Dipeptidyl aspartyl fluoromethylketones as potent caspase inhibitors: SAR of the N-protecting group

Article abstract of DOI:10.1016/j.bmcl.2004.08.027

The synthesis and biological evaluation of a group of N-protected Val-Asp-fmk as caspase inhibitors is reported. This article describes the synthesis and biological evaluation of a group of N-protected Val-Asp-fmk as caspase inhibitors. The protecting group was found to contribute to caspase-3 inhibiting activity, and compounds with a large group such as Cbz are more active than compounds with a small group such as Ac. Compounds with more hydrophobic protecting groups were found to be more active in cell apoptosis protection assays, probably due to increased cell permeability. MX1122, 2,4-di-Cl-Cbz-Val-Asp-fmk, is identified as a potent broad-spectrum caspase inhibitor and is selective for caspases versus other proteases, with good activity in the cell apoptosis protection assays as well as good efficacy in the mouse liver apoptosis model.

Full text of DOI:10.1016/j.bmcl.2004.08.027

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5295–5300
Dipeptidyl aspartyl fluoromethylketones as potent
caspase inhibitors: SAR of the N-protecting group
Sui Xiong Cai,* Lufeng Guan, Shaojuan Jia, Yan Wang, Wu Yang,
Ben Tseng and John Drewe
Maxim Pharmaceuticals, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
Received 16July 2004; revised 11 August 2004; accepted 11 August 2004
Available online 9 September 2004
Abstract—This article describes the synthesis and biological evaluation of a group of N-protected Val-Asp-fmk as caspase inhibi-
tors. The protecting group was found to contribute to caspase-3 inhibiting activity, and compounds with a large group such as Cbz
are more active than compounds with a small group such as Ac. Compounds with more hydrophobic protecting groups were found
to be more active in cell apoptosis protection assays, probably due to increased cell permeability. MX1122, 2,4-di-Cl-Cbz-Val-Asp-
fmk, is identified as a potent broad-spectrum caspase inhibitor and is selective for caspases versus other proteases, with good activity
in the cell apoptosis protection assays as well as good efficacy in the mouse liver apoptosis model.
Ó 2004 Elsevier Ltd. All rights reserved.
Apoptosis is a highly regulated process of cell death and
caspases play a pivotal role in apoptosis by mediating a
series of proteolytic reactions that cleave many impor-
tant proteins within the cells, and lead to cell death.1
Caspases are cysteine proteases with strict substrate speci-
ficity requiring aspartic acid as P1 amino acid.2 It is well
known that excessive apoptosis is responsible, at least in
part, for a variety of diseases, including liver diseases,3
brain ischemia,4 myocardial infarction,5 and neuro-
degenerative disorders such as HuntingtonÕs disease
and AlzheimerÕs disease.6 Therefore the discovery and
development of caspase inhibitors could potentially lead
to effective anti-apoptotic drugs for the treatment of vari-
ous diseases.7–9
caspases, including many of the dipeptide based inhibi-
tors. Since apoptosis plays an important function for
the elimination of billions of unwanted and excessive
cells daily,16 it might be more preferable to use a specific
caspase inhibitor for the treatment of chronic diseases,
such as HuntingtonÕs disease and AlzheimerÕs disease.
However, because multiple caspases are activated in
the apoptosis pathway, a broad-spectrum caspase inhib-
itor should be more effective to inhibit all the caspases
and to achieve maximum inhibition of apoptosis, espe-
cially for the treatment of acute diseases, such as stroke,
sepsis and myocardial infarction.
We have reported the identification of MX1013 (Cbz-
Val-Asp-fmk) as a potent and broad-spectrum caspase
inhibitor, with potent in vivo activities (IV or IP admin-
istration) in several animal models of apoptosis, includ-
ing rodent liver failure model, rat middle cerebral artery
occlusion (MCAO) re-perfusion brain ischemia model,
myocardial infarction model,17,18 and a rodent endo-
toxin shock model.19 These data suggest that the dipep-
tide structure of MX1013 provides a good combination
of in vitro and in vivo activities for a caspase inhibitor,
which might be useful for the design of novel caspase
inhibitors.
