Synthesis and biological evaluation of novel piperidine carboxamide derived calpain inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00430-3

Calpain inhibitors which are derived from piperidine carboxamides in the P² region were prepared and evaluated for μ-calpain inhibition. In particular, the keto amides 11f and 11j have K(i) of 30 and 9 nM and display a more than 100-fold selectivity over the closely related cysteine protease cathepsin B. Furthermore, these compounds inhibit NMDA induced convulsions in mice indicating that calpain inhibition in brain results in some anticonvulsive properties. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00430-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2187±2191
Synthesis and Biological Evaluation of Novel Piperidine
Carboxamide Derived Calpain Inhibitors
W. Lubisch,* H. P. Hofmann, H. J. Treiber and A. Moller
Department of Discovery Research, KNOLL AG, 67008 Ludwigshafen, Germany
Received 8 May 2000; revised 14 July 2000; accepted 25 July 2000
AbstractÐCalpain inhibitors which are derived from piperidine carboxamides in the P2 region were prepared and evaluated for m-
calpain inhibition. In particular, the keto amides 11f and 11j have Ki of 30 and 9 nM and display a more than 100-fold selectivity over
the closely related cysteine protease cathepsin B. Furthermore, these compounds inhibit NMDA induced convulsions in mice indi-
cating that calpain inhibition in brain results in some anticonvulsive properties. # 2000 Elsevier Science Ltd. All rights reserved.
Calpains represent a class of intracellular cysteine pro-
teases of which in most mammalian cells, at least one
individual member is found.1 Calpains are proposed to
be involved in many intracellular processes and their
enhanced activation would result in serious cellular
damage or even cell death.2 In a number of pathological
conditions, excessive calpain activities have been observed
and calpains have been suggested to be involved in the
progress of certain diseases.3 Indeed, calpain inhibition
has shown to have bene®cial e€ects in experimental
models, for example stroke,4 myocardial infarction,5
brain trauma6 and muscular dystrophy.7 Consequently,
calpain inhibitors have attracted some attention in drug
research and development with the purpose of proving
their therapeutic bene®ts in the clinic.
as building blocks into the P2±P3 region which led to the
piperidine keto amides 3 as novel calpain inhibitors
(Scheme 1).
The calpain inhibitors 11 were synthesized using two
di€erent routes outlined in Scheme 2. Route A corre-
sponds to a method by Li et al.12 which includes a
Dakin±West-type reaction (steps c and d) to build up the
keto ester moiety. Several examples could be prepared
according to this route but only with low or moderate
yields. The amino alcohol 9 was synthesized from 8 by a
multi-step procedure.13 Compound 9 was coupled to the
piperidine carboxylate 5 using the convenient EDC/
HOBT method to obtain 10 which subsequently was
converted into the envisaged keto amide 11 by oxidation
.
with either DMSO/pyr SO3 or DMSO/EDC. Since 9 and
A number of reversible and irreversible calpain inhibi-
tors have been discovered such as MDL 28170 1 and the
proline derivative 2.8 But most of them have problems
regarding selectivity, water-solubility, metabolic stability
in vivo or cellular penetration.9 Recently, several novel
potent nonpeptidic calpain inhibitors have been repor-
ted.10 We have also addressed some e€orts to obtain
novel calpain inhibitors, in particular inhibitors for
calpain I or m-calpain. Our initial e€orts focused on
replacing an amino acid moiety within the peptidic
inhibitors by residues which may improve stability.
Some variations are tolerated within the P2 region and
even cyclic amines such as proline 2 are potent calpain
inhibitors.11 We incorporated piperidine 4-carboxylates
10 were employed as single diastereomers, an enantiomeric
keto amide 11 should be obtained after oxidation. In all
cases 11 had been isolated as racemate which may be
attributed to immediate racemization in solution.13
Route A gave the racemic compounds in any case since
the Dakin±West reaction destroyed the chirality in the
amino acid derivative 4.l2
The biological activities of the prepared compounds 11
were evaluated in a common enzyme assay14 using
human m-calpain isolated from erythrocytes and Suc-
Leu-Tyr-AMC as ¯uorogenic substrate. The inhibition
of cathepsin B was tested in a corresponding assay using
human cathepsin B.15 The results are shown in Table 1.
Several potent calpain inhibitors have been discovered
within this piperidine set. The most potent derivatives,
the naphthalene 11f and the benzothiophene 11j, show
*Corresponding author. Fax: +49-621-6020440; e-mail: wilfried.lubisch@
knoll-ag.de
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00430-3

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W. Lubisch et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2187±2191
Scheme 1.
