Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: Peptidomimetic replacement of the P2 amino acid by 2-aminoaryl acids and other non-natural amino acids

Article abstract of DOI:10.1016/j.bmcl.2007.09.030

As a continuation of our SAR studies of dipeptidyl aspartyl-fmk as caspase inhibitors, we explored the replacement of the P₂ amino acid by a 2-aminoaryl acid or other non-natural amino acids. Several of these compounds, such as 6l and 6p, were

Full text of DOI:10.1016/j.bmcl.2007.09.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6178–6182
Dipeptidyl aspartyl fluoromethylketones as potent
caspase inhibitors: Peptidomimetic replacement of the P2
amino acid by 2-aminoaryl acids and other non-natural amino acids
Yan Wang, Shaojuan Jia, Ben Tseng, John Drewe and Sui Xiong Cai*
EpiCept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
Received 3 August 2007; revised 30 August 2007; accepted 5 September 2007
Available online 8 September 2007
Abstract—As a continuation of our SAR studies of dipeptidyl aspartyl-fmk as caspase inhibitors, we explored the replacement of the
P2 amino acid by a 2-aminoaryl acid or other non-natural amino acids. Several of these compounds, such as 6l and 6p, were found to
have good activities with inhibition potencies of around 100 nM in a caspase-3 enzyme assay. EP1113, Z-Val-(2-aminobenzoyl)-
Asp-fmk (9b), is identified as a potent broad-spectrum caspase inhibitor with IC50 values of 6–60 nM in different caspases.
EP1113 also has good activity in a cell apoptosis protection assay.
Ó 2007 Elsevier Ltd. All rights reserved.
Caspases are a family of structurally related cysteine
proteases that cleave their substrates with strict sub-
strate specificity for aspartic acid as the P1 amino acid.1
One member of the caspase family, represented by cas-
pase-1, activates interleukin-1 and plays an important
function in cytokine maturation and inflammation.2
The other group of caspases, including caspase-3, -6,
-7, -8, and -9, are key mediators of apoptosis via cleav-
ing numerous important proteins leading to apoptotic
cell death.3 Excessive apoptosis has been established to
play an important role in many pathological conditions,
including liver diseases,4 brain ischemia,5 myocardial
infarction,6 and neurodegenerative disorders such as
Huntington’s disease and Alzheimer’s disease.7 There-
fore the discovery and development of caspase inhibitors
could result in novel anti-inflammatory and anti-apop-
totic treatments for a variety of diseases.8,9
libraries.15 Some of these are selective for specific casp-
ases, including VX-740 which is a peptidomimetic,
reversible, and a selective caspase-1 inhibitor.16 Others
are broad-spectrum caspase inhibitors, such as IDUN-
6556 which is a dipeptide based, irreversible, and
broad-spectrum caspase inhibitor.17
We have reported the discovery of the dipeptide-fmk
EP1013 (MX1013, Cbz-Val-Asp-fmk) as a potent and
broad-spectrum caspase inhibitor, with potent in vivo
activities in several animal models of apoptosis.18–21
Similar to IDUN-6556, MX1013 and other peptide-
fmk molecules are competitive and irreversible inhibi-
tors of caspase-3.18 To maintain the preferred dipeptide
scaffold of EP1013 but to reduce the peptide character-
istics of the inhibitor, we have replaced the P2 a-amino
acid by an a-hydroxy acid, which led to the discovery
of a series of potent caspase inhibitors including
EP1153 (MX1153).22 We also explored the replacement
of the P2 amino acid by non-natural amino acids such as
2-aminobenzoic acids (Chart 1). Herein we report the
synthesis and evaluation of a group of non-natural ami-
no acid derivatives as caspase inhibitors.
Many caspase inhibitors have been designed and synthe-
sized.10 These include peptide-based inhibitors,11 pepti-
domimetic-based inhibitors such as those incorporated
with a P2–P3 conformationally constrained dipeptide
mimetic12 and a thiazepine ring as P2–P3 mimetic,13
aza-peptide,14 as well as non-peptide inhibitors such as
isatins discovered through screening of compound
2-(Benzyloxycarbonylamino)benzoyl-Asp-fmk (6a) was
prepared using procedures similar to the synthesis of
dipeptide-fmk inhibitors as reported previously.19
2-(Benzyloxycarbonylamino)benzoic acid (2a) was pre-
pared via reaction of 2-aminobenzoic acid (1a) with
benzyl chloroformate in pyridine. Compound 2a was
Keywords: Caspases; Inhibitors.
*
Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202
4000; e-mail: scai@epicept.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.030

