Design, Synthesis, Molecular Modeling Studies, and Calpain Inhibitory Activity of Novel α-Ketoamides Incorporating Polar Residues at the P 1′-Position

Article abstract of DOI:10.1021/jm0301336

A series of novel α-ketoamides incorporating stereoisomeric residues with different electronic properties at the P₁′-position were synthesized to study the electronic requirements for inhibitor binding to the S₁′-subsite of calpain I

Full text of DOI:10.1021/jm0301336

72
J . Med. Chem. 2004, 47, 72-79
Design , Syn th esis, Molecu la r Mod elin g Stu d ies, a n d Ca lp a in In h ibitor y Activity
of Novel r-Ketoa m id es In cor p or a tin g P ola r Resid u es a t th e P 1′-P osition
Isaac O. Donkor,* J ie Han, and Xiaozhang Zheng†
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Received March 24, 2003
A series of novel R-ketoamides incorporating stereoisomeric residues with different electronic
properties at the P1′-position were synthesized to study the electronic requirements for inhibitor
binding to the S1′-subsite of calpain I. The results of the study suggested the presence of an
acidic amino acid residue at the S1′-subsite of calpain I. For example, ester 1a (Cbz-L-Leu-L-
Phe-CO-D-Phe-OMe) was over 450-fold more potent than its carboxylic acid derivative 2a (Cbz-
L-Leu-L-Phe-CO-D-Phe-OH). Additionally, amidino derivative 3a (Cbz-L-Leu-L-Phe-CONH-D-
CH[C(NH)NH2]Bn) was about 6000-fold more potent than 2a . Furthermore, 4a (Cbz-L-Leu-L-
Phe-CONHCH2Bn) was 12-fold less potent than its aza analogue 4b (Cbz-L-Leu-L-Phe-
CONHNHBn). The results are consistent with the presence of an acidic amino acid residue at
the S1′-subsite of calpain I. The acidic amino acid residue was found to be Glu261 via molecular
modeling studies.
Ta ble 1. Structure, Diastereomeric Purity, and Calpain I
Inhibitory Activity of Novel R-Ketoamides 1-4
In tr od u ction
Calpain is an intracellular cytoplasmic nonlysosomal
cysteine endopeptidase that is activated by calcium ions.
Several calpain isoenzymes have been discovered1-12 of
which calpain I and calpain II (also known as µ-calpain
and m-calpain, respectively) are the most ubiquitous in
mammalian cells. The structure, mechanism of activa-
tion, and pharmacological actions of calpain have been
reviewed.12-20 The enzyme has been implication in
several pathological conditions including stroke, cere-
bral ischemia, and cataract.18,19,21 This has led to the
search for calpain inhibitors as potential therapeutic
agents.18-21
Structural information regarding the active site pocket
of calcium-activated calpain is pivotal to the discovery
of potent and specific inhibitors of the enzyme. Unfor-
tunately, such information was not available until the
recent disclosure of the X-ray crystal structure of the
calcium-bound protease core of calpain I.22 Our labora-
tory23-25 and that of others21,26-29 have employed struc-
ture-activity relationship (SAR) studies to probe the
S- and S′-subsites of calpain in attempts to understand
the structural requirements for inhibitor binding to the
active site of the enzyme. As a part of this effort, we
report in this paper the synthesis, molecular modeling
a
Ki values are the averages of triplicate determinations ob-
tained by Dixon plots where 1/v were plotted against inhibitor
studies, and calpain inhibitory activity of a novel series
of R-ketoamides incorporating polar residues at the P1′-
position of the inhibitors (Table 1). The compounds were
developed as molecular probes to study the significance
of electrostatic interaction between an inhibitor and the
S1′-subsite of calpain I.
concentration at two different substrate concentrations (0.2 mM
and 1.0 mM) to give intersecting lines with correlation coefficient
b
g0.95. DP ) diastereomeric purity. The diastereomeric purity
of the compounds was determined using a Shimadzu HPLC system
and a Macrosphere RP 300 C18 column. c The mobile phase was
d
acetonitrile (55%)/TFA (0.15%)/water. The mobile phase was
acetonitrile (30%)/diethylamine (0.15%)/water. e The mobile phase
was acetonitrile (25%)/TFA (0.15%)/water.
Ch em istr y
Compound 5 was synthesized as described previ-
ously28 and coupled with D-phenylalanine methylester
using EDC and HOBT as the coupling agent to give
R-hydroxylamide dipeptide 6a (Scheme 1). Cleavage of
the Boc protecting group of 6a followed by coupling with
Cbz-L-leucine and oxidation of the hydroxyl group with
Dess-Martin reagent gave 1a . Basic hydrolysis of the
ester group of 1a afforded carboxylic acid derivative 2a .
Compound 2b was synthesized in a similar manner
* Corresponding author. Phone: (901) 448-7736. Fax: (901) 448-
6828. E-mail: idonkor@utmem.edu.
† Currently at Neurogen Corporation, Bradford, CT 06405.
