Discovery of phenyl alanine derived ketoamides carrying benzoyl residues as novel calpain inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00176-2

Novel calpain inhibitors derived from phenyl alanine aldehydes or ketoamides carrying a benzoyl residue were prepared and evaluated for their biological potency. A brief structure-activity relationship elucidated the importance of ortho-substitutents in t

Full text of DOI:10.1016/S0960-894X(02)00176-2

Bioorganic & Medicinal ChemistryLetters 12 (2002) 1335–1338
Discovery of Phenyl Alanine Derived Ketoamides Carrying
Benzoyl Residues as Novel Calpain Inhibitors
W. Lubisch* and A. Moller
Department of CNS Discovery Research, Abbott GmbH&Co. KG, PO Box210805, 67008 Ludwigshafen, Germany
Received 17 December 2001; revised 25 February2002; accepted 20 March 2002
Abstract—Novel calpain inhibitors derived from phenyl alanine aldehydes or ketoamides carrying a benzoyl residue were prepared
and evaluated for their biological potency. A brief structure–activity relationship elucidated the importance of ortho-substitutents in
the benzoyl moiety. The most potent derivative, the ketoamide 19c, exhibited a Ki of 6 nM and represents a novel class of reversible,
highlypotent and non-peptidic calpain inhibitors. # 2002 Elsevier Science Ltd. All rights reserved.
Calpains represent a class of intracellular cysteine pro-
teases whose numbers have rapidlygrown in recent
years.1 The well-known calpain I or m-calpain is ubi-
quitouslyfound in man and its phsyiological role is
attributed to manyintracellular processes such as the
degradation of both cytoskeleton proteins and signal
transduction proteins.2 Excessive activation of calpains
contributes to serious cellular damage or even cell
death3 and calpains are thought to be involved in the
progress of a number of diseases.4 Indeed, inhibition of
calpain, in particular of m-calpain, has revealed bene-
ficial effects in experimental models, for example, on
stroke,5 myocardial infarction,6 brain trauma,7 multiple
sclerosis8 and muscular dystrophy.9 For years, con-
siderable efforts have been focused on calpain inhibitors
as a novel therapeutic principle.10
Our aim was to identifynew scaffolds for reversible
calpain inhibitors in order to overcome the inadequate
pharmacokinetic properties. Since most reported rever-
sible calpain inhibitors are transiton state mimics and
were derived from aldehydes and ketons, we also deci-
ded to use these moieties as a warhead in the envisaged
inhibitors. Recently, we reported an approach demon-
strating that substituted naphthoyl piperidines in the P2/
P3 region which are derived from ketoamides as war-
heads are potent calpain inhibitors.13 Proceeding these
efforts we discovered phenyl alanine derivatives 2 car-
rying benzoyl residues as novel calpain inhibitors (see
Fig. 1). The present paper focuses on the importance of
ortho-substitution at the benzoyl residue moiety and the
discoveryof the stilbene moietyas substituent at the
phenyl alanine scaffold such as 19c.
A number of reversible and irreversible calpain inhibi-
tors have been reported. Most of them are derived from
peptides, but meanwhile nonpeptidic calpain inhibitors
have been discovered.11 However, no calpain inhibitors
are reported to show pharmacodynamic and pharma-
cokinetic properties, which are required to prove the
principle in animals and man. Their use is generally
limited by poor selectivity, poor metabolic stability, low
cellular penetration, poor kinetics or, depending on the
envisaged therapeutic indication, low oral availability
and low water solubility.12
The syntheses of the compounds are outlined in Figures
2 and 3.
The benzoic acids (e.g., 5, 8, and 13) were either pur-
chased or prepared according to the following routes,
also shown in Figure 2. Alkylation of the phenols 4 with
chloromethyl derivatives 3 gave the phenolic ethers 5.
The Heck reaction was used to prepare the stilbene and
tolane derivatives. For example, the styrol 7 was added
to o-bromo-benzoic carboxylate 6 in the presence of
Pd[(Ph3P)2Cl]2 at 100 ꢀC to obtain a good yield of the
ester 8a. O-alkylated benzoic acid 13 was prepared from
4,4-dimethyl-2-phenyl-2-oxazolidine 9 in 3 steps. Com-
pound 9 was deprotonated by n-BuLi at À78 ꢀC and
2-naphthaldehyde 10 was added to produce the alcohol
11. Hydrolysis of the oxazolidine 11 byHCl generated
*Corresponding author. Tel.: +49-621-60-42-016; fax: +49-621-589-
68936; e-mail: wilfried.lubisch@abbott.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00176-2

