are the product concentrations at t and t ) ∞,
respectively, A is the apparent rate constant in the presence of the
substrate. The apparent rate constants were determined from the
slopes of plots of ln([P∞] - [P]) versus time (t) in seconds as
previously described, where A ) slope/[I].
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-methylphenylethylcarbamoylacryloyl)hy-
drazine (Cbz-Leu-Glu-Thr-AAsp-CHdCH-CON(CH3)CH2-
CH2Ph, 21p). 1H NMR (acetone-d6): 0.94 (m, 6H, Leu CH3), 1.20
(m, 3H, Thr CH3), 1.65 (m, 2H, Leu CH2), 1.75 (m, 1H, Leu CH),
2.00 (m, 2H, Glu CH2), 2.46 (m, 2H, Glu CH2), 2.88 (m, 4H, CH2-
Ph and AAsp CH2), 2.98 and 3.11 (d, 3H, NCH3), 3.68 (m, 2H,
NCH2), 4.30 (m, 2H, R-H), 4.50 (m, 2H, R-H and Thr CH), 5.09
(m, 2H, Z), 5.65 (b, 1H, NH), 6.75 (b, 1H, NH), 7.10-7.40 (m,
12H, CHdCH and Ph), 7.85 (b, 1H, NH), 7.95 (b, 1H, NH). HRMS
(FAB, M+1) Calcd. for C38H51N6O12: 783.3565; Observed m/z
783.3493. Anal. (C38H50N6O12‚2H2O) C, H, N.
For competitive and irreversible inhibition, the apparent rate
constant is converted to the second-order rate constant k2 by taking
into consideration the effect of the substrate concentration on the
apparent rate constant. The second-order rate constant is described
as in the equation:
k2 ) A × (1 + [S]/KM)
Assays using the fluorogenic substrates Ac-DEVD-AMC (λex
) 360 nm, λem ) 465 nm), Z-VDVAD-AFC (λex ) 430 nm, λem
) 535 nm), and Ac-LEHD-AFC were carried out on a Tecan
Spectra Fluor microplate reader at 37 °C. The KM values for Ac-
N2-(N-Benzyloxycarbonylleucylglutamylthreonyl)-N1-carbox-
ylmethyl-N1-trans-(3-dibenzylcarbamoylacryloyl)hydrazine (Cbz-
Leu-Glu-Thr-AAsp-CHdCH-CON(CH2Ph)2, 21r). 1H NMR
(DMSO-d6): 0.85 (t, 6H, Leu CH3), 1.06 (m, 3H, Thr CH3), 1.45
(m, 2H, Leu CH2), 1.60 (m, 1H, Leu CH), 1.80 and 1.90 (d of m,
2H, Glu CH2), 2.25 (m, 2H, Glu CH2), 3.32 (s, 2H, AAsp CH2),
4.03 (m, 2H, R-H), 4.25 (m, 1H, R-H), 4.40 (m, 1H, Thr CH),
4.60 (d, 4H, CH2Ph), 5.00 (s, 2H, Z), 7.10-7.35 (m, 12H, CHd
CH and Ph), 7.40 (b, 1H, NH), 7.80 (b, 1H, NH), 8.07 (b, 1H,
DEVD-AMC with caspase-3 (KM ) 9.7 µM), caspase-6 (KM )
236.35 µM), caspase-7 (KM ) 23.0 µM), caspase-8 (KM ) 6.79
µM), and caspase-10 (KM ) 20.2 µM) were determined in the
laboratory of Guy Salvesen. The KM value for Ac-LEHD-AFC with
caspase-9 (KM ) 114 µM) was also determined in the laboratory
of Guy Salvesen. The KM value for Z-VDVAD-AFC with caspase-2
was found to be 80.5 µM. The k2 values are 2.24-fold higher than
the apparent rate for caspase-2 because of the 100 mM [S] and KM
) 80.5 µM. The k2 values are 11.31-fold higher than the apparent

5746 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
rate for caspase-3 because of the 100 mM [S] and KM ) 9.7 µM.
