Dipeptidyl aspartyl fluoromethylketones as potent caspase-3 inhibitors: SAR of the P2 amino acid

Article abstract of DOI:10.1016/j.bmcl.2003.12.065

This article describes the synthesis and biological evaluation of a series of dipeptidyl aspartyl fluoromethylketones as caspase-3 inhibitors. Structure-activity relationship (SAR) studies showed that for caspase-3 inhibition, Val is the best P₂ amino acid. The SAR studies also showed that the Asp free carboxylic acid in P₁ is important for caspase inhibiting activities, as well as for selectivity over other proteases.

Full text of DOI:10.1016/j.bmcl.2003.12.065

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1269–1272
Dipeptidyl aspartyl fluoromethylketones as potent caspase-3
inhibitors: SAR of the P2 amino acid
Yan Wang,a Jin-Chen Huang,b Zhang-lin Zhou,b Wu Yang,a John Guastella,a,y
John Drewea and Sui Xiong Caia,*
aMaxim Pharmaceuticals, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
bCoCensys, Inc., 213 Technology Drive, Irvine, CA 92718, USA
Received 26 October 2003; accepted 9 December 2003
Abstract—This article describes the synthesis and biological evaluation of a series of dipeptidyl aspartyl fluoromethylketones as
caspase-3 inhibitors. Structure–activity relationship (SAR) studies showed that for caspase-3 inhibition, Val is the best P2 amino
acid. The SAR studies also showed that the Asp free carboxylic acid in P1 is important for caspase inhibiting activities, as well as for
selectivity over other proteases.
# 2003 Elsevier Ltd. All rights reserved.
The caspases are a family of cysteine proteases that
require aspartic acid residues at P1 of substrates for
cleavage. Twelve enzymes in this family have been
identified,1 and the structures of caspase-1, -3, -7 and -8
have been determined by X-ray crystallography.2 Based
on its biological function, caspases can be divided into
two groups. One of the groups, represented by caspase-
1, also known as interleukin-1b converting enzyme
(ICE), plays an important role in cytokine maturation
and inflammation.3 The other group, represented by
caspase-3, -8 and -9, are critical for death receptor sig-
naling and execution of apoptosis. Among them, Cas-
pase-3 has been identified as a key member of the
executioner caspases.4 Due to the important role of
caspases in both inflammation and apoptosis, the dis-
covery and development of caspase inhibitors has
become a focus of research in recent years.5
strained compounds, using heterocycles to replace the
peptide backbone,7 or the discovery of non-peptide
inhibitors.8 Another approach is to reduce the number
of amino acids in the inhibitor, truncating the tetra-
peptide to either a tripeptide, dipeptide,9 or mono-
peptide, that is, substituted aspartic inhibitor.10 We
have reported that, starting with the DEVD tetrapep-
tide caspase-3 recognition site of PARP,11 by reducing
the number of amino acids to the VD dipeptide scaffold
with a fluoromethylketone warhead, MX1013 (Z-Val-
Asp-fmk) has been identified as a potent and broad-
spectrum caspase inhibitor with IC50 values ranging
from 5 to 20 nM for caspase-1, -3, -6, -7, -8 and -9.12,13
MX1013 is more cell permeable than the corresponding
tetrapeptide and tripeptide-fmk, as well as the widely
used Z-VAD(OMe)-fmk, and shows good potency in
protecting cells against apoptosis. MX1013 also is
highly efficacious in several animal models of apoptosis,
including rodent liver failure model, rat middle cerebral
artery occlusion (MCAO) re-perfusion brain ischemia
model and myocardial infarction model by IV adminis-
tration,13 and rodent endotoxin shock model.14 Herein
we report the synthesis and SAR studies of dipeptidyl
aspartyl fluoromethylketones as caspase-3 inhibitors.
Many potent caspase inhibitors have been prepared
based on the tetrapeptide substrate structures of cas-
pases. However, due to the peptidyl structure and the
presence of carboxylic acid group(s), many of these
inhibitors do not have good cell penetration and are not
very active in cell assays.6 One approach to address this
problem is the preparation of conformationally con-
Scheme 1 shows the synthesis of the dipeptides Z-Val-
Asp-fmk (4) and its methyl ester 5. The intermediate 1
was prepared according to Revesz et al.15 from t-Bu 3-
nitropropionate and 2-fluoroethanol. Coupling of 1
with Z-Val gave amide 2, which was oxidized by Dess–
* Corresponding author. Tel.: +1-858-202-4006; fax: +1-858-202-
4000; e-mail: scai@maxim.com
Current address: Insert Therapeutics, Inc. Pasadena, CA 91107,
USA.
y
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.065