10.1021/jm0301336 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/02/2003

Novel R-Ketoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 73
Sch em e 1a
a
Reagents: (a) D-Phenylalanine methyl ester, EDC, HOBT; (b) HCl/dioxane; (c) Cbz-L-leucine, EDC, HOBT; (d) Dess-Martin reagent;
(e) 1 N HaOH.
Sch em e 2a
a
Reagents: (a) TEA, ClCO2Et, NH3(g); (b) TEA, (CF3CO)2O; (c) 50% TFA; (d) EDC, HOBt, NMM, 5; (e) 50% TFA; (f) Cbz-L-leucine,
EDC, HOBT, NMM; (g) N-acetylcysteine, NH4OAc; (h) DMSO, pyridiniium trifluoroacetate, EDC.
using L-phenylalanine methyl ester as the starting
material.
Compound 4a was synthesized as described previ-
ously.26 The synthesis of 4b is shown in Scheme 3.
Compound 5 was Boc-deprotected using 2 N HCl to give
R-hydroxy-â-amino acid 15, which was esterified by
treatment with SOCl2/MeOH to give 16. Compound 16
was coupled with Cbz-L-leucine to afford pseudodipep-
tide methyl ester 17. Treatment of 17 with hydrazine
gave hydrazide 18, which was reacted with benzalde-
hyde to obtain hydrazone 19. Reduction of 19 with
NaBH3CN gave N′-benzyl hydrazide 20. Boc-protection
of 20 afforded 21, which was transformed to 22 by
treatment with Dess-Martin reagent. Removal of the
Boc protecting group of 22 gave hydrazide 4b.
The synthesis of amidine 3a commenced with the
transformation of Boc-protected D-phenylalanine to car-
boxamide 9a using ClCO2Et in the presence of TEA
followed by treatment of the resulting acid chloride with
NH3 gas (Scheme 2). Dehydration of carboxamide 9a
with (CF3CO)2O in the presence of TEA afforded nitrile
10a , which was deprotected with 50% TFA to give 2(R)-
(+)-amino-3-phenylpropionitrile (11a ). Coupling of 11a
with 5 in the presence of EDC, HOBT, and NMM
afforded pseudodipeptide 12a . Deprotection of 12a with
50% TFA followed by coupling with Cbz-L-leucine gave
pseudotripeptide 13a . Compound 13a was treated with
N-acetylcysteine and NH4OAc to give amidine 14a , the
hydroxyl group of which was oxidized with pyridinium
trifluoroacetate and EDC in DMSO to give R-ketoamide
3a . Compound 3b was synthesized in a similar manner
starting with Boc-L-phenylalanine.
Resu lts a n d Discu ssion
SAR Stu d ies. SAR studies have suggested that the
S′-subsites of calpain I can accommodate polar func-
tional groups.24,26-29 We therefore synthesized R-keto-
amides 1-4 (Table 1) as molecular probes to study the

74 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Donkor et al.
Sch em e 3a
a
Reagents: (a) 2 N HCl; (b) SOCl2, MeOH; (c) Cbz-L-leucine, EDC, HOBT, NMM; (d) NH2NH2 (e) BnCHO; (f) NaBH3CN, HCl; (g)
(t-BuOCO)2O, NaOH; (h) Dess-Martin reagent.
significance of electrostatic interaction at the S1′-subsite
of calpain I. The compounds were evaluated as inhibi-
tors of porcine erythrocyte calpain I at 25 °C using two
different concentrations (0.2 and 1.0 mM) of Suc-Leu-
Tyr-AMC as the substrate. The Ki values of the inhibi-
tors were determined using Dixion plots.30 Table 1
displays the inhibitory data for the compounds.
We also synthesized stereoisomeric compounds 3a
and 3b to further study the S1′-subsite of calpain I.
Compound 3a with a basic (R)-1-amidino-2-phenyleth-
ylamine substituent at the P1′-position was about 6000-
fold more potent than 2a with an acidic (R)-phenylala-
nine residue at the P1′-position. This is consistent with
an acidic amino acid residue at the S1′-subsite of caplain
I, because 3a would undergo electrostatic attraction and/
or hydrogen-bonding interaction with the carboxylate
group at the S1′-subsite of the enzyme, while 2a would
experience electrostatic repulsion at this site (Figure 1).