The k2 values are 1.42-fold higher than the apparent rate for
caspase-6 because of the 100 mM [S] and KM ) 236.35 µM. The
k2 values are 5.35-fold higher than the apparent rate for caspase-7
because of the 100 mM [S] and KM ) 23.0 µM. The k2 values are
15.73-fold higher than the apparent rate for caspase-8 because of
the 100 mM [S] and KM ) 6.79 µM. The k2 values are 2.32-fold
higher than the apparent rate for caspase-9 because of the 150 mM
[S] and KM ) 114 µM. The k2 values are 8.43-fold higher than the
The progress curve data of all the ester derivatives (16h, 17h,
18g, 18h, 19h, 20h, 20i, 21h, 21i) was observed for a period of 20
min. However, only the data points corresponding to the first 10
min of the incubation were used for the kinetic calculations, during
which time the inhibitors were still stable and the progress curve
data had already reached its plateau.
S. mansoni Legumain. Conor R. Caffrey in James McKerrow’s
laboratory performed the S. mansoni legumain kinetic assays at
the Sandler Center for Basic Research in Parasitic Diseases,
University of California at San Francisco, San Francisco, CA. The
zymogen form of schistosome legumain SmAE was expressed in
Pichia. The lyophilized enzyme (50-100 mg) was reconstituted
in 1.5 mL 0.5 M sodium acetate, pH 4.5 containing 4 mM DTT,
and left to stand at 37 °C for 3-4 h to allow for auto-activation of
the zymogen. In a black 96-well microtiter plate, 50 µL of activated
enzyme was added to an equal volume of 0.1 M citrate-phosphate
buffer pH 6.8 containing 4 mM DTT. The inhibitors, added as 1
µL aliquots (serial water dilutions of DMSO stock solutions, 2 to
0.00002 µM [final]), were preincubated with the protease at room
temperature for 20 min. After incubation, 100 µL of the same buffer
containing 20 µM substrate (Cbz-Ala-Ala-Asn-AMC) was added
to the wells and the reaction monitored for 20 min. A plot of the
RFU/min versus the inhibitor concentration [µM] permitted calcula-
tion of an IC50 value.
Pichia cell culture medium containing recombinant S. mansoni
pro-legumain was incubated overnight at room temperature in 0.3
M sodium acetate, pH 4.5, 2 mM DTT, to allow autoactivation of
the endogenous zymogen. Inhibitor (1 µL) at 6 concentrations (to
yield 0 to 1 µM [final]) was spotted into a 96-well black microtiter
plate. To this was added 180 µL 0.1 M citrate-phosphate buffer,
pH 6.8, containing 2 mM DTT and 20 µM Cbz-AAN-AMC
substrate. Activated legumain (20 µL) was added to the mix and
the progress of inhibition followed every 2 s for 30 min at 25 °C
(Molecular Devices Flex Station fluorometer in the injection mode;
ex 355/em 460).
Clostripain. Clostripain was purchased from Sigma Chemical
Co. (St. Louis, MO) as a solid which was dissolved in an activation
solution of 8 mM DTT at a concentration of 5.962 µM and stored
at -20 °C prior to use. The inhibition of clostripain began with
the addition of 25 µL of stock inhibitor solution (concentration
varies by inhibitor) in DMSO to a solution of 250 µL of 20 mM
Tris/HCl, 10 mM CaCl2, 0.005% Brij 35, 2 mM DTT buffer, at
pH 7.6 (clostripain buffer) and 5 µL of the stock enzyme solution.
Aliquots (25 µL) of this incubation mixture were taken at various
time points and added to a solution containing 100 µL of the
clostripain buffer and 5 µL of Z-Phe-Arg-AMC substrate solution
(0.139 mM) in DMSO. The enzymatic activity was monitored by
following the change in fluorescence at 465 nm. All data obtained
was processed by pseudo-first-order kinetics.
apparent rate for caspase-10 because of the 150 mM [S] and KM
20.2 µM.
)
The concentration of the caspase-3 stock solution was 2 nM in
the assay buffer. Assay buffer is a 1:1 mixture of caspase buffer
(40 mM Pipes, 200 mM NaCl, 0.2% (w/v) CHAPS, sucrose 20%
(w/v)) and 20 mM DTT solution in H2O at pH 7.2. The concentra-
tion of the substrate stock solution was 2 mM in DMSO. The
enzyme was preactivated for 10 min at 37 °C in the assay buffer.
