Novel dual inhibitors of calpain and lipid peroxidation

Article abstract of DOI:10.1016/j.bmcl.2004.04.105

A series of molecules with dual inhibitory activities on calpain and lipid peroxidation were synthesized. These hybrid compounds were built on the calpain pharmacophore 2-hydroxytetrahydrofuran linked to a set of antioxidants via a L-leucine linker. Compo

Full text of DOI:10.1016/j.bmcl.2004.04.105

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3825–3828
Novel dual inhibitors of calpain and lipid peroxidation
Serge Auvin,a,* Bernadette Pignol,b Edith Navet,a Dominique Pons,a Jean-G. Marin,b
Dennis Bigga and Pierre-E. Chabrierb
aDepartment of Medicinal Chemistry, Ipsen Research Laboratories, Institut Henri Beaufour,
5, Avenue du Canada, 91966 Les Ulis Cedex, France
bDepartment of Neurobiology, Ipsen Research Laboratories, Institut Henri Beaufour,
5, Avenue du Canada, 91966 Les Ulis Cedex, France
Received 2 March 2004; revised 9 April 2004; accepted 22 April 2004
Available online 2 June 2004
Abstract—A series of molecules with dual inhibitory activities on calpain and lipid peroxidation were synthesized. These hybrid
compounds were built on the calpain pharmacophore 2-hydroxytetrahydrofuran linked to a set of antioxidants via a L-leucine
linker. Compound 7, the most potent in cellular calpain and lipid peroxidation inhibitions, provided effective protection against glial
cell death induced by maitotoxin.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
furan group, a masked aldehyde, was selected as a cal-
pain pharmacophore5a suitable for our purposes. In this
context, the biologically active species was hypothesized
to be the hydroxyalkyl-aldehyde (the opened semi-
acetal),5b which is in equilibrium with the cyclic semi-
acetal. Thus, the available hydroxyalkyl-aldehyde forms
a hemithioacetal with the cysteine residue of the calpain
active site.
Calpains, members of the thiol protease superfamily, are
implicated in a number of patho-physiological pro-
cesses. In particular, the role of calpain 1 in central
nervous system conditions such as stroke, Parkinson’s
and Alzheimer’s diseases, subarachnoid hemorrhage or
head trauma, as well as in peripheral pathologies like
muscular dystrophy, cataract, cardiac ischemia, reste-
nosis or arthritis has been extensively studied.1 Most of
these pathologies are associated with inflammatory
processes and the production of free radical species such
as reactive oxygen species (ROS).
On the other hand, the selection of the antioxidant part
of these hybrid compounds was facilitated by our pre-
vious work in the field of dual NOS-ROS inhibitors.6
Furthermore, we reasoned that these hybrid compounds
should have balanced calpain inhibitory and free radical
scavenging properties. Attention was therefore focused
on a series of potent antioxidants (1a–d) as illustrated in
Scheme 1.
In pioneering experiments, we demonstrated that the
combination of a calpain inhibitor (Z-LL-H)2 and an
antioxidant (BHT)3 led synergistically to protection
against glial cell death.4 Consequently, we set up a
chemical program aimed at designing molecules pos-
sessing both activities.
As a first approach to the design of these dual inhibitors,
the connection between the calpain pharmacophore and
the antioxidant was inspired by the peptidic backbone of
classical calpain inhibitors. Thus, the optimization of
the molecules focused on the nature of the antioxidant
and its impact on the calpain and lipid peroxidation
The free aldehyde group of Z-LL-H was regarded as a
chemically reactive function incompatible with in vivo
administration. Therefore the 2-hydroxy-tetrahydro-
inhibitory activities.
A 4-step synthetic pathway
(Scheme 1) was used to convert commercial (S)-leucine
methyl esterÆHCl, 2, into compounds 6a–d. Compound
2
Keywords: Calpain inhibition; Antioxidants.
* Corresponding author. Tel.: +33-16092-2003; fax: +33-16907-3802;
e-mail: serge.auvin@ipsen.com
was either condensed with troloxâ 1a–CO2H,
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.105