Scheme 2. (a) EDC, HOBT; (b) LiOH, rt; (c) EtOOC-COCl, THF, re¯ux; (d) EtONa, EtOH, rt; (e) (CH2SH)2, BF3 Â Et2O, CH2Cl2, rt; (f) R4-NH2,
rt; (g) 1. DMSO, py  SO3, rt; 2. KCN; 3. HCl, Et2O, re¯ux; (h) EDC, HOBT; (i) DMSO, py  SO3, rt.
potency comparable to that of the aldehyde derived
inhibitor MDL 28170 1, which is one of the most potent
calpain inhibitors known. We focused our interests on
keto amides which we expected to show improved in
vivo stability compared to aldehydes. Nevertheless, sev-
eral naphthalene derivatives of 11 had been prepared as
aldehydes which show potencies in calpain inhibition
comparable to that of the keto amides (data not shown).
series, we limited the R2 variations to hydrocarbon resi-
dues. In the cinnamoylic set 11b, 11c and 11d, in which
the methyl group of 11b was replaced by isopropyl or
phenyl, the potency increases 20- and 70-fold, respec-
tively, demonstrating the bene®cial e€ects of larger resi-
dues in R2.
A simple benzoyl moiety attached to the piperidino
group in the P3 region results in only a moderate activity
11a (Ki=0.56 mM). Incorporation of an ethylene bridge
as spacer between phenyl ring and amide bond has no
in¯uence on potency, which may indicate that there is
an extended liphophilic pocket at the enzyme binding
site that is not optimally occupied by these residues.
The set of the synthesized derivatives indicate a struc-
ture±activity relationship (SAR) of the piperidines. The
nature of the lipophilic side chain R2 may have con-
siderable importance since this P1 region represents the
recognition site of calpain substrates.16 In the present

W. Lubisch et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2187±2191
2189
Table 1. Synthesized novel calpain inhibitors and the results of testing calpain inhibition
Compound
R1
R2
R4
Synthetic routea
Calpain inhibition
Ki (mM)b
11a
11b
11c
11d
11e
11f
11g
11h
Phenyl-CO-
E-Ph-CHˆCH-CO-
E-Ph-CHˆCH-CO-
E-Ph-CHˆCH-CO-
E-(4-Py)-CHˆCH-CO-
2-Naphthyl-CO-
2-Naphthyl-CH2-
3-Quinolyl-CO
Ph
CH3
CH(CH3)2
Ph
H
H
H
H
H
H
H
H
B
A
A
A
B
B
B
B
B
B
A
A
A
A
0.56
33
1.35
0.46
13.5
0.030
13.6
0.67
0.16
0.009
0.96
0.45
800
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
11i
11j
11k
11l
11m
11n
MDL 28170 1
6-Quinolyl-CO-
H
H
2-Benzothienyl-CO-
2-Naphthyl-CO-
2-Naphthyl-SO2-
2-Naphthyl-SO2-
2-Naphthyl-SO2-
(CH2)3-1-Morpholine
H
CH2CH3
Ph
Ph
(CH2)3-1-Morpholine
0.81
0.015
aSee Scheme 2.
bThe values of the inhibition constants Ki are mean values for two or more independent experiments.
Likewise, the replacement of the benzoyl residue by
naphthalene (11f) caused an 18-fold improvement of the
Ki as compared to 11a. Also, the benzothiophen deri-
vative 11j has a Ki of 9 nM, which is the most potent
inhibitor shown in this study. Incorporation of N-hetero-
cycles such as a pyridine (11e) and quinolines (11h, 11i)
has no advantage, indicating that lipophilic residues are
preferred in this region.
The amido group in P2/P3 is supposed to enter a
hydrogen bond interaction with enzyme residues. The
importance of this amido group is demonstrated by the
sulfonamide 11l and amino methyl derivative 11g which
Scheme 3. Calpain inhibitors MDL 28170 1 (grey line), the proline
aldehyde 2 (black dotted line) and the piperidine keto amide 11e (black
bold line) have been built up by Corina. Finally all three compounds were
superimposed to give an optimal overlap of the P1 region.
are >10- and >100-fold less active, respectively.
Furthermore, to explore the importance of substitution
at the keto amido group we selected residues which had
been demonstrated to be bene®cial in peptidic inhibi-
tors.12,17 We examined an alkyl morpholine (11k and
11n) as well as an ethyl residue (11m), but all derivatives
have no advantage over the unsubstituted compounds
in respect to enzyme inhibition (Table 1).
with the phenyl ring in 1. Both carbonyl groups,
attached either to the naphthalene in 11f and incorpo-
rated in the urethane moiety of 1, are also distinctly
orientated. It is not excluded that rotations, which are
quite possible, may enable hydrogen bond interaction
between the carbonyl groups and the enzyme. Never-
theless, the bene®cial e€ects of the naphthalene ring or
even more the benzothienyl residue in 11j suggest a further
lipophilic hole at the enzyme peptide backbone which
can be utilized for designing novel inhibitors.