Many caspase inhibitors have been designed and synthe-
sized based on substrate specificity of caspases. These
include tetrapeptide,10 tripeptide,11 dipeptide,12 or
monopeptide based inhibitor,13 as well as conformation-
ally constrained compounds using heterocycles to re-
place the peptide backbone,14 and the discovery of
non-peptide inhibitors.15 Some of these are specific to
different caspases, including many of the tetrapeptide
based inhibitors; while others are active against all the
It has been known that for inhibition of caspase enzyme,
caspase inhibitors based on tetrapeptide are more potent
than the corresponding tripeptides, and tripeptides are
Keywords: Caspase inhibitor; Apoptosis.
*
Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202
4000; e-mail: scai@maxim.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.027

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S. X. Cai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5295–5300
aqueous solution to produce 1g. N-protected Val 1h–t
were prepared similarly in two steps starting from the
corresponding commercial available alcohols.
Ph
CO2H
CH2F
O
H
N
O
N
H
O
O
Benzyl-glutaryl-Val-Asp-fmk (5u) and glutaryl-Val-Asp-
fmk (5v) was prepared as shown in Scheme 4. Glutaric
acid monobenzyl ester (8)24 was prepared from reaction
of glutaric anhydride with benzyl alcohol. Acid 8 was
coupled with Val-O-t-Bu in THF with EDC, HOBT
and DMAP to produce benzyl-glutaryl-Val-O-t-Bu (9),
and the t-Bu protecting group was removed by TFA
to produce benzyl-glutaryl-Val (10). Benzyl-glutaryl-
Val-Asp-fmk (5u) was prepared in three steps by cou-
pling of acid 10 with amine 2 to give amide 11, followed
by oxidation to ketone 12 and then deprotection. Glut-
aryl-Val-Asp-fmk (5v) was prepared from benzyl-glut-
aryl-Val-Asp(O-t-Bu)-fmk (12) by deprotection of the
benzyl and t-Bu groups in a AcOH solution of HBr.25
MX1013
CO2H
O
R3
O
H
N
CH2F
Ph
O
N
H
N
H
O
O
Chart 1.
more potent than the corresponding dipeptides.20 To
maintain the preferred Val-Asp dipeptide scaffold and
to explore the P3 subsite, we examined the replacement
of the Cbz protecting group in MX1013 with other
groups. As shown in Chart 1 comparing the structure
of the dipeptide inhibitor MX1013 with a tripeptide
inhibitor, the benzyl of the Cbz protecting group of
MX1013 could potentially bind to the same subsite as
the side chain (R3) of P3 amino acid of a tripeptide
inhibitor. Herein we report the synthesis and biological
evaluation of a group of N-protected Val-Asp-fmk as
caspase-3 inhibitors.
The activity of the dipeptides to inhibit human recomb-
inant caspase-3 was determined using a standard fluoro-
metric assay,17,26 and the results are summarized in
Table 1. Compound 5a (Ac-Val-Asp-fmk) had an IC50
value of 250nM in caspase-3, which was about eight-
times less active than MX1013 (Cbz-Val-Asp-fmk), indi-
cating that the Cbz protecting group contributes to the
activity of MX1013. Compound 5b and 5c were found
to be about 3- and 4-fold more potent than 5a, suggest-
ing that a larger group is preferred as the protecting
group. Interestingly, compound 5d is about 2-fold more
potent than 5b, and MX1013 is 3-fold more potent than
compound 5e, suggesting that an oxygen might be pre-
ferred over a CH2 group as the linker. Compound 5f
and 5g both have similar activity as that of MX1013,
indicating that the Ph group can be replaced by a non-
aromatic ring and a non-ring structure. Interestingly,
compound 5h, with a phenethyloxy group, was >3-fold
less active than MX1013; while compound 5i, with a
phenylpropyloxy group, was found to be only slightly
less potent than MX1013.