3826
S. Auvin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3825–3828
i
O
O
O
O
H
N
H
N
H
N
iv
v
HCl.H2N
R
R
O
R
O
OR1
OR1
N
H
N
H
O
O
O
OH
O
ii
3a-d R1 = Me
4a-d R1 = H
2 R1 = Me
5a-d
6a-d
iii
vi
HO
R =
S
1a
1b
S
1c
O
O
NH
H
O
N
N
H
N
H
S
O
O
H
N
Ac
1d
N
H
N
7
BN 82270
Scheme 1. Reagents and conditions: (i) 1a–CO2H, 1b–CO2H or 1c–CO2H, EDC, HOBT, Et3N, DMF, 20 °C, 15 h; (ii) triphosgene, DIEA, 1d (NH2),
20 °C, 4 h, 70% ; (iii) LiOH, THF, H2O, 20 °C, 2 h; (iv) (S)-a-amino-c-butyrolactoneÆHBr, EDC, HOBT, Et3N, DMF, 20 °C, 15 h; (v) DIBAL 3 equiv
THF, )60 °C, 1 h; (vi) 6b, Ac2O, DMAP, 3 h, 20 °C, 70%.
phenothiazines 1b–CO2H7 or 1c–CO2H8 to afford
carboxamides 3a–c, or converted to the urea 3d by
reaction with 1d (NH2) in the presence of triphosgene
and DIEA. The methyl esters of 3a–d were saponified
and the resulting carboxylic acids 4a–d were condensed
with commercial (S)-a-amino-c-butyrolactone using
EDC/HOBT to yield the lactones 5a–d which, on
reduction with DIBAL at )60 °C, afforded the hybrid
molecules 6a–d. It should be noted that each compound
of the 6a–d series exists as an equilibrated two-diaste-
reomeric mixture. This is attributed to the reversible and
nonstereoselective closing and opening of the semi-ace-
tal ring, with intermediate formation of a transient hydr-
oxyalkyl aldehyde, with traces of water.
potency assessed by their ability to inhibit Fe2þ induced
lipid peroxidation (LPO) in rat brain microsomes.11
Z-LL-H and BHT (2,6-di-tert-butyl-4-methylphenol)
were chosen as reference compounds for calpain and
LPO test, respectively. The results are shown in Table 1.
This study on dual calpain and LPO inhibition began
with the synthesis of the Troloxâ based compound 6a,
which confirmed the synthetic feasibility of these hybrid
compounds. The promising calpain inhibitory activity
(IC50 ¼ 147 nM) of 6a demonstrated that, despite the
Table 1. Calpain and lipid peroxidation inhibitions for 6a–d
The aminoindoline 1d (NH2)9 was accessible (Scheme 2)
through a short straightforward strategy from com-
mercial 5-nitroindoline. Alkylation with benzyl bromide
and reduction of 8 by a mixture of Raney Ni and
hydrazine hydrate in EtOH readily gave 1d (NH2).
O
H
R
N
O
N
H
O
OH
Compds
R
Calp Inh.
IC50, nM
LPO Inh.
IC50, nM
6a
6b
1a
1b
1c
1d
147
22.5
82
1700
70
6c
6d
3600
2340
na
2. Results and discussion
20
Z-LL-H
BHT
4.9
na
The biological activities of compounds 6a–d were eval-
uated in a human calpain 1 enzyme assay,10 with Suc-
Leu-Tyr-AMC as substrate, and the antioxidant
3250
na, not active.
NO2
NO2
NH2
i
ii
N
N
N
H
8
1d(NH2)
Scheme 2. Reagents and conditions: (i) Benzyl bromide, NaH, DMF, 15 h, 20 °C, 92%; (ii) N2H4, H2O, Raney Ni, EtOH, reflux, 4 h, 78%.