The standard 100 µL reaction was started by adding 40 µL of assay
buffer, 5 µL of various amounts of inhibitor (stock solution
concentrations varied from 5 × 10-3 M to 4.84 × 10-7 M in
DMSO), and 5 µL of substrate in DMSO (100 µM final concentra-
tion) at 37 °C. 50 µL of 2 nM enzyme stock solution (final
concentration: 1 nM) was added to the mixture after 1 min and
reading started immediately for 20 min at 37 °C. Inhibition
experiments were repeated in duplicate and standard deviations
determined.
Caspase-2 kinetic assays were performed using Z-VDVAD-AFC
as the substrate (2 mM stock solution in DMSO) and with the same
conditions as caspase-3. The concentration of the caspase-2 stock
solution was 86.7 nM in the assay buffer (final concentration in
the well: 43.3 nM). The inhibitor stock solution concentrations
varied from 5 × 10-3 M to 1 × 10-4 M in DMSO.
Caspase-6 kinetic assays were performed using the same condi-
tions and the same substrate (Ac-DEVD-AMC, 2 mM stock solution
in DMSO). The enzyme stock solution was 10 nM (final concentra-
tion in the well: 5 nM) in the assay buffer. The inhibitor stock
solution concentrations varied from 5 × 10-3 M to 2.42 × 10-6
M
in DMSO.
Caspase-7 kinetic assays were performed using the same condi-
tions and the same substrate (Ac-DEVD-AMC, 2 mM stock solution
in DMSO). The enzyme stock solution was 10 nM (final concentra-
tion in the well: 5 nM) in the assay buffer. The inhibitor stock
solution concentrations varied from 5 × 10-3 M to 2.5 × 10-6
M
in DMSO.
Caspase-8 kinetic assays were performed using the same condi-
tions and the same substrate (Ac-DEVD-AMC, 2 mM stock solution
in DMSO). The enzyme stock solution was 100 nM (final
concentration in the well: 50 nM) in the assay buffer. The inhibitor
stock solution concentrations varied from 5 × 10-3 M to 2.42 ×
10-6 M in DMSO.
Gingipain K. Gingipain K stock solution was obtained from
Jan Potempa’s lab (University of Georgia, Athens, GA) in a buffer
containing 20 mM Bis-Tris, 150 mM NaCl, 5 mM CaCl2, 0.02%
NaN3, at pH 8.0 at a concentration of 9 µM, which was stored at
-20 °C prior to use. Before using the enzyme, an aliquot (1 µL)
of the stock enzyme was diluted to a concentration of 4.61 nM in
1.951 mL of a solution of 0.2 M Tris/HCl, 0.1 M NaCl, 5 mM
CaCl2, 2 mM DTT at pH 8.0 (gingipain K buffer) and kept at 0
°C. This solution was used only for 1 day, as freezing the enzyme
at this concentration destroyed all activity. The inhibition of
gingipain K began with the addition of 25 µL of stock inhibitor
solution (concentration varies by inhibitor) in DMSO to 244 µL of
the diluted enzyme solution (4.61 nM) in gingipain K buffer warmed
to room temperature. Aliquots (20 µL) of this were taken at various
time points and added to a solution containing 100 µL of the
gingipain K buffer and 5 µL of Suc-Ala-Phe-Lys-AMC‚TFA as
the substrate (0.910 mM stock) in DMSO. The enzymatic activity
was monitored by following the change in fluorescence at 465 nm.
The data for gingipain K was processed by pseudo-first-order
kinetics.
Caspase-9 kinetic assays were performed using Ac-LEHD-AFC
as the substrate (3 mM stock solution in DMSO) and with the
following conditions. The concentration of the caspase-9 stock
solution was 150 nM in the assay buffer (final concentration in the
well: 75 nM). Assay buffer is a 1:1 mixture of buffer (200 mM
HEPES, 100 mM NaCl, 0.01% (w/v) CHAPS, sucrose 20% (w/v))
and 20 mM DTT solution in H2O at pH 7.0. The assay buffer was
supplemented with 0.7 M sodium citrate. The enzyme was
preactivated for 10 min at 37 °C in the assay buffer. The inhibitor
stock solution concentrations varied from 5 × 10-3 M to 2.5 ×
10-5 M in DMSO.
