A novel series of urea-based peptidomimetic calpain inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.12.068

A series of peptide aldehyde derivatives in which the P₂ chiral carbon has been replaced with nitrogen were synthesized as urea-based peptidomimetic inhibitors of μ-calpain. The compounds mirrored the general SAR of peptidyl aldehyde calpain inhibitors but displayed greater selectivity for μ-calpain over cathepsin B.

Full text of DOI:10.1016/j.bmcl.2005.12.068

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1965–1968
A novel series of urea-based peptidomimetic calpain inhibitors
M. Lee Sanders and Isaac O. Donkor*
Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163, USA
Received 22 November 2005; revised 19 December 2005; accepted 20 December 2005
Available online 18 January 2006
Abstract—A series of peptide aldehyde derivatives in which the P2 chiral carbon has been replaced with nitrogen were synthesized as
urea-based peptidomimetic inhibitors of l-calpain. The compounds mirrored the general SAR of peptidyl aldehyde calpain
inhibitors but displayed greater selectivity for l-calpain over cathepsin B.
Ó 2006 Elsevier Ltd. All rights reserved.
Calpain is of considerable interest because of its implica-
tion in numerous physiological1,2 and pathological
events.1,3–6 This has led to the search for calpain inhib-
itors as pharmacological agents for modulating calpain
action.7,8 For example, calpain inhibitors are of interest
as potential therapy for cardiac ischemia,1,9,10 cerebral
ischemia,1,8,9,11 cancer,12,13 and cataracts.14,15 Most cal-
pain inhibitors are peptide substrate analogues in which
the scissile amide bond of the substrate has been
replaced with an electron-deficient center (e.g., an alde-
hyde) for covalent interaction with the catalytic site cys-
teine of calpain.7,8,16 The inhibitors generally display
poor selectivity for calpain.7 To overcome this problem,
several groups have embarked on the search for
peptidomimetic inhibitors of calpain.7,8 As part of this
effort, we herein report the synthesis, l-calpain inhibito-
ry activity, and selectivity of a series of urea-based
peptidomimetic compounds 1–12 (Table 1). Our objec-
tive was to study the effect on l-calpain inhibition and
selectivity when the geometry of the a-carbon of the
P2 amino acid residue of a peptidyl calpain inhibitor is
changed from chiral and tetrahedral to achiral and
trigonal planar via replacement of the P2 chiral carbon
with nitrogen (Fig. 1). The R1–R3 substituents were also
varied to study the general SAR of the urea-based
compounds.
(Scheme 1). Deprotection of 16 followed by reacting
with isopropyl isocyanate17 afforded urea 17 (94%),
which was reduced with LAH to give urea-based pepti-
domimetic aldehyde 1 (63%). Compounds 2–12 were
synthesized as outlined in Scheme 2. Reductive amina-
tion of the appropriate aldehyde and the corresponding
amine with NaBH3CN at rt gave secondary amine 20
(41–63%). The secondary amine that was used for the
synthesis of 5 was obtained by refluxing a mixture of ex-
cess isopropyl amine and 1-chloro-4-phenylbutane in
anhydrous EtOH for 48 h.18 The secondary amines were
purified by column chromatography and reacted with
the appropriate methyl ester isocyanate 21 to afford urea
methyl ester 22 (83–96%).17 Following purification via
column chromatography, the methyl ester functional
group was hydrolyzed (2 N NaOH) to give acid 23
(82–99%), which was transformed to Weinreb amide
24 (43–68%) followed by LAH reduction to give urea-
based peptidomimetic aldehydes 2–12 (74–96%) in over
95% ee.
Earlier SAR studies of calpain inhibitors suggested that
for potent inhibition of calpain the P2 residue of the
inhibitor must be either L-valine or L-leucine. However,
recent studies from our laboratory19 and those of oth-
ers20,21 have demonstrated that peptidyl aldehydes with
D-amino acids at the P2-position are effective inhibitors
of calpain. To further study the significance of the geom-
etry of the P2 residue on the potency and selectivity of
peptidyl calpain inhibitors, we replaced the P2 a-carbon
with a nitrogen atom thus changing the geometry of the
P2-position from chiral and tetrahedral to achiral and
trigonal planar to afford compounds 1–12. The com-
pounds were studied as inhibitors of porcine erythrocyte
l-calpain (Calbiochem) and human liver cathepsin B
Compound 1 was synthesized by transformation of
Boc-L-phenylalanine 15 to Weinreb amide 16 (89%)
Keywords: Calpain inhibitors; Peptidomimetic; Urea-based calpain
inhibitors; Peptidyl aldehyde inhibitors.
*
Corresponding author. Tel.: +1 901 448 7736; fax: +1 901 448
6828; e-mail: idonkor@utmem.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.068