Y. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6178–6182
6179
CO2H
CO2H
CH2F
O
O
H
H
Ph
O
N
CH2F
N
O
N
H
Ph
N
H
O
O
O
O
EP1013
EP1153
CO2H
CH2F
CO2H
O
O
A
H
N
Ph
O
Ph
O
N
CH2F
N
H
N
H
A
O
O
O
O
Chart 1.
coupled with amine 323 to give amide 4a, which was
oxidized by Dess–Martin reagent to produce the
corresponding ketone 5a. The t-Bu ester was then cleaved
by TFA to give the free acid 6a (Scheme 1). Other non-
natural amino acid-based Asp-fmks 6b–6o were prepared
similarly from the corresponding substituted 2-aminoben-
zoic acids (1b–1k), 3-aminothiophene-2-carboxylic acid 1l,
cis- and trans-2-aminocyclohexanecarboxylic acids 1m
and 1n, and cis-2-aminocyclopentanecarboxylic acid 1o.
Compounds 6p and 6q were prepared similarly using ben-
zyloxycarbonyl protected (S)-azetidine-2-carboxylic acid
2p and proline 2q, respectively.
tection to remove the two t-Bu groups to produce
Z-Glu-(2-aminobenzoyl)-Asp-fmk (9a). Z-Val-(2-amino-
benzoyl)-Asp-fmk was prepared similarly in four steps
starting from benzyloxycarbonyl-Val-OH (7b).
The activity of these compounds to inhibit human
recombinant caspase-3 was determined using a standard
fluorometric assay,18,24 and the results are summarized
in Table 1. Compound 6a, with a 2-aminobenzoic acid
as P2, had good activity with an IC50 value of 200 nM
for caspase-3. Compounds 6b–6d, with a Me group
substituted in different positions of the phenyl ring,
except 5-Me analog 6c, were 3- to 4-fold less active than
6a, indicating that substitution at those positions is not
preferred. The 3,5-dimethyl analog 6e was the least
active one among the Me-substituted analogs, confirm-
ing the above observation. Similarly, the Cl-substituted
analogs 6f–6i were all less active than 6a. Interestingly,
the 5-substituted analogs 6c, 6g, and 6j all were only
slightly less active than 6a, indicating that substitution
at the 5-position is more tolerated. In comparison,
Compound 9a was prepared in four steps similar to 6a
(Scheme 2). Benzyloxycarbonyl-Glu(O-t-Bu)-2-amino-
benzoic acid 8a was prepared from reaction of
benzyloxycarbonyl-Glu(O-t-Bu)OH (7a) with isobutyl
chloroformate and N-methylmorpholine in THF, fol-
lowed by addition of 2-aminobenzoic acid. Compound
8a was reacted with amine 3 to produce the amide,
followed by oxidation to produce the ketone and depro-
CO2t-Bu
O
O
NH2
O
3
H2N
F
O
a
CO2t-Bu
+
OH
b
O
NH O
O
NH O
OH
O
Cl
OH
O
N
F
H
OH
2a
1a
4a
O
CO2t-Bu
c
CO2H
O
NH O
O
NH O
d
N
H
F
N
F
H
O
O
5a
6a
Scheme 1. Reagents: (a) pyridine; (b) 3, EDCI, HOBT, DMAP, THF; (c) Dess–Martin, dichloromethane; (d) TFA, dichloromethane.
O
O
H
O
H
CO2H
CH2F
H
Ph
O
N
Ph
O
N
a
d
O
b
c
O
NH
Ph
O
N
NH
OH
R1
O
R1
O
O
R1
N
OH
H
O
7a, R1 = CH2CH2CO2t-Bu
7b, R1 = 2-Pr
9a, R1 = CH2CH2CO2H
9b, R1 = 2-Pr
8a, R1 = CH2CH2CO2t-Bu
8b, R1 = 2-Pr
Scheme 2. Reagents: (a) 1—isobutyl chloroformate, N-methylmorpholine/THF; 2—anthranilic acid; (b) 3, EDCI, HOBT, DMAP, THF; (c) Dess–
Martin, dichloromethane; (d) TFA, dichloromethane.