Additionally, 3a was about 60-fold more potent than 3b
due to the difference in the stereochemistry of their P1′-
substituents. The results are consistent with previous
reports that the S′-subsites of calpain tolerate basic
functional groups.27-29
On the basis of our hypothesis that the S1′-subsite of
calpain I contains an acidic amino acid residue, 3a
should be more potent than 4a . However, 3a and 4a
were almost equipotent. We reasoned that steric in-
teraction between the amidine group of 3a and the
carboxylate group of the S1′-acidic amino acid resi-
due negatively affected the calpain I inhibitory po-
tency of 3a . To investigate this, we synthesized 4b
(which is the aza derivative of 4a ) and found it to be
12-fold more potent than 4a , in support of the presence
Compound 2b with (S)-phenylalanine at the P1′-
position was over 500-fold more potent than 2a with (R)-
phenylalanine at the P1′-position. The greater calpain
I inhibitory potency of 2b compared to its stereoisomer
2a led us to propose the presence of an acidic amino
acid residue (aspartic acid or glutamic acid) at the S1′-
subsite of calpain I. We attributed the poor calpain I
inhibitory potency of 2a to electrostatic repulsion be-
tween the carboxylate group of its P1′-substituent and
the carboxylate group of the proposed acidic amino acid
residue at the S1′-subsite of the enzyme (Figure 1).
Compound 1a , which is the methyl ester derivative of
2a , was also over 450-fold more potent than 2a , in
support of the presence of an acidic amino acid residue
at the S1′-subsite of calpain I. Esterification of the
carboxylate group (as in 1a ) relieved the unfavorable
electrostatic repulsion between the carboxylate of the
inhibitor and that of the enzyme; hence, the greater
potency of 1a compared to 2a .
F igu r e 1. Proposed electrostatic interaction between the inhibitors and an active site acidic amino acid residue of calpain I. The
amino acid residue is either aspartic acid (n ) 1) or glutamic acid (n ) 2).

Novel R-Ketoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 75
Ser115 was released.22 We docked 3a , 3b, and 4b into
the active site pocket of the enzyme to study their
binding modes and to identify the acidic amino acid
residue that the SAR studies predicted to be present at
the S1′-subsite of calpain I. The docking studies were
performed after in silico mutation of Ser115 to Cys115
to mimic the wild-type enzyme. The compounds were
constructed using Sybyl 6.831 and energetically mini-
mized using the Tripos force field with Gasteiger-
Hu¨ckel charges.32 The binding site for docking the
inhibitors was defined using Cys115 as the center and
all amino acid residues within 10 Å radius of it were
included.
The docking studies showed that the stereoisomeric
compounds 3a and 3b exhibit different binding modes
in the active site pocket of calpain I. Compound 3a
adopted a conformation that allowed the formation of
an intramolecular hydrogen bond between one of the
amidine NH groups and the amide carbonyl of the
ketoamide functionality. This conformation facilitated
the formation of three hydrogen bonds between the
amidine of 3a and the carboxylate of Glu261 (Figure 2,
panel A). Thus, Glu261 was identified as the acidic
amino acid residue that was predicted by the SAR
studies to be present at the S1′-subsite of calpain I. Four
other hydrogen bonds, including a hydrogen bond
between the NH of the P1-P2 amide bond of 3a and
Gly271, were also observed. Angelastro et al.33 replaced
the P1-P2 amide bond of a calpain inhibitor with a
ketomethylene moiety and noticed a 250-fold loss of
calpain inhibitory potency. The decrease in potency was
attributed to the lack of a critical hydrogen bond
between the normally occurring NH of the P1-P2 amide
bond and calpain. Our docking studies identified Gly271
as the active residue of calpain that is involved in the
formation of this critical hydrogen bond.
Compound 3b adopted a different binding conforma-
tion in the active site pocket of calpain I compared to
its stereoisomer 3a . Unlike 3a , no intramolecular
hydrogen bonding was observed in the binding of 3b to
calpain I (Figure 2, panel B). Additionally, only three
hydrogen-bonding interactions were observed between
3b and calpain I. Furthermore, the amidine group of
3b was not juxtapositioned to Glu261, thus precluding
electrostatic interaction between 3b and the enzyme.
The docking studies also showed that 4b is capable of
electrostatic interaction with calpain I via its protonated
hydrazine nitrogen and the carboxylate group of Glu261
(Figure 2, panel C). The results suggest that the calpain
I inhibitory potency of the compounds is influenced by
their ability to interact electrostatically with Glu261.
F igu r e 2. Hydrogen-bonding interactions between calpain I
and 3a (panel A), calpain I and 3b (panel B), and calpain I
and 4b (panel C). Hydrogen atoms are removed for clarity,
except those involved in hydrogen bonding. Hydrogen bonds
are shown as black dashed lines. The atoms are colored as
follows: all the protein residues are orange, except those
involved in the hydrogen-bonding interactions; carbon ) green;
hydrogen ) cyan; nitrogen ) blue; oxygen ) red; sulfur )
yellow.
Con clu sion
In this paper, we have described the account of our
work on a series of novel R-ketoamides incorporating
polar functional groups at the P1′-position of the inhibi-
tors. SAR studies of the compounds suggested the
presence of an acidic amino acid residue at the S1′-
subsite of calpain I. Using molecular modeling studies
the acidic amino acid residue was identified as Glu261.
Compounds that were capable of electrostatic and/or
hydrogen-bonding interaction with this residue (Glu261)
were more potent inhibitors of calpain I than those that
were not capable of undergoing such interactions (e.g.,
of an acidic amino acid residue at the S1′-subsite of
calpain I.