S. Auvin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3825–3828
3827
steric bulk of the antioxidant moiety, this compound
was capable of binding to the calpain active site. Pre-
vious work in the domain of hybrid compounds, showed
that Troloxâ based NOS inhibitors,6 exhibited strong
lipid peroxidation inhibition, however, in 6a, the
Troloxâ moiety conferred a weaker activity than
expected. Thus, the antioxidant potency of a molecule
not only depends on the nature of the antioxidant
moiety used, but also on the molecular structure as a
whole. In the phenothiazine series, the advantageous
contribution of the relatively flat heterocyclic ring was
highlighted by the performance of compounds 6b and 6c
in the calpain test. The 2-subsituted phenothiazine 6b
had better affinity than the 1-substituted phenothiazine
6c for the calpain active site. The superior inhibitory
potency of 6b in the calpain test (IC50 ¼ 22.5 nM) was
accompanied by a dramatic improvement in the free
radical scavenging activity with an IC50 ¼ 70 nM. A final
attempt to modify the antioxidant moiety of the mole-
cule was illustrated by the use of the benzyl-aminoind-
oline 1d. The compound 6d proved to be as active as 6b
in the calpain test, however with lower antioxidant
activity. A structural comparison of 6b and 6d demon-
strated that a nearly planar heterocyclic system was
required at the N-terminal position to achieve a high
level of calpain inhibition.
in the C6 cell test was a consequence of moderate cell
permeation.
Accordingly, the free hydroxyl group was masked in
order to enhance the cellular activity. Compound 6b
was transformed into 7 with acetic anhydride in the
presence of DMAP (Scheme 1). This acetylation of the
semi-acetal ring generated a non-equilibrated two-
diastereomeric mixture of 7 with a ratio of (83/17) as
determined by NMR experiments. In the human calpain
1 assay, as expected, 7 did not show any significant
activity (IC50 > 1 lM). However, to our satisfaction, 7
was 1.8-fold more active than 6b in the C6 cell test and
2.5-fold in the necrosis test, which demonstrated that 7
was able to regenerate 6b inside the cell. Furthermore,
the LPO inhibitory activity was conserved for 7.
3. Conclusion
This study demonstrates the synthetic feasibility of
obtaining dual calpain/LPO inhibitors in spite of the
steric bulk of the antioxidant moiety. Calpain was
indeed very sensitive to the nature of the antioxidant
group and 2-substituted phenothiazines proved to be
superior to the other antioxidants in the calpain and
LPO tests. Compound 6b was not only a potent inhib-
itor of isolated calpain but also a powerful free radical
scavenger, with comparable activities in both tests.
Compound 7, a prodrug of 6b, proved to be superior to
6b in cellular assays and furthermore, these hybrid
compounds were much more active than Z-LL-H in the
protection against C6 glial cell death. Compound 7 is
currently undergoing further in vivo evaluation.
In the light of these results compounds 6b and 6d, were
chosen for further in vitro studies involving cellular
calpain inhibition12 in C6 glial cells and protection
against C6 glial cell death induced by maitotoxin.13 The
latter requires calpain inhibition associated with free
radical scavenging activity to protect the cells. Z-LL-H
and BHT were used as reference compounds (Table 2).
Surprisingly, 6d was devoid of activity in the glial cell
test. Whether 6d was unable to penetrate into the cell or
whether it was degraded inside the cell remains unclear.
On the contrary, 6b was active in the C6 calpain inhi-
bition test, although less potent than Z-LL-H. The
superiority of 6b versus Z-LL-H was revealed in the
protection against cell death test, where 6b had an
IC50 ¼ 38.5 lM whereas Z-LL-H, and BHT were weakly
active (100 lM). Since 6b showed good enzyme inhibi-
tion (Table 1), we hypothesized that its modest activity
Acknowledgements
ꢀ
The authors would like to thank Jose Camara and team
for their helpful discussions and advice.
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R
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Compds
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nt
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7
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