1966
M. L. Sanders, I. O. Donkor / Bioorg. Med. Chem. Lett. 16 (2006) 1965–1968
Table 1. l-Calpain inhibitory activity of urea-based compounds 1–12 and peptidyl aldehyde calpain inhibitors 13 and 14
R1
O
R3
H
X
R2
N
H
O
Compound
X
R1
R2
R3
l-Calpaina, Kib (lM)
Cathepsin Bc, Kib (lM)
SRd
1
2
N
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
Me
H
PhCH2
13% inh.e
6.82
2.35
N
3
N
Ph(CH2)2
Ph(CH2)3
Ph(CH2)4
4
N
1.16
0.40
5
N
6
N
PhCH2O(CH2)2
PhCH2O(CH2)2
PhCH2O(CH2)2
PhCH2O(CH2)2
PhCH2O(CH2)2
PhCH2O(CH2)2
PhCH2O(CH2)2
PhCH2OCO
0.23
6.69
22.1
33.3
96.1
7
N
8
N
Et
1.15
0.15
9
N
i-Bu
Bn
222
10
11
12
13f
14f,g
N
6.65
88.7
N
i-Bu
i-Bu
i-Bu
i-Pr
N
i-Pr
Bn
Bn
9.27
0.015
0.01
CH
CH
0.12
0.02
8
2
PhCH2OCO
a l-Calpain is porcine erythrocyte calpain (Calbiochem).
b The Ki values are the average of duplicate determinations.
c Cathepsin B is from human liver.
d SR = selectivity ratio, which was determined by dividing the Ki value for cathepsin B inhibition by the Ki value for l-calpain inhibition.
e 100 lM of 1 inhibited l-calpain activity by 13%.
f Peptidyl calpain inhibitors with L-amino acid residues at the P2-position.
g MDL28170 (calpain inhibitor III) was purchased from Calbiochem.
R1
O
R1
(Calbiochem). Assays using synthetic fluorogenic
substrates were employed to determine the Ki values
for inhibition of the enzymes.22,23 The results of the
study are shown in Table 1. Compound 1, which lacks
an R3 substituent for interaction with the S3 subsite of
l-calpain, was a poor inhibitor of the enzyme. Introduc-
tion of an R3 benzyl group (as in 2) enhanced calpain
O
R3
H
R3
H
N
N
N
H
H
R2
O
R2
O
Peptidylaldehyde
Urea derivative
Figure 1. General structures of peptidyl aldehyde calpain inhibitors
and the proposed urea derivatives.
Ph
O
Ph
O
Ph
O
Ph
O
O
O
b, c
a
d
N
H
OH
N
HN
N
O
HN
N
BocHN
BocHN
O
H
H
15
16
17
1
Scheme 1. Synthesis of compound 1. Reagents: (a) EDC, HClÆNH(OCH3)CH3, TEA, CH2Cl2; (b) TFA, CH2Cl2; (c) TEA, isopropyl isocyanate,
toluene; (d) LAH, THF.
R1
O
R1
O
R2
a
b
OMe
H2N R2
R3
N
OMe
+
+
O
C N
R3
N
R2
N
H
R3
H
H
O
O
19
18
20
21
22
c
R1
O
R1
O
R1
O
e
d
N
H
OH
R3
N
R2
N
O
R3
N
N
R3
N
R2
N
H
H
H
R2
O
O
O
2-12
23
24
Scheme 2. Synthesis of compounds 2–12. Reagents and condition: (a) MeOH, NaBH3CN; (b) TEA, toluene, 30 °C; (c) 2 N NaOH, MeOH; (d) CDI,
HClÆNH(OCH3)CH3, DIEA, THF; (e) LAH, THF.