6180
Y. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6178–6182
Table 1. Caspase-3 inhibition activity of 2-amino-benzamides and
related compounds
Table 1 (continued)
Entry
Caspase-3 inhibition IC50 (lM)
A
CO2H
O
H
CH2F
Ph
O
N
N
6m
0.25
H
A
O
O
b
Entry
Caspase-3 inhibitiona IC50 (lM)
A
6n
6o
1.9
6a
0.20
0.21
6b
6c
6d
6e
6f
6g
6i
0.86
0.26
0.72
1.7
CO2H
O
Me
B
Ph
O
CH2F
N
N
H
O
O
B
Caspase-3 inhibition IC50 (lM)
N
Me
6p
6q
0.10
N
N
Me
0.41
Me
O
Me
H
CO2H
Ph
O
N
O
NH
O
R1
CH2F
N
0.45
0.32
0.55
0.27
H
Cl
O
R1
Caspase-3 inhibition IC50 (lM)
9a
9b
CH2CH2CO2H
2-Pr
0.34
0.033
0.030
EP1013
NAc
Cl
a IC50 is determined as described in Ref. 18.
b Enzyme IC50 results are expressed as 20% or less.
c NA, not applied, please see Chart 1 for the structure of EP1013.
Cl
substitution at the 3-position, such as those in com-
pounds 6d, 6e, and 6k, led to a 3- to 12-fold reduction
in activity, indicating that substitution at the 3-position
is the least tolerated, probably due to unfavorable inter-
action with the enzyme. Interestingly, replacing the 6-
membered benzene ring by a 5-membered thiophene
ring led to 6l, which had an IC50 value of 100 nM and
was 2-fold more potent than 6a.
6j
F
6k
6l
2.5
MeO
We then explored the replacement of the aromatic ring
with a saturated ring. Compound 6m, with a cis-substi-
tuted cyclohexane ring, was about as active as 6a. Inter-
estingly, the corresponding trans-isomer 6n was almost
8-fold less active than 6m. The cis-cyclopentane analog
6o was about as active as 6m. The (S)-azetidine-2-car-
boxylic acid derivative 6p was found to be highly active
0.10
S

Y. Wang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6178–6182
6181
showing an IC50 value of 100 nM, 4-fold more potent
than the corresponding 5-membered ring analog
Z-Pro-Asp-fmk 6q.
(Table 3),19 compound 9b is
caspase inhibitor.
a
broad-spectrum
In conclusion, we have designed and synthesized a
group of novel peptidomimetic-based caspase inhibitors
by replacement of the P2 amino acid with non-natural
amino acid such as 2-aminobenzoic acid. Several of
these compounds were found to have good activity with
IC50 values in the sub 100 nM range. Interestingly,
incorporation of Val at the P3 position led to compound
9b which is a potent and broad-spectrum caspase
inhibitor, with IC50 values of 6–61 nM for caspase-1,
-3, -6, -7, -8, and -9. Compound 9b is also highly active
in the cell apoptosis protection assays. Although pepti-
domimetics such as 9b might be more stable in vivo than
peptide-based caspase inhibitors, additional in vitro and
in vivo studies are needed to determine whether 9b has
any true advantages over dipeptide caspase inhibitors
such as EP1013 and is suitable for future development.
Since it is known that glutamate is the preferred P3 ami-
no acid for caspase-3,25 we explored the incorporation
of Glu to the N-terminal of 6a and prepared 9a. How-
ever, 9a was found to be about 2-fold less active than
6a, suggesting that the Glu side chain did not fit into
the S3 pocket of caspase-3. Interestingly, compound
9b, with a Val added to the N-terminal of 6a, was found
to be highly active with an IC50 value of 33 nM, 6-fold
more potent than 6a and as active as the dipeptide
EP1013.
Selected compounds were tested in the HeLa cell apop-
tosis protection assay,18 which measures the protecting
effects of caspase inhibitors against apoptosis induced
by TNF-a. The viability of the cells was quantified by
calcein AM uptake, and the concentration of inhibitor
that provided 50% of cell protection is summarized in
Table 2. Compound 6a was found to provide 50% cell
protection at a concentration of 4000 nM. Compound
9b was the most active one, provided 50% protection
at a concentration of 450 nM, approaching that of
EP1013.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.09.030.
Compound 9b (EP1113) was then tested against other
caspases and the results are summarized in Table 3.
Compound 9b was found to have high activity in
caspase-1, -3, -6, -7, -8, and -9, with IC50 values
between 6 and 61 nM. Therefore similar to EP1013
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