Molecu la r Mod elin g Stu d ies. During the course of
this study the crystal structure of the calcium-bound
protease core of calpain I in which the catalytic site
Cys115 of the wild-type enzyme has been mutated to

76 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Donkor et al.
1
obtain 7b in 76% yield: mp 180-182 °C; H NMR (CDCl3) δ
0.88 (d, 6H), 1.36 (m, 1H), 1.61 (m, 4H), 3.13 (m, 3H), 3.62 (s,
2H), 3.62 (s, 2H), 3.74 (s, 1H), 4.11 (m, 3H), 4.71 (q, 0.5H),
4.90 (m, 0.5H), 5.14 (m, 2H), 5.53 (d, 1H), 6.20 (d, 1H), 6.85
(m, 1H), 7.27 (m, 15H).
4b vs 4a , and 3b vs 3a ). Gly271 was also identified as
the active site residue with which the NH group of the
P
1-P2 amide bond of peptidyl calpain inhibitors must
hydrogen bond if they are to be potent inhibitors of the
enzyme.
2(R)-[3(S)-(N-Cb z-L-leu cyla m in o)-4-p h en yl-2-oxo-b u -
tyr yla m in o]-3-p h en ylp r op ion ic Acid Meth yl Ester (1a ).
The hydroxyl group of 7a (1.15 g, 1.9 mmol) was oxidized using
Dess-Martin reagent (3.23 g, 7.6 mmol). The crude product
was recrystallized from EtOAc to afford 1a in 79% yield: mp
159-161 °C; 1H NMR (CDCl3) δ 0.91 (d, 6H), 1.45 (m, 1H),
1.68 (m, 3H), 3.12 (dd, 1H), 3.19 (d, 2H), 3.32 (dd, 1H), 3.78
(s, 3H), 4.16 (m, 1H), 4.86 (q, 1H), 5.01 (d, 1H), 5.12 (s, 1H),
5.51 (q, 1H), 6.56 (d, 1H), 7.26 (m, 15H). Anal. (C34H39N3O7)
C, H, N.
Exp er im en ta l Section
Ch em istr y. Gen er a l Meth od s. Melting points were de-
termined on a Fisher-J ohns melting point apparatus and are
uncorrected. Optical rotations were measured with a DigiPol
781 automatic polarimeter (Rudolph Instruments). Molecular
masses were determined with Bruker-HP Esquire-LC-MS. 1H
NMR spectra were recorded on Bruker ARX-300 MHz and
Varian Inova-500 MHz. Deuterated solvents were purchased
from Cambridge Isotope Laboratories, Inc. The chemical shifts
(δ) are reported in ppm relative to TMS. Elemental analyses
(C, H, N) were performed by Atlantic Microlab Inc., Norcross,
GA and are within (0.4% of the theoretical values. Chemicals
and solvents were purchased from either Aldrich Chemical Co.
or Fisher Scientific.
Gen er a l P r oced u r e 1. Syn th esis of 6, 7, 12, 13, a n d 17.
EDC (3.5 mmol) and HOBT (3.5 mmol) were added to an ice
cooled solution of the appropriate acid in anhydrous DMF (45
mL) followed by the slow addition of NMM (3.5 mmol) and
the amine hydrochloride (3.5 mmol). The mixture was stirred
overnight at room temperature and the DMF was removed in
vacuo. The residue was dissolved in EtOAc (60 mL); washed
with saturated NaHCO3 solution, 0.5 N HCl, water, and brine;
and dried over MgSO4. The solvent was removed and the
residue was recrystallized from EtOAc/hexane.
Gen er a l P r oced u r e 2. Dess-Ma r tin Oxid a tion . Dess-
Martin reagent (0.38 mmol) was slowly added to an ice cooled
solution of the appropriate alcohol (0.19 mmol) in anhydrous
CH2Cl2 (10 mL). After 10 min, the ice bath was removed and
the milky reaction mixture was stirred at room temperature
for 4 h. A solution of Na2S2O3 (2 mmol) in saturated NaHCO3
was added and the mixture was stirred for additional 10 min
at room temperature. After separation, the aqueous layer was
extracted with CH2Cl2 (3 × 20 mL) and the combined organic
layer was washed successively with NaHCO3 solution, water,
and brine. It was dried (MgSO4), concentrated, and purified
either by flash chromatography or crystallization.
2(S)-[3(S)-(N-Cb z-L-leu cyla m in o)-4-p h en yl-2-oxo-b u -
tyr yla m in o]-3-p h en ylp r op ion ic Acid Meth yl Ester (1b).
Compound 7b was oxidized with Dess-Martin reagent and
the crude product was recrystallized from EtOAc/hexane to
give 1b in 95% yield: mp 167-168 °C; 1H NMR (CDCl3) δ 0.90
(d, 6H), 1.43 (m, 1H), 1.58 (m, 4H), 3.12 (m, 2H), 3.25 (dd, 2H),
3.77 (s, 3H), 4.91 (m, 1H), 5.12 (m, 2H), 5.52 (q, 1H), 6.50 (d,
1H), 6.95 (br. s, 1H), 7.26 (m, 15H). Anal. (C34H39N3O7) C, H,
N.