1336
W. Lubisch, A. Moller / Bioorg. Med. Chem. Lett. 12 (2002) 1335–1338
¨
Figure 1.
Figure 2. Routes for synthesis of the benzene carboxylates used as center building block. (i) DMF, K2CO3, 2. NaOH; (ii) PdCl2, Ph3P, DMF; (iii)
NaOH; (iv) (1) n-Buli, À78 ꢀC, (2) 2-naphthyl-carbaldehyde 10; (v) aq HCl, 80ꢀC; (vi) H2, Pd/BaSO4.
Figure 3. Routes for the preparation of aldehydes 17 and 18 as well as ketoamides 19. (i) EDC, HOBT, rt; (ii) DMSO, pyÂSO3, rt.
the carboxylate which immediately formed the lactone
12. After that, 12 was hydrogenated in the presence of
Pd/BaSO4 to provide the desired carboxylate 13. In a
final step, all esters were hydrolyzed by diluted NaOH
or KOH to give the carboxylates 14.
All carboxylates were coupled to either (2S )-2-amino-3-
phenyl-1-propanol 15a or to 3-amino-2-hydroxy-4-
phenyl-butyramide 15b14 byconvenient methods (e.g.,
EDC, HOBT) to obtain the amides 16. Finally, these
amides 16, which carrythe alcohol moiety, were oxi-

W. Lubisch, A. Moller / Bioorg. Med. Chem. Lett. 12 (2002) 1335–1338
¨
1337
.
dized byDMSO/py SO3 at ambient temperature to give
either the aldehydes 17 and 18 or the ketoamides 19.
displayed calpain inhibition (Ki=0.23 mM) but this was
onlyslightlysuperior to 17.
The biological activities of the prepared compounds 17,
18 and 19 were evaluated in a common enzyme assay
using human m-calpain isolated from erythrocytes and
Suc-Leu-Tyr-AMC as the fluorogenic substrate.15 The
inhibition of cathepsin B and cathepsin L was tested in
corresponding assays using commercially available
enzymes.16 The results are summarized in Tables 1 and
2.
To verifythe relevance of the carbonyl group within the
bridge as the hydrogen bond acceptor we looked for the
corresponding methylene derivative 18c and, surpris-
ingly, 18c retained calpain inhibition (Ki=0.37 mM). To
elucidate this unexpected result we addressed additional
efforts on variations of both the bridge and the distal
aromatic ring.
The biphenyl 18b demonstrates the importance of a
distal aromatic ring. Merelya phenyl ring in the ortho-
position at the benzoyl moiety (18b) resulted in a
10-fold increase in calpain inhibition (compared to 17)
and 18b had a Ki of 90 nM. Incorporation of a methyl-
ene bridge (18c, 18e) or an ethylene bridge (18d) into the
biphenyl moiety slightly diminished the inhibition
potency. Insertion of a heteroatom, such as an oxygen
atom, into the bridge produced controversial effects in
potency. The biphenylether 18f showed a Ki of 50 nM
and represented one of the most potent inhibitors in the
present series. On the other hand, prolongation of the
bridge with a methylene group (see 18g, 18h), favoring
the flexibilityof the side chain, was unfavorable which is
shown bythe Ki of 0.45 mM. A bulkynaphthalene resi-
due as the distal aromate was well tolerated (see 18e and
18h) indicating the considerable size of this lipophilic
cave at the enzyme-binding site.
MDL 28170 1 represents a small-peptidic aldehyde-
derived calpain inhibitor and is widelyused as a tool
compound. According to the reported structure–activity
relationship the Z-Val moietycontributes considerably
17a
to the inhibitorypotenc.y
The replacement of this
moietywith a benzoyl residue resulted in a more than
90-fold drop of affinityand the Ki is roughly1 mM (see
17). Recentlyit was reported that halogen substitution
at this benzoyl residue may be favorable.17b Therefore,
we decided to employthe phenyl ring as the spacer car-
rying substituents selected to interact with the P2/P3
region of the enzyme. Superimposition of MDL 28170 1
and 17 bymolecular modeling revealed that a substitu-
tion of 17 bybenzoyl residues might be favorable in the
ortho-position. Indeed, the ortho-benzophenone 18a
Table 1. Synthesized compounds and their results in a common
enzyme inhibition assay using purified human calpain and Suc-Leu-
Tyr-AMC as the substrate
In order to optimize the conformation of the central
and distal aromate, we examined the stilbene 18i and
the acetylene derivative 18j, both representing more
rigid structures. Both the E-stilbene 18i and the acetyl-
ene 18j were as potent as the alkyl derivative 18d, which
mayraise doubts as to the significance of such rigidity.
On the contrary, substitution of the distal aromate
opened up additional opportunities for optimizations.
The stilbene 18k, which carries two methoxygroups at
the distal phenyl ring, disclosed a Ki of 40 nM. Further-
more, the naphthalene 18l exhibited a Ki of 15 nM and
is roughlyone and two orders of magnitude more
potent than the stilbene 18i and the alkyl derivative 18d,
respectively.
R1
R2
Calpain Ki/mM
17
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Phenyl-CO
Phenyl
1.08
0.23
0.09
0.37
0.28
0.20
0.05
0.45
0.05
0.14
0.45
0.04
0.015
0.04
0.08
0.006
18a
18b
18c
18d
18e
18f
18g
18h
18i
18j
18k
18l
19a
19b
19c
Phenyl-CH2
Phenyl-CH2CH2
2-Naphthyl-CH2
Phenyl-O
Since all of the above inhibitors were derived from
aldehydes, which were used as a warhead to the active
center of the calpain enzyme, we looked for more stable
derivatives and selected ketoamides as the aldehyde
surrogate.18 Ketoamides corresponding to three potent
aldehydes, 18b, 18k and 18l were prepared. Interest-
ingly, all three ketoamides 19a, 19b and 19c retained the
potencyof the corresponding aldehydes within a two-
fold range in calpain inhibition. In particular, the
Phenyl-OCH2
2-Naphthyl-CH2O
E-Phenyl-CH=CH
Phenyl-CꢁC
E-(3,4-MeO)2-phenyl-CH=CH
E-2-Naphthyl-CH=CH
Phenyl
CONH2
CONH2
CONH2
E-(3,4-MeO)2-phenyl-CH=CH
E-2-Naphthyl-CH=CH
Table 2. Results of selected compounds in enzyme inhibition using commercially available cathepsin B and L and Suc-Leu-Tyr-AMC as the sub-
strate. Inhibition of the tyrosine kinase pp60src was determined in human thrombocytes19
Calpain (Ki/nM)
Cathepsin B (Ki/nM)
Cathepsin L (Ki/nM)
pp60src (IC50/mM)
19c
MDL 28170 1
6
15
99
nda
6100
nda
1.4
0.7
aNot determined.