Caspase-10 kinetic assays were performed using the same
substrate as caspase-3 (Ac-DEVD-AMC, 3 mM stock solution in
DMSO) and with the following conditions. The concentration of
the caspase-10 stock solution was 50 nM in the assay buffer (final
concentration in the well: 25 nM). Assay buffer is a 1:1 mixture
of buffer (200 mM HEPES, 0.2% (w/v) CHAPS, PEG 20% (w/v))
and 20 mM DTT solution in H2O at pH 7.0. The enzyme was
preactivated for 10 min at 25 °C in the assay buffer. The inhibitor
stock solution concentrations varied from 5 × 10-3 M to 2.5 ×
10-5 M in DMSO.
Cathepsin B. Irreversible kinetic assays were performed by the
incubation method with human liver cathepsin B. Enzymatic

Inhibitors of Caspases-2, -3, -6, -7, -8, -9, and -10
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5747
activities of cathepsin B were measured in 0.1 M KHPO4, 1.25
mM EDTA, 0.01% Brij, pH 6.0 buffer and at 23 °C using Cbz-
Arg-Arg-AMC as the substrate. To a freshly prepared enzyme stock
solution of cathepsin B (approximately 6.98 × 10-3 µg/µL) in
enzyme buffer containing DTT (0.1 mM) was added an additional
300 µL of enzyme buffer and 30 µL of a stock inhibitor solution
in DMSO. At various time intervals 50 µL aliquots were withdrawn
from the incubation mixture and added to 200 µL enzyme buffer
containing Cbz-Arg-Arg-AMC (500 µM). Substrate hydrolysis was
(Met-Ala-Glu376-Asp479) were inserted in the NcoI/BamHI sites of
pET11d plasmids (Novagen). For caspase-8, the cloned genes for
the large p18 subunit (Met-Gly-Glu218-Asp374) and the small p12
subunit (Met-Ala-Glu376-Asp479) were inserted in the NcoI/BamHI
sites of pET11d plasmids (Novagen). For separate expression of
both subunits, E. coli BL21-CodonPlus (DE3)-RIL cells (Strat-
agene), containing one of the two plasmids, were grown to a density
of A600 ) 0.5 at 37 °C in a 0.5-L LB medium. Expression was
induced by the addition of IPTG (1 mM), and the culture was
shaken at 37 °C for 4 h post induction. Cells were harvested, washed
in PBS buffer, and lysed using a French press. Due to overexpres-
sion, the protein was localized in the inclusion body portion of the
cells. Refolding was achieved by rapid mixing of equimolar amounts
of each subunit to a final concentration of about 100 µg of subunit/
mL in the refolding buffer (100 mM HEPES, pH 7.5, 10% sucrose,
1% CHAPS, 100 mM NaCl and 10 mM DTT) and was incubated
overnight at room temperature with continuous stirring. Misfolded
and aggregated protein was removed by centrifugation (5000g, 30
min), and the supernatant was concentrated using an Amicon-stirred
cell. To reduce the salt concentration and to facilitate binding to
an anion exchange column (ResQ 1 mL, Amersham Biosciences),
the protein solution was dialyzed against the anion exchange buffer
(20 mM Tris pH 8.0, 30 mM NaCl and 10 mM DTT) prior to
chromatography. The protein was eluted using a 300 mM NaCl
gradient. The pure and active caspase-3 and caspase-8 were further
purified by size exclusion chromatography on an analytical S200
column with buffer containing 20 mM Tris pH 8.0, 50 mM NaCl,
and 10 mM DTT. Fractions containing pure and active caspase were
pooled and concentrated by ultrafiltration (centricon, NMWL 10000
Da) to a final concentration of 10 mg/mL and the final yield was
about 5-7 mg/L culture. Eventually, the purified caspases were
subsequently inhibited with a 3-fold molar excess of the inhibitor
in a buffer containing 20 mM Tris pH 8.0 and 10 mM DTT.
monitored using a Tecan Spectra Fluor microplate reader (λex
)
360 nm, λem ) 465 nm). Pseudo first-order rate constants (kobs
)
were obtained from plots of ln Vt/V0 versus time.