The dipeptide Ac-Val-Asp-fmk (5a) was prepared simi-
lar to MX1013 as reported previously.18 Ac-Val (1a)
was coupled with amine 221 to give amide 3a, which
was oxidized by Dess–Martin reagent to produce the
corresponding ketone 4a. Trifluoroacetic acid catalyzed
cleavage of the t-Bu ester gave the free acid 5a (Scheme
1). Other dipeptides 5b–t were prepared similarly from
N-protected Val 1b–t.
Propionyl-Val (1b)22 was prepared by reaction of Val
with propionyl chloride (7b) in NaOH aqueous solution
(Scheme 2). N-protected Val 1c–f were prepared simi-
larly by reaction of Val with the corresponding commer-
cial available acyl chloride.
Since the preferred P3 amino acid for caspase-3 is gluta-
mic acid,27 we also explored the incorporation of the
side chain of glutamic acid into the protecting group.
Compound 5v, with the protecting group matching the
side chain of glutamic acid, was found to be 5-fold more
potent than 5a, but slightly less active than MX1013,
Cyclopentylmethoxycarbonyl-Val (1g) was prepared in
two steps (Scheme 3). Reaction of cyclopentylmethanol
(6g) and phosgene in diethyl ether with N,N-diisopro-
pylethyl amine produced cyclopentylmethyl chlorofor-
mate (7g),23 which was reacted with Val in NaOH
O
CO2-t-Bu
CH2F
OH
CO2-t-Bu
H
N
O
EDCI
H
N
OH
CH2F
OH
+
H2N
N
H
HOBt, DMAP
O
O
1a
2
3a
CO2-t-Bu
CH2F
CO2H
CH2F
O
O
TFA
H
H
Dess-Martin
N
N
N
H
N
H
O
O
O
O
5a
4a
Scheme 1.

S. X. Cai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5295–5300
5297
We then explored MX1013 analogs with chloro and
fluoro substituted on the Ph of the Cbz group, with
the goal of increasing the hydrophobicity of the com-
pounds, which might make them more cell permeable.
As shown in Table 1, the mono and dichloro, and mono
and difluoro substituted analogs 5j–t all have similar po-
tency as MX1013 in the caspase-3 assay, with IC50 val-
ues ranged between 15 and 38nM, indicating that the
chloro and fluoro groups have little effects on caspase-
3 activity.
O
O
H
N
NaOH/H2O
+
H2N
Cl
OH
OH
O
O
7b
1b
Scheme 2.
Ether
O
Cl
+
ClCOCl
Several of these compounds were tested in the HeLa cell
apoptosis protection assays.17 The assays measure the
protecting effects of caspase inhibitors against apoptosis
induced by TNF-a in HeLa cells. The viability of the
cells was quantified by uptake of calcein AM, and re-
ported as the concentration of inhibitor that provides
50% of cell protection (Table 2). Compound 5d, which
is about as potent as MX1013 in the caspase-3 asaay,
was found to be about half as potent as MX1013 in
the cell protection assay, suggesting that the additional
hydrophobicity of the Ph group in MX1013 might make
the compound more cell permeable relative to com-
pound 5d. Compound 5g, which has similar caspase-3
enzyme activity as that of 5d, and has an additional
cyclopentyl group comparing to 5d, also was found to
be more potent than 5d in the cell protecting assay. Sev-
eral compounds with fluoro or chloro substituted on the
Ph of the Cbz group were found to be more active in the
cell protecting assay than MX1013. Compound 5p
(MX1122), with a di-chloro substituted Ph group, was
found to be >2-fold more potent than MX1013 in the
cell protection assay, confirming that increasing hydro-
phobicity can increase cell permeability of these
OH
O
O
7g
6g
H2N
OH
O
H
N
O
OH
O
NaOH/H2O
1g
Scheme 3.
suggesting that the glutamic acid side chain may not be
able to bind effectively to the P3 subsite without part of
the tripeptide backbone, probably due to the important
hydrogen bond formed between the P3 amide NH and
the backbone carbonyl of Arg207
benzyl protected compound 5u was found to be about
as potent as MX1013, indicating that a large group
can be tolerated in that position.