The above results indicate important di€erences in the
SAR of the peptidic inhibitors and the piperidines,
which may be attributed to distinct orientations of the
inhibitors within the competitive binding site at the
enzyme. To elucidate this we modeled three inhibitors,
MDL 28170 1, the proline derivative 2 and the naph-
thalene 11f. The result is shown in Scheme 3. For the
piperidine we considered several conformations and
only the most favored chair conformation is depicted in
Scheme 3. The superimposition was performed to obtain
an optimal overlap of the active carbonyl group and the
structural fragment in P1. Scheme 3 discloses the di€erent
orientations of the naphthalene residue in 11f compared
It is well known that MDL 28170 1 and many other
aldehydes derived calpain inhibitors also inhibit closely
related cysteine proteases such as cathepsin B.18 There-
fore, the above-mentioned novel inhibitors were tested
for their potency to inhibit cathepsin B. The results are
shown in Table 2. Both inhibitors, 11f and 11j, were
only moderate inhibitors of cathepsin B with Ki values

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W. Lubisch et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2187±2191
Table 2.
tyrosine kinase pp60src. Finally, we have shown that
two calpain inhibitors are able to suppress seizures
induced by NMDA which also indicates that these
inhibitors penetrate into brain when administered sys-
temically. Altogether, the piperidines represent novel
calpain inhibitors which are suitable tools to investigate
the importance of calpain in animals.
Calpaina Cathepsin Ba pp60srcb
NMDA
Ki/mM
Ki/mM
IC50/mM convulsionsc
ED50/mg/kg
11f
11j
0.030
0.009
0.015
7.300
3.200
0.02518
1.8
ntd
0.7
1.3
1.0
nt
MDL 28170 1
aThe values of the inhibition constants Ki are mean values of two or
more independent experiments.
Acknowledgements
bInhibition of the tyrosine kinase pp60src degradation.
cInhibition of the lethal convulsions induced by NMDA in mice.
dNot tested.
We thank A. Finck, F. Regner and M. Vierling for
supporting chemical synthesis and biological testings
and J. Sadowski for supporting molecular modeling
experiments.
in the mM range. Thus, 11f and 11j represent potent
calpain inhibitors which show a >100-fold selectivity
over a closely related protease.
References and Notes
Since calpain is an intracellular enzyme, inhibitors have
to be evaluated for their ability to penetrate into cells.
This could be estimated in cellular assays in which inhi-
bitors block the intracellular calpain mediated protein
degradation. In platelets, calpain which was activated
by the calcium ionophore A23187 cleaves the tyrosine
kinase pp60src in a speci®c manner.19 Calpain inhibitors
block this degradation and, indeed, MDL 28170 and the
tested piperidine 11f inhibit this degradation with IC50s
of 0.7 and 1.8 mM, respectively, indicating that both
compounds penetrate into cells and have comparable
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It had been reported that calpain inhibitors attentuate
cellular toxicity induced by glutamate receptors agonists
such as AMPA and glutamate.20 This has raised the
question whether calpain inhibitors also modify the
glutamate receptor mediated e€ects in vivo. Therefore,
we have evaluated the ability of calpain inhibitors to
suppress convulsion induced by the glutamate receptor
subtype agonist N-methyl-d-aspartic acid (NMDA) in
vivo, a model which is generally used to characterize
glutamate receptor antagonists.21 Both piperidines 11f
and 11g were tested in this model and, remarkably, both
compounds exert potent anticonvulsive properties (see
Table 2).22 Their ED50s were determined to be 1.3 mg/
kg and 1.0 mg/kg which are rather low dosages in this
model even compared with many NMDA antagonists.
Nevertheless, whether calpain inhibition is responsible
for this anticonvulsive property is not conclusive but, so
far tested, there are no hints that these compounds act
via other molecular targets such as glutamate receptors
(data not shown). On the other hand, this result may
indicate that these calpain inhibitors reached sucient
levels in brain even when administered systemically
which makes these compounds suitable as tools for fur-
ther testing.
In summary, we have outlined the synthesis and a brief
SAR of novel piperidines as m-calpain inhibitors. In
particular, the piperidines 11f and 11j both derived from
ketoamides are potent calpain inhibitors and disclose
high selectivity versus the closely related cysteine pro-
tease cathepsin B. It was also shown that 11f is e€ective
in inhibiting the calpain mediated degradation of the
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W. Lubisch et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2187±2191
2191
15. The assay was performed corresponding to the calpain
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22. In this model, severe convulsions were induced in un-
anesthetized mice by intracerebroventricular injection of 10 mL
of a 0.035% NMDA solution resulting in severe convulsions
and death of animals. The calpain inhibitors were adminis-
tered intraperitoneally (ip) 120 min prior to NMDA and the
ED50 values were calculated as the dose which protected 50%
of the animals.
20. Rami, A.; Ferger, D.; Krieglstein, J. Neurosci. Res. 1997,