2(R)-[-3(S)-(N-Cbz-L-leu cyla m in o)-4-p h en yl-2-oxo-b u -
tyr yla m in o]-3-p h en ylp r op ion ic Acid (2a ). Compound 1a
(0.46 g, 0.765 mmol) was suspended in MeOH (5 mL) and 1 N
NaOH (1 mL) and stirred at room temperature for 90 min.
The solvent was removed and the residue was recrystallized
from Et2O/hexane to afford 2a in 60% yield: mp 167.5-168.3
°C; 1H NMR (CDCl3) δ 0.91 (d, 6H), 1.45 (m, 3H), 2.95 (m, 2H),
3.25 (m, 2H), 4.38 (dd, 1H), 5.00 (m, 3H), 5.42 (d, 1H), 5.71 (d,
1H), 6.71 (m, 1H), 6.93 (d, 1H), 7.25 (m, 15H), 7.64 (d, 1H).
Anal. (C33H37N3O7) C, H, N.
2(S)-[3(S)-(N-Cbz-L-leu cyla m in o)-4-p h en yl-2-oxobu tyr -
yla m in o]-3-p h en yl-p r op ion ic Acid (2b). Compound 2b was
synthesized in 52% yield from 1b as described for the synthesis
of 2a : mp 166.8-167.7 °C; 1H NMR (DMSO-d6) δ 0.76 (m, 6H),
0.85 (m, 2H), 1.22 (t, 1H), 1.56 (m, 1H), 2.66 (m, 1H), 3.11 (m,
2H), 4.05 (q, 1H), 4.45 (m, 1H), 5.00 (m, 2H), 5.17 (m, 1H),
7.26 (m, 15H), 8.17 (d, 1H), 8.27 (d, 1H), 8.98 (d, 1H), 13.00 (s,
1H). Anal. (C33H37N3O7) C, H, N.
2(R)-(+)-Am in o-3-p h en ylp r op ion itr ile (11a ). N-tert-Boc-
D-phenylalanine (8a ) (8 g, 30.15 mmol) was dissolved in THF
(80 mL) and cooled to -10 °C. TEA (4.46 mL, 32 mmol,) and
ClCO2Et (3.4 g, 31 mmol) were added successively. The
reaction was stirred at -10 °C for 30 min before a stream of
anhydrous ammonia gas was introduced while the tempera-
ture was maintained below 0 °C. After 90 min the THF was
removed, water (50 mL) was added, and the mixture was
extracted with Et2O (3 × 80 mL). The combined Et2O extract
was washed with saturated NaHCO3 solution, 0.5 N HCl,
water, and brine, followed by drying (MgSO4), and the solvent
was removed to give 9a as a white solid in 80% yield: 1H NMR
(CDCl3) δ 1.44 (s, 9H), 3.11 (d, 2H, J ) 6.8 Hz), 4.39 (d, 1H, J
) 6.3 Hz), 5.07 (s, 1H), 5.50 (s, 1H), 5.84 (s, 1H), 7.21-7.33
(m, 5H). Compound 9a (5.86 g, 22.2 mmol) was dissolved in
THF (200 mL) and the solution was cooled on an ice bath
followed by the successive addition of TEA (6.27 mL, 45 mmol)
and (CF3CO)2O (3.2 mL, 22.2 mmol). The reaction mixture was
stirred for 30 min at room temperature and the THF was
removed. The residue was dissolved in EtOAc (200 mL) and
washed with saturated NaHCO3 solution, 0.5 N HCl solution,
water, brine, and dried (MgSO4). Evaporation of the solvent
3(S)-(N-ter t-Bu tyloxyca r bon yla m in o)-2(R,S)-h yd r oxy-
4-p h en ylbu tyr ic Acid (5). Compound 5 was synthesized as
described previously:24,28 yield 96%; mp 123.1-124.7 °C; 1H
NMR (CDCl3) δ 1.38 (s, 9H), 2.96 (s, 2H), 3.88-4.25 (m, 2H),
4.36 (s, 1H), 4.48 (s, 1H), 6.21 (s, 1H), 7.02-7.31 (m, 5H).
2(R)-[3(S)-(N-ter t-Bu tyloxyca r bon yla m in o)-2(R,S)-h y-
d r oxy-4-p h en ylbu tyr yl-a m in o]-3-p h en ylp r op ion ic Acid
Meth yl Ester (6a ). Compound 5 was coupled with D-phenyl-
alanine methyl ester hydrochloride to generate 6a in 87%
1
yield: mp 164-166 °C; H NMR (CDCl3) δ 1.40 (s, 9H), 3.08
(m, 4H), 3.70 (s, 3H), 3.98 (m, 1H), 4.12 (dd, 1H), 4.28 (br. s,
1H), 4.91 (m, 2H), 5.24 (br. s, 0.5H), (br. s, 0.5H), 7.28 (m, 10H).