28
.
Interestingly, the
O
O
Ph
O
O
H2N
O-t-Bu
H
N
O
Ether
Ph
O
OR
Ph
OH
+
CO2H
O
O
O
EDCI/HOBt/DMAP
9, R = t-Bu
10, R = H
O
TFA
8
CO2-t-Bu
CH2F
OH
O
O
Ph
O
O
O
Ph
O
H2N
CO2-t-Bu
CH2F
O
CO2-t-Bu
CH2F
OH
H
N
H
N
Dess-Martin
N
N
H
H
EDCI/HOBt/DMAP
O
O
12
11
HBr/AcOH
O
O
Ph
O
O
OH
CO2H
TFA
CO2H
CH2F
O
O
H
N
H
N
CH2F
N
H
N
H
O
O
O
5u
5v
Scheme 4.

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S. X. Cai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5295–5300
Table 1. Caspase-3 inhibiting activity of the dipeptide inhibitors
Table 2. Cell apoptosis protection activity and Caspase-3 inhibiting
activity of the dipeptide inhibitors
CO2H
Entry
R
Caspase-3 IC50
(nM)
50% Cell
protectiona
(nM)
O
H
R
N
CH2F
N
H
O
O
5d
5g
CH3O
37
30
500
O
a
IC50 (nM)
300
Entry
R
5a
5b
5c
5d
5e
CH3
250
81
5o
5p
5q
5r
4-F–PhCH2O
28
25
21
15
150
100
150
100
CH3CH2
PhCH2
CH3O
2,4-di-Cl–PhCH2O
3,4-di-Cl–PhCH2O
2,5-di-Cl–PhCH2O
61
37
98
PhCH2CH2
HO
5v
50
30
>2000
250
O
5f
35
30
O
PhCH2O
MX1013
O
5g
a Concentration of inhibitor that provided 50% of cell protection is
determined as described in Ref. 17.
5h
5i
PhCH2CH2O
110
46
36
36
34
38
29
28
25
21
15
35
30
PhCH2CH2CH2O
2-Cl–PhCH2O
3-Cl–PhCH2O
5j
Table 3. Inhibiting activity of compound 5p (MX1122) against
different proteases
5k
5l
4-Cl–PhCH2O
2-F–PhCH2O
a
IC50 (nM)
5m
5n
5o
5p
5q
5r
5s
5t
Enzyme
3-F–PhCH2O
4-F–PhCH2O
Caspase-1
Caspase-3
Caspase-7
Caspase-8
Caspase-9
Calpain-1
Cathepsin B
Factor Xa
26
25
10
2,4-di-Cl–PhCH2O
3,4-di-Cl–PhCH2O
2,5-di-Cl–PhCH2O
2,4-di-F–PhCH2O
3,4-di-F–PhCH2O
6
9
ꢀ5000
ꢀ10,000
>100,000
Ph
O
5u
33
a IC50 is determined as described in Ref. 17.
O
HO
are expected to be broad-spectrum and inhibit all the
caspases.
5v
50
30
O
MX1013
PhCH2O
Compound 5p also was tested in a mouse liver apoptosis
model.17,29 The liver apoptosis model, which is based on
the fact that hepatocytes express high levels of cell death
receptor Fas and can be activated by agonistic antibod-
ies, is useful for the determination of the in vivo anti-
apoptotic efficacy of caspase inhibitors. In this model,
mice were injected IV with anti-Fas monoclonal anti-
body, followed 5min later by IV administration of var-
ious doses of compound 5p formulated in an aqueous
vehicle containing 50mM Tris–HCl. The number of sur-
viving animals in each group was determined at 1, 3, 6,
24h, and 5days post-injection, and was expressed as a
percentage of the total number of animals in each group.