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W. Lubisch, A. Moller / Bioorg. Med. Chem. Lett. 12 (2002) 1335–1338
¨
naphthalene 19c displayed a Ki of 6 nM and represents
one of the most potent calpain inhibitors, which are not
derived from aldehydes, reported so far.
3. Squier, M. K. T.; Cohen, J. J. Death and Differentiation
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1994, 15, 412.
Ketoamides are supposed to represent reversible enzyme
inhibitors.18 Indeed, according to its enzyme kinetics,
the ketoamide derived inhibitor 19c represents a rever-
sible calpain inhibitor (data not shown). Manyof the
reported calpain inhibitors were found to be lacking
owing to poor or modest selectivityversus related
cysteine proteases such as cathepsines B and L.11b
Accordingly, 19c inhibited cathepsin B and cathepsin L
with Ki’s of 99 and 6100 nM, respectively, demonstrating
moderate selectivityversus cathepsin B (15-fold), whereas
selectivityversus cathepsin L is excellent (1000-fold).
Compound 19c did not block non-cysteine proteases
even up to higher mM concentrations (data not shown).
In comparison, the commonlyused tool compound for
calpain inhibition, MDL 28170 1, exhibited no selectiv-
ityversus these cysteine proteases and blocks calpain as
well as cathepsines B and L at low nanomolaric con-
centrations.19
5. Markgraf, C. G.; Velayo, N. L.; Johnson, M. P.; McCarty,
D. R.; Medhi, S.; Koehl, J. R.; Chmielewski, P. A.; Linnik,
M. D. Stroke 1998, 29, 152. Bartus, R. T.; Hayward, N. J.;
Elliott, P. J.; Sawyer, S. D.; Baker, K. L.; Dean, R. L.;
Akiyama, A.; Straub, J. A.; Harbenson, S. L.; Li, Z. Z.; Pow-
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6. Iwamoto, H.; Miura, T.; Okamura, T.; Shirakawa, K.;
Iwatate, M.; Kawamura, S.; Tatsuno, H.; Ikeda, Y.; Matsu-
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9. Baghiguian, S.; Martin, M.; Richard, I.; Pons, F.; Astier,
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I. O. Curr. Med. Chem. 2000, 7, 1171.
To determine the penetration of cellular membranes we
evaluated the inhibition of the calpain-mediated degra-
dation of the tyrosine kinase pp60src in human thro-
mobocytes.20 Both compounds, MDL 28170 1 and 19c,
blocked pp60src degradation at low mM-concentrations
with an IC50 of 0.7 and 1.4 mM, respectively.
12. Wells, G. J.; Bihovsky, R. Exp. Opin. Ther. Pat. 1998, 8,
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In summary, we used the phenyl alanine carrying a
benzoyl residue 17, which showed onlypoor calpain
inhibition, as the starting point for our investigations.
Optimization of substitution in the ortho-position in the
benzoyl moiety resulted in a novel class of potent cal-
pain inhibitors. The most active inhibitor in this series,
the naphthyl derivative 19c, represents a reversible, cell
penetrating inhibitor which also displays selectivity ver-
sus cathepsin B and L.
13. Lubisch, W.; Hofmann, H. P.; Treiber, H. J.; Moller, A.
Bioorg. Med. Chem. Lett. 2000, 10, 2187.
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R., III; Carroll, R. M.; Straub, J. A.; Tkaacz, J. N.; Wu, C. C.;
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