2(S)-[3(S)-(N-ter t-Bu tyloxyca r bon yla m in o)-2(R,S)-h y-
d r oxy-4-p h en ylbu tyr yl-a m in o]-3-p h en ylp r op ion ic Acid
Meth yl Ester (6b). Compound 5 was coupled with L-phenyl-
alanine methyl ester hydrochloride to give 6b in 91% yield:
mp 168-170 °C; 1H NMR (CDCl3) δ 1.39 (s, 9H), 3.10 (m, 4H),
3.71 (s, 3H), 3.95 (m, 1H), 4.11 (dd, 1H), 4.30 (br. s, 1H), 4.97
(m, 2H), 5.37 (br. s, 0.5H), 5.51 (br. s, 0.5H), 7.29 (m, 10H).
2(R)-[3(S)-(N-Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph e-
n ylbu tyr yla m in o]-3-p h en ylp r op ion ic Acid Meth yl Ester
(7a ). Compound 6a was Boc-deprotected with a 4N HCl/
dioxane (1:1) mixture and coupled with N-Cbz-L-leucine to
1
gave 10a as a white solid in 92% yield: mp 195-210 °C; H
1
afford 7a in 73% yield; mp 168.3-170.2 °C; H NMR (CDCl3)
NMR (CDCl3) δ 1.4 (s, 9H), 3.07 (d, 2H, J ) 6.6 Hz), 4.30 (t,
1H, J ) 6.3 Hz), 5.80 (s, 1H), 7.29-7.39 (m, 5H). Compound
10a (5 g, 20.3 mmol) was dissolved in 50% TFA in CH2Cl2 (50
mL) and stirred at room temperature for 30 min, followed by
solvent removal. The residue was dried overnight in vacuo to
give a quantitative yield of 11a as the TFA salt: mp 203-210
°C (dec); [R]20D + 5.32° (c ) 0.5, MeOH); 1H NMR (D2O) δ 3.14
(dd, 1H, J 1 ) 7.95 Hz, J 2 ) 7.8 Hz), 3.36 (dd, 1H, J 1 ) 13.5
δ 0.86 (d, 6H), 1.47 (m, 5H), 3.04 (m, 3H), 3.68 (s, 2H), 3.73 (s,
1H), 4.18 (m, 3H), 4.81 (q, 1H), 5.03 (m, 3H), 5.53 (br. s, 1H),
6.82 (br. s, 1H), 7.30 (m, 15H).
2(S)-[3(S)-(N-Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph e-
n ylbu tyr yla m in o]- 3-p h en ylp r op ion ic Acid Meth yl Ester
(7b). Compound 6b was Boc-deprotected with a 4 N HCl/
dioxane (1:1) mixture and coupled with N-Cbz-L-leucine to

Novel R-Ketoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 77
Hz, J 2 ) 5.5 Hz), 4.82 (dd, 1H, J 1 ) 10.05 Hz, J 2 ) 9.9 Hz),
7.24-7.42 (m, 5H).
NaHCO3 solution, water, and brine, followed by drying over
MgSO4. The organic layer was then concentrated under
reduced pressure to give a brown solid, which was dissolved
in EtOH (60 mL) and purified by ion-exchange chromatogra-
phy with 2-propanol as the eluant. The solvent was removed
to give 3a as a brown solid in 60% yield: mp 195-197°C (dec)
(HCl salt);1H NMR (DMSO-d6) δ 0.88 (d, 6H, J ) 6.1 Hz),
1.20-1.33 (m, 3H), 2.69 (d, 2H, J ) 5.7 Hz), 3.19 (d, 2H, J )
6.9 Hz), 3.22 (s, 1H), 4.03 (m, 1H), 4.61 (m, 1H), 4.80 (m, 1H),
4.93 (s, 2H), 6.01 (s, 1H), 6.95-7.52 (m, 15H), 8.81-9.10 (s,
4H); MS (ESI) m/z 618.3 [M + Na]+, 586.3 [M + H]+. Anal.
(C33H39N5O5) C, H, N.
2(S)-[3(S)-(N-Cb z-L-leu cyla m in o)-4-p h en yl-2-oxo-b u -
tyr yla m in o]-1-a m id in o-3-p h en ylp r op a n e (3b). Compound
14b was converted to 3b in 62% yield as described for the
synthesis of 3a : mp 175-176°C (dec) (HCl salt); 1H NMR
(DMSO-d6) δ 0.89 (d, 6H, J ) 6.0 Hz), 1.18-1.32 (m, 3H), 2.73
(d, 2H, J ) 5.7 Hz), 3.18 (d, 2H, J ) 6.8 Hz), 3.28 (s, 1H), 3.95
(m, 1H), 4.55 (m, 1H), 4.82 (m, 1H), 4.90 (s, 2H), 5.97 (s, 1H),
6.96-7.61 (m, 15H), 8.8-9.4 (br, s, 4H); MS (ESI) m/ z m/z
618.4 [M + Na]+, 586.5 [M + H]+. Anal. (C33H39N5O5) C, H, N.