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Y. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1269–1272
Scheme 1.
Martin reagent to produce the corresponding ketone 3.
Trifluoroacetic acid catalyzed cleavage of the t-Bu ester
gave the free acid 4. Acid 4 was then converted to
methyl ester 5 by treatment with methanol in the pre-
sence of HCl. Other dipeptides were prepared similarly
from the Z protected amino acid. The activity of the
dipeptides to inhibit human recombinant caspase-3 was
determined using a standard fluorometric assay.13,16
thiophene ring, also is more active than 10. These results
are in agreement with the analysis of crystal structure of
caspase-3 in complex with an Ac-DEVD tetrapeptide
inhibitor, which suggested that a small and hydrophobic
group in the P2 position is best for caspase-3 inhibitors.18
Introduction of an extra methyl group in the a-position
of P2 amino acid, as in compounds 19 and 20, resulted
in reduction of potency. Compound 20 is >70-fold less
active than 4. This large reduction of potency suggests
that either the extra methyl could not be fit into the S2
pocket, or the extra methyl group forces the isopropyl
group into an unfavorable position, which could not
interact with the S2 pocket properly.
The caspase-3 inhibition activity (IC50) of the dipeptides
are summarized in Table 1. Compound 4 (Z-Val-Asp-
fmk) showed an IC50 value of 0.03 mM in caspase-3. The
P2 position was explored by substituting the Val with
other amino acids. When the side chain (isopropyl) of
Val in 4 was replaced by a smaller group, such as a
methyl (Ala, 11) and a proton (Gly, 12), the potencies
decreased 20- and 63-fold, respectively. More bulky
groups, such as an isobutyl (Leu, 9) and a phenylmethyl
(Phe, 10), also resulted in compounds with reduced
inhibition activities of 6–13 fold. Compound 6, with a 2-
butyl group (Ile), retains good activity and is about half
as active as that of 4, probably because the size of a
2-butyl (Ile) group is close to that of isopropyl (Val).
The results showed that for caspase-3 inhibition, Val is
the best amino acid at the P2 position for these dipep-
tide inhibitors. This is consistent with the results of a
combinatorial approach to define specific caspase tetra-
peptide substrates, which showed that for caspase-3, Val
is the best P2 amino acid, and Ile is a good replacement
for Val at the P2 position.17 The polar and large Lys
(13) and Glu (14) as P2 amino acid gave the least potent
compound, indicating that polar and large size groups
are not tolerated at P2 position. This is also consistent
with the results from the combinatorial approach, which
showed that Lys and Glu are not preferred amino acid
at the P2 position for caspase-3.17
When the P1 free carboxylic acid (4) was converted to a
methyl ester (5), the potency in caspase-3 was reduced
>35-fold, confirming that the carboxylic acid of Asp in
the P1 position is essential for the binding of the inhi-
bitor to the caspase enzyme. Similarly, when 6 was
converted to the methyl ester 7, it also showed ꢀ35-fold
drop in potency.
Compounds 4 and 5 were then tested against other cas-
pases and proteases for selectivity and the results are
summarized in Table 2. Free acid 4 was found to have
similar activity in caspase-1, -3, -6, -7, -8 and -9, but
inactive in non-caspase proteases. Therefore, compound
4 is a broad-spectrum caspase inhibitor and is selective
for caspases. Interestingly, the methyl ester 5 was found
to be more active in non-caspase cysteine proteases cal-
pain I and cathepsin B than in caspase-1 and -3, thus
ester 5 is not selective for caspases. Both compounds 4
and 5 are not active (>100 mM) against serine proteases
thrombin and factor Xa, indicating that the conversion
of the acid 4 to ester 5 only affects their activity as
cysteine protease inhibitors. These results indicate that
for these dipeptides, the free carboxylic acid in the P1 is
not only important for the caspase inhibiting activity, but
also important for their selectivity versus other cysteine
proteases. Since ester prodrugs of caspase inhibitors
have been used to increase cell permeability,19 including
Z-VAD(OMe)-fmk, Z-D(OMe)E(OMe)VD(OMe)-fmk,
and Z-YVAD(OMe)-fmk, which have been reported to
We also explored the replacement of P2 Val by non-
natural amino acids. When the isopropyl side group of
Val was replaced by an ethyl (15), phenyl (16) and
cyclohexyl (17), the compounds retain good activity and
are about 1/3 as active as that of 4. These compounds
are all more active than 10, which has a more bulky
phenylmethyl side group. Compound 18, with a 5-member

Y. Wang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1269–1272
1271
Table 1. Caspase-3 activity of the dipeptide inhibitors
Table 2. Activity of compound 4 and 5 in different proteases
IC50 (mM)a
Compd
4
5
Caspase-1
Caspase-3
Caspase-6
Caspase-7
Caspase-8
Caspase-9
Cathepsin B
Calpain-1
Thrombin
Factor Xa
0.020
0.030
0.018
0.007
0.007
0.005
0.9
1.1
ND
ND
ND
ND
0.3
0.12
>100
>100
Entry
Name
R1
R2
Caspase-3
IC50 (mM)a
4
5
6
7
9
Z-Val-Asp-fmk
Z-Val-Asp(OMe)-fmk
Z-Ile-Asp-fmk
H
Me
H
0.030
1.1
>10
>10
>100
>100
0.070
2.6
a IC50 is determined as described in ref 13.
Z-Ile-Asp(OMe)-fmk
Z-Leu-Asp-fmk
Me
H
In conclusion, we have synthesized a group of dipepti-
dyl aspartyl fluoromethylketones and evaluated their
activity as caspase-3 inhibitors. We found that for cas-
pase-3 inhibition, Val is the best in the P2 position
among the natural and non-natural amino acids
explored for these dipeptidyl inhibitors. The introduc-
tion of an extra methyl group in the a-position of P2
amino acid was found to result in large reduction in
potency. We also found that the free carboxylic acid
in the P1 Asp is important not only for their activity as
caspases inhibitors, but also for their selectivity over
non-caspase proteases. It is therefore critical to distin-
guish the free acid from the ester prodrug when using
caspase inhibitors for biological studies.
0.20
10
11
Z-Phe-Asp-fmk
Z-Ala-Asp-fmk
H
0.40
0.60
Me
H
H
1.9
12Z-Gly-Asp-fmk
H
13
Z-Lys-Asp-fmk
H
1.6
14
15
Z-Glu-Asp-fmk
Z-Abu-Asp-fmk
H
H
14.
Et
0.10
16
17
Z-Phg-Asp-fmk
Z-Chg-Asp-fmk
H
H
0.10
0.10
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