Papain. Irreversible kinetic assays were performed by the
incubation method with papaya latex papain. An enzyme stock
solution for the papain assays was freshly prepared from 330 µL
of enzyme storage solution (1.19 mg/mL) diluted with 645 µL
papain buffer (50 mM HEPES, and 2.5 mM EDTA at pH 7.5) and
25 µL of DTT (0.1 M). The incubation mixture was prepared from
300 µL buffer, 30 µL of inhibitor stock solution (DMSO), and 30
µL of enzyme stock solution. At various times aliquots (50 µL)
were withdrawn and added to 200 µL of Cbz-Phe-Arg-pNA
substrate in buffer (53.7 µM). The absorbance of released p-
nitroaniline was monitored at 405 nm on a Molecular Devices
Thermomax plate reader.
Calpain I. Irreversible kinetic assays were performed by the
incubation method with calpain I from porcine erythrocytes.
Enzymatic activities of calpain I were measured at 23 °C in 50
mM HEPES buffer (pH 7.5) containing 10 mM cysteine and 5 mM
CaCl2, using Suc-Leu-Tyr-AMC as the substrate. To 30 µL of an
enzyme stock solution (1 mg/mL) of calpain I were added 300 µL
of incubation buffer and 30 µL of a stock inhibitor solution in
DMSO. At various time intervals 50 µL aliquots were withdrawn
from the incubation mixture and added to 200 µL of enzyme buffer
containing Suc-Leu-Tyr-AMC (1.6 mM). Substrate hydrolysis was
Crystallization and Data Collection. Cocrystals of the complex
between recombinant human caspase and the Michael acceptor
inhibitors were grown from 2 µL hanging drops formed by mixing
equal volumes of protein (10 mg/mL in 20 mM Tris/HCl pH 8.0,
10 mM DTT) and reservoir solution. The reservoir solution consists
of 15% (w/v) poly(ethylene glycol) 6000, 100 mM sodium citrate,
pH 5.0 in case of caspase-3 and 1.4 M sodium citrate, 100 mM
HEPES/NaOH pH 8.0 in case of caspase-8. Crystals (∼100 × 100
× 50 µm) grew within 2-3 days. For data collection, crystals were
frozen in a nitrogen stream after a short soak in reservoir solution
containing 20% glycerol for caspase-3. The crystals of caspase-3
complexes belong to the space group I222 and the typical unit cell
dimensions were, a ) 67.2 Å, b ) 83.3 Å, c ) 96.0 Å. Diffraction
data were collected at the rotating anode generator (Bruker-Nonius,
FR591) at 100 °K. Images were integrated with MOSFLM43 and
scaled with SCALA44 from the CCP4 suite of programs.
monitored using a Tecan Spectra Fluor microplate reader (λex
)
360 nm, λem ) 465 nm). Pseudo first-order rate constants (kobs
)
were obtained from plots of ln Vt/V0 versus time.
Determination of the Stability of the Inhibitors in Buffer and
in the Presence of DTT. The stability of a representative group of
inhibitors was studied by monitoring the UV spectrum of solutions
at 250 nm, at 25 °C. The concentrations of the inhibitors were 2.5-
4.0 mM and of DTT was 10 mM. The pH of the buffer solution
was 7.2 and it contained about 50% (v/v) DTT. The half-lives (t1/2
)
were obtained from first-order rate plots of ln(At/A0) versus time
(15 h) and the correlation coefficients were greater than 0.99.
An aliquot (50 µL) of 4 mM Cbz-Asp-Glu-Val-AAsp-CHdCH-
COOEt was added to 450 µL of assay buffer, and the stability of
the inhibitor was monitored spectrophotometrically at 250 nm for
every 30 min for 4 h. A half-life of 10 min was obtained from the
first-order rate plot from absorbance (250 nm) versus time (15 h).
An aliquot (50 µL) of 2.5 mM Cbz-Asp-Glu-Val-AAsp-CHd
CH-CONHCH2Ph was added to 450 µL of assay buffer, and the
stability of the inhibitor was monitored spectrophotometrically at
250 nm for every 30 min for 4 h. A half-life of 58 min was obtained
from the first-order rate plot from absorbance (250 nm) versus time
(15 h).
An aliquot (50 µL) of 2.5 mM Cbz-Asp-Glu-Val-AAsp-CHd
CH-CON(CH3)CH2Ph was added to 450 µL of assay buffer, and
the stability of the inhibitor was monitored spectrophotometrically
at 250 nm for every 30 min for 4 h. A half-life of 116 min was
obtained from the first-order rate plot from absorbance (250 nm)
versus time (15 h).