3(S)-(N-Cbz-L-leu cylam in o)-4-ph en yl-2-oxobu tyr ic Acid
P h en yleth yl Am id e (4a ). Compound 4a was synthesized as
described previously:24,28 1H NMR (CDCl3) δ 0.91 (d, 6H), 1.43
(m, 1H, J ) 8.3 Hz), 1.60 (m, 2H), 2.90 (m, 2H, J ) 6.7 Hz),
3.11 (dd, 0.5H, J ) 6.9 Hz), 3.14 (dd, 0.5H, J ) 6.9 Hz), 3.31
(dd, 0.5H, J ) 5.3 Hz), 3.35 (dd, 0.5H, J ) 5.4 Hz), 3.63 (m,
2H), 4.17 (m, 1H), 5.05 (d, 1H, J ) 7.3 Hz), 5.11 (s, 2H), 5.57
(q, 1H, J ) 6.8 Hz), 6.60 (d, 1H, J ) 6.4 Hz), 6.95 (d, 1H),
7.05-7.37 (m 15H). Anal. (C32H37N3O5) C, H, N.
2(S)-(-)-Am in o-3-p h en ylp r op ion itr ile (11b). N-tert-Boc-
L-phenylalanine was transformed to 11b as described for the
synthesis of 11a : mp 208 °C (dec); [R]20 - 5.38° (c ) 0.5,
D
MeOH); 1H NMR (D2O) δ 3.05 (dd, 1H, J 1 ) 13.2 Hz, J 2
)
10.5 Hz), 3.42 (dd, 1H, J 1 ) 13.2 Hz, J 2 ) 10.5 Hz), 4.86 (dd,
1H, J 1 ) 10.5 Hz, J 2 ) 10.2 Hz), 7.17-7.53 (m, 5H).
2(R)-[3(S)-(N-ter t-Bu tyloxyca r bon yla m in o)-2(R,S)-h y-
dr oxy-4-ph en ylbu tyr ylam in o]-3-ph en ylpr opion itr ile (12a).
Compound 5 was coupled with 11a to give 12a in 72% yield:
1H NMR (CDCl3) δ 1.42 (s, 9H), 3.08 (d, 2H, J ) 6.0 Hz), 3.13
(d, 2H, J ) 6.8 Hz), 4.05 (m, 1H), 4.10 (m, 1H), 4.26 (d, 1H, J
) 3.9 Hz), 4.93 (s,1H), 5.10 (m, 1H), 5.93 (m, 1H), 7.15-7.39
(m, 10H); MS (ESI) m/z 446.1 [M + Na]+, 422.0 [M - H]-.
2(S)-[3(S)-(N-ter t-Bu tyloxyca r bon yla m in o)-2(R,S)-h y-
dr oxy-4-phenylbutyr yl-am ino]-3-ph enylpr opionitr ile (12b).
Compound 5 was coupled with 11b to afford 12b in 90%
yield: 1H NMR (CDCl3) δ 1.40 (s, 9H), 2.99 (d, 2H, J ) 6.1
Hz), 3.09 (d, 2H, J ) 6.8 Hz), 3.99 (m, 1H), 4.13 (d, 1H, J )
2.4 Hz), 4.26 (s, 1H), 4.90 (s,1H), 5.13 (m, 1H), 5.99 (m, 1H),
7.13-7.59 (m, 10H); MS (ESI) m/z 446.0 [M + Na]+, 422.1 [M
- H]-.
2(R)-[3(S)-(N-Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph e-
n ylbu tyr yla m in o]-3-p h en ylp r op ion itr ile (13a ). Compound
12a was Boc-deprotected and coupled with Cbz-L-leucine to
obtain 13a in 84% yield: 1H NMR (CDCl3) δ 0.92 (d, 6H, J )
6.0 Hz), 1.28 (s, 1H), 1.40 (m, 2H), 2.72 (d, 2H, J ) 7.2 Hz),
2.98 (d, 2H, J ) 6.6 Hz), 3.77 (s, 1H), 4.05 (m, 1H), 4.13 (s,
1H), 4.71 (m, 1H), 5.12 (s, 1H), 5.30 (s, 2H), 5.52 (s, 1H), 6.95-
7.81 (m, 15H); MS (ESI) m/z 593.3 [M + Na]+, 569.1 [M - H]-.