M. L. Sanders, I. O. Donkor / Bioorg. Med. Chem. Lett. 16 (2006) 1965–1968
1967
inhibition. Increasing the alkyl chain to four methylene
Acknowledgments
groups (as in 5) further increased l-calpain inhibition.
We attribute the increase in potency with increased
chain length to favorable interaction with the S3 subsite
of l-calpain. Introduction of a heteroatom into the alkyl
chain appeared to enhance potency (5 vs. 6) presumably
due to hydrogen bonding in the S3 pocket of the enzyme
and/or a change in the conformation of the molecule. A
benzyl group was preferred as the R1 substituent (e.g.,
9 > 12 > 11), while the iso-butyl group was the preferred
R2 substituent (e.g., 9 > 6 > 8 > 7). Thus, the SAR of the
urea-based calpain inhibitors mirrored that of peptidyl
aldehyde calpain inhibitors.7,8,16 However, the urea-
based inhibitors were generally less potent than the cor-
responding peptide-based analogues. For example, 9
was 10-fold less potent than the closely related peptidyl
aldehyde inhibitor 13. The decrease in potency is consis-
tent with the observation that peptide substrates of cal-
pain that bind to the enzyme with L-Leu residue
occupying the S2 subsite are efficiently cleaved by the en-
zyme24–26 and that peptide aldehydes with P2 L-Leu
residues are potent inhibitors of calpain.25–28
The study was supported in part by NIH Grant
R21CA104982 and American Heart Association (South-
east Affiliate) Grant 0255066B.
References and notes
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Most peptide calpain inhibitors lack selectivity for the
enzyme because they equally inhibit other cysteine pro-
teases such as the cathepsins. Only a few active site-di-
rected calpain inhibitors with good selectivity for the
enzyme compared to the closely related cathepsins are
known.7 We therefore tested compounds 6 and 9,
which were the most potent members of the series
against human liver cathepsin B to determine if chang-
ing the geometry at the P2-position from chiral/tetrahe-
dral to achiral/trigonal planar will favor binding to l-
calpain over cathepsin B. As shown in Table 1, com-
pounds 6 and 9 were markedly selective for l-calpain
over cathepsin B. Compound 9, which was the most
potent member of the series, was also the most selective
inhibitor. It was over 220-fold selective for l-calpain
compared to cathepsin B, while the equivalent peptidyl
aldehyde inhibitor 13 was only 8-fold selective for l-
calpain over cathepsin B. Thus, despite the apparent
10-fold decrease in l-calpain inhibitory potency of
the urea-based compounds compared to their peptide-
based analogues, the urea backbone appears to be a
good scaffold for the discovery of active site-directed
l-calpain inhibitors with enhanced selectivity for the
enzyme.
19. Donkor, I. O.; Korukonda, R.; Huang, T. L.; LeCour, L.,
Jr. Bioorg. Med. Chem. Lett. 2003, 13, 783.
20. Chatterjee, S.; Gu, Z.-Q.; Dunn, D.; Tao, M.; Josef, K.;
Tripathy, R.; Bihovsky, R.; Senadhi, S. E.; O’Kane, T. M.;
Mckenna, B. A.; Mallaya, S.; Ator, M. A.; Bozyczko-
Coyne, D.; Siman, R.; Mallamo, J. P. J. Med. Chem. 1998,
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In summary, our results suggest that transformation of
the geometry of the a-carbon of the P2 amino acid res-
idue of peptide aldehyde calpain inhibitors from chiral/
tetrahedral to achiral/trigonal planar by replacement of
the P2 chiral carbon with nitrogen maintains the gener-
al SAR of peptide aldehyde calpain inhibitors. The
change in geometry at the P2-position also improves
the selectivity of the inhibitors for l-calpain over
cathepsin B albeit a 10-fold decrease in l-calpain
inhibitory potency. We are currently in the process
of synthesizing active site-directed l-calpain inhibitors
that incorporate the urea scaffold with the objective
of improving l-calpain inhibitory potency, while
retaining the high selectivity of the inhibitors for the
enzyme.
21. Tripathy, R.; Gu, Z.-Q.; Dunn, D.; Senadhi, S. E.; Ator,
M. A.; Chatterjee, S. Bioorg. Med. Chem. Lett. 1998, 19,
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22. Calpain assay: l-Calpain activity was monitored in a
reaction mixture containing 50 mM Tris–HCl (pH 7.4),
50 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 1 mM
EGTA, 0.2 mM or 1.0 mM Suc-Leu-Tyr-AMC (Calbio-
chem), 2 lg porcine erythrocyte l-calpain (Calbiochem),
varying concentrations of inhibitor dissolved in DMSO
(2% total concentration), and 5 mM CaCl2 in a final
volume of 250 lL in a polystyrene microtiter plate. Assays
were initiated by addition of CaCl2 and the increase in
fluorescence (kex = 370 nm, kem = 440 nm) was moni-
tored at ambient temperature using a SPECTRAmax
Gemini fluorescence plate reader (Molecular Devices).
The Ki values were estimated from the semi-reciprocal

1968
M. L. Sanders, I. O. Donkor / Bioorg. Med. Chem. Lett. 16 (2006) 1965–1968
plot of the initial velocity versus the concentration of the
24. Murachi, T.; Hatanaka, M.; Hamakubo, T. In Neuropep-
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inhibitor according to the method of Dixon.29 The
correlation coefficients for the Dixon plots were above
0.95. No other attempt was made to correct for slow
binding or autolysis. The reported Ki values are the
average of duplicate determinations.
23. Cathepsin B assay: Ki values for inhibition of human liver
cathepsin B were determined as described for calpain
using a reaction mixture containing 14 ng human liver
cathepsin B (Calbiochem), 20 mM NaOAc (pH 6.0),
1 mM EDTA, 0.5 mM DTT, 50 lM or 250 lM Z-Arg-
Arg-AMC, and varying concentrations of inhibitor dis-
solved in DMSO (2% total concentration) in a final
volume of 200 lL.
29. Dixon, M. Biochem. J. 1972, 129, 197.