An aliquot (50 µL) of 2.5 mM Cbz-Val-AAsp-CHdCH-CHd
CH-CH3 was added to 450 µL of assay buffer, and the stability
of the inhibitor was monitored spectrophotometrically at 250 nm
for every 30 min for 4 h. The inhibitor was essentially stable over
the course of the study.
Crystal Structures. Caspase Purification. Human recombinant
caspase-3 and caspase-8 were produced in E.coli as inclusion bodies,
refolded, and purified. Briefly, for caspase-3, the cloned genes for
the large p17 subunit (Met-Ser29-Asp175) and the small p12 subunit
Diffraction data for caspase-8:inhibitor complex was collected
at the Swiss Light Source synchrotron (Paul Scherrer Institute,
Villigen, Switzerland). The X-ray data was collected without
utilizing any cryo protectant. The crystals of caspase-8 complex
belongs to P3′21 space group and the unit cell dimensions are a )
67.2 Å, b ) 83.3 Å, c ) 96.0 Å. The data reduction were done
with program XDS45 and scaled with XSCALE.46
Structure Solution and Refinement. The structure was solved
by the difference Fourier technique. Structure refinement and
automated water addition were performed using the program CNS47
and the model building was done with the program “O”. Iterative
cycles of refinement and model building was performed. Manual
adjustments were done with program “O”. The final crystallographic
R and free R factors for caspase-3 complexes are in the range of
16-19% and 19-22%, respectively. The final model for caspase-3
complexes consisted of residues 29-174 of R subunit and 176-
277 of â subunit. For the caspase-8 complex the final R and free
R factors are 16.8% and 20.1%, respectively. The final model shows
residues 223-371 of the R subunit and residues 390-479 of the â
subunit, 326 water molecules and 1 DTT molecule.

5748 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Ekici et al.
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Inhibitors Containing Vinyl Groups Conjugated to Electron With-
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Coordinates. The atomic coordinates for caspase-inhibitor
complexes have been deposited with the Protein Data Bank
(accession codes: 2C1E, 2C2K, 2C2M, 2C2O, and 2C2Z).
(18) Matthews, D. A.; Dragovich, P. S.; Webber, S. E.; Fuhrman, S. A.;
Patick, A. K.; Zalman, L. S.; Hendrickson, T. F.; Love, R. A.; Prins,
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T. J.; Fuhrman, S. A.; Meador, J. W.; Zalman, L. S.; Matthews, D.
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F. C.; Fuhrman, S. A.; Zalman, L. S.; Tuntland, T.; Lee, C. A.; Patick,
A. K.; Matthews, D. A.; Hendrickson, T. F.; Kosa, M. B.; Liu, B.;
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Acknowledgment. The work was supported by a grant from
the National Institute of General Medical Sciences (GM61964).
K.J. acknowledges a fellowship from the Center for the Study
of Women, Science, and Technology (WST) at Georgia Tech,
and J. A. acknowledges a fellowship from the Molecular Design
Institute under prime contract from the Office of Naval
Research. We thank Conor R. Caffrey and James McKerrow at
the Sandler Center for Basic Research in Parasitic Diseases,
University of California at San Francisco, San Francisco, CA,
for the S. mansoni legumain kinetic assays. We also thank Jan
Potempa and James Travis of the University of Georgia, Athens,
GA for the gingipain K. We gratefully acknowledge the Swiss
Light Source, Paul Scherrer Institute, Villigen, Switzerland, for
providing synchrotron beam time and T. Tomizaki, A. Wagner,
and C. Schulze-Briese for their excellent support during data
collection. J. Tschopp from the University of Lausanne is
acknowledged for providing the caspase-3 cDNA. The financial
support from the Swiss National Science Foundation and the
Baugartenstiftung (Zurich, Switzerland) is gratefully acknowl-
edged.
Supporting Information Available: Analytical data (C, H, N
analyses) for target componds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(22) Sirois, S.; Wei, D. Q.; Du, Q.; Chou, K. C. Virtual Screening for
SARS-CoV Protease Based on KZ7088 Pharmacophore Points. J.
Chem. Inf. Comput. Sci. 2004, 44, 1111-1122.
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