As shown in Table 4, mice injected with 8lg of anti-Fas
antibody and without treatment were all dead by the 1h
time point. In contrast, the lowest dose of compound 5p
tested (0.25mg/kg) protected 100% of the mice from the
lethal effects of anti-Fas antibody at the 1 and 3h time
point, and 83% (5/6), 33% (2/6), and 16% (1/6) at the
6, 24h, and 5days time point, respectively. At 0.5mg/
kg dose, the 24h and 5days time point protection was
increased to 50% (3/6). At 1 and 10mg/kg dose, 5p pro-
tected 100% of the mice at all the time points, indicating
that 5p truly protected the mice from the initial injury
caused by anti-Fas antibody and is highly active in this
in vivo model of apoptosis. The activity of compound 5p
a IC50 is determined as described in Ref. 17.
dipeptide inhibitors. Interestingly, compound 5v, which
has an extra carboxylic acid group and is slightly less ac-
tive than MX1013 in the caspase-3 assay, was not active
in the cell protection assays up to 2000nM, at least 8-fold
less active than MX1013, indicating that the extra acidic
group results in major reduction of cell permeability.
Compounds 5p was then tested against other caspases
and proteases for selectivity and the results are summa-
rized in Table 3. Compound 5p was found to have sim-
ilar activity in caspase-1, -3, -7, -8, and -9. Testing
against other proteases, compound 5p was found to be
about 200-fold, 500-fold, and >5000-fold selective for
caspase-3 versus Calpain-1, caspase-3 versus Cathepsin
B, and caspase-3 versus Factor Xa, respectively. There-
fore, similar to MX1013,18 compound 5p is a broad-
spectrum caspase inhibitor and is selective for caspases
versus other proteases. Since it is known that the P4 ami-
no acid in tetrapeptide caspase substrates is the key
determinant of specificity between caspases,27 dipeptide
based caspase inhibitors, including MX1013 and com-
pound 5p, which do not have the P4 and P3 amino acids,

S. X. Cai et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5295–5300
5299
Table 4. Survival of mice with compound 5p (MX1122) treatment after induction of apoptosis by anti-Fas antibody
Dose of 5p (mg/kg)
% Survival (n = 6 )
1h
3h
6h
24h
5days
0
100
100
100
100
100
0
0
83
0
33
0
16
0.25
0.5
1
100
100
100
100
83
50
50
100
100
100
100
100
100
10
Mice were injected intravenously (IV) with 8lg of anti-Fas antibody (clone JO-2; Pharmingen), followed 5min later with IV injection of compound
5p at the indicated doses or by vehicle. Six mice were treated in each group and the number of surviving mice was monitored for up to 5days.
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in this model is similar to that of MX1013, which also
provides 100% protection at all time points at dose of
1mg/kg.
In conclusion, we have synthesized a group of N-pro-
tected Val-Asp-fmk and evaluated their activity as cas-
pase-3 inhibitors. We found that the N-protecting
group contributes to the potency of these caspase-3
inhibitors and many different groups are tolerated in
that position. The introduction of chloro and fluoro
groups in the phenyl ring of Cbz protecting group did
not affect their potency as caspase-3 inhibitors. In the
cell apoptosis protecting assays, compounds with more
hydrophobic protecting group were found to be more
active, suggesting that the increased hydrophobicity
makes them more cell permeable. Compound 5p
(MX1122), 2,4-dichloro-Cbz-Val-Asp-fmk, was found
to be a potent and broad-spectrum caspase inhibitor.
It is selective for caspases among the proteases tested
and is highly active in the cell apoptosis protection as-
says. MX1122 also was found to be highly efficacious
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