2(S)-[3(S)-(N-Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph e-
n ylbu tyr yla m in o]-3-p h en ylp r op ion itr ile (13b). Compound
12b was Boc-deprotected and coupled with Cbz-L-leucine to
afford 13b in 88% yield: 1H NMR (CDCl3) δ 0.92 (d, 6H, J )
6.0 Hz), 1.28 (s, 1H), 1.42 (m, 2H), 2.87 (d, 2H, J ) 7.8 Hz),
3.08 (d, 2H, J ) 6.6 Hz), 3.62 (s, 1H), 4.10 (m, 1H), 4.13 (s,
1H), 4.72 (m, 1H), 5.04 (s, 1H), 5.25 (s, 2H), 5.66 (s, 1H), 7.10-
7.79 (m, 15H); MS (ESI) m/z 593.2 [M + Na]+, 569.2 [M - H]-.
2(R)-[3(S)-(N-Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph e-
n ylbu tyr ylam in o]-1-am idin o-3-ph en ylpr opan e (14a). Com-
pound 13a (1 g, 1.75 mmol) and N-acetylcysteine (287 mg, 1.75
mmol) were dissolved in MeOH (2 mL), and NH4OAc (154 mg,
0.2 mmol) was added. The reaction vessel was tightly sealed
and heated at 50 °C under N2 for 16 h, after which the solvent
was removed. The residue was dissolved in CH2Cl2 (150 mL)
and washed with water. The crude solid was dissolved in
i-PrOH and passed through SBG1-OH ion-exchange resin
(ResinTech, Inc.) at a flow rate of 1 cm/s. Evaporation of
solvent gave 14a as a brown solid in 92% yield: 1H NMR
(CDCl3) δ 0.95 (d, 6H, J ) 6.0 Hz), 1.25-1.36 (m, 3H), 2.96 (d,
2H, J ) 5.5 Hz), 3.10 (d, 2H, J ) 6.6 Hz), 3.70 (m, 1H), 4.05
(d, 1H, J ) 5.5 Hz), 4.38 (br, 1H), 4.71 (m, 1H), 5.15 (s, 2H),
6.98 (s, 1H), 7.08-7.69 (m, 15H). MS (ESI) m/z 610.4 [M +
Na]+, 588.2 [M + H]+.
3(S)-Am in o-2(R,S)-h yd r oxy-4-p h en ylbu tyr ic Acid (15).
Compound 5 (2.75 g, 14 mmol) was dissolved in 2 N HCl (15
mL) and MeOH (30 mL). The mixture was stirred at room
temperature for 2 h. The solvent was evaporated to give 15 as
a white solid, which was used in the next reaction without
1
further purification. H NMR (D2O) δ 2.92 (dd, 1H, J 1 ) 6.9
Hz, J 2 ) 13.95 Hz), 3.13 (dd, J 1 ) 6.9 Hz, J 2 ) 13.95 Hz), 3.75
(m, 1H), 4.02 (d, 1H, J ) 3.6 Hz), 7.21-7.40 (m, 3H), 7.40-
7.55 (m, 2H); 13C NMR (D2O) δ 33.96, 36.15, 56.19, 56.55,
128.43, 130.01, 130.27, 136.34, 177.23, 177.75.
3(S)-Am in o-2(R,S)-h ydr oxy-4-ph en ylbu tyr ic Acid Meth -
yl Ester (16). SOCl2 (3.7 g, 30.7 mmol) was slowly added
(under an atmosphere of nitrogen) to an ice cooled solution of
MeOH (40 mL) and stirred for 5 min followed by the addition
of 15 (1.5 g, 7.7 mmol). The mixture was stirred at room
temperature overnight and the solvent was evaporated in
vacuo to give a brown solid in 93% yield: 1H NMR (D2O) δ
2.93 (dd, 1H, J 1 ) 7.5 Hz, J 2 ) 14.75 Hz), 3.01 (dd, 1H, J 1
)
7.5 Hz, J 2 ) 14.75 Hz), 3.65 (s, 3H), 3.86 (br s, 1H), 4.29 (d,
1H, J ) 2.5 Hz), 7.20-7.29 (m, 3H), 7.30-7.42 (m, 2H).
3(S)-(N-Cbz-L-leu cyla m in o)-2(R,S)-h yd r oxy-4-p h en yl-
bu ta n oic Acid Meth yl Ester (17). Coupling of 16 with Cbz-
L-leucine afforded 17 as a yellow solid in 92% yield after
purification by flash chromatography [silica gel, hexane:EtOAc
1
(2:1)]: H NMR (CDCl3) δ 0.90 (d, 6H, J ) 6.5 Hz), 1.38 (m,
1H), 1.55 (m, 2H), 2.90 (d, 2H, J ) 7.5 Hz), 3.20 (s, 1H), 3.69
(s, 3H), 4.09 (m, 2H), 4.54 (dd, 1H, J 1 ) 9.5 Hz, J 2 ) 16.75
Hz), 4.99 (m, 1H), 5.11 (s, 2H), 6.22 (d, 1H, J ) 8.5 Hz), 7.18-
7.47 (m, 10H); MS (ESI) m/ z 479.3 [M + Na]+.
2(S)-[3(S)-(N-Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph e-
n ylbu tyr ylam in o]-1-am idin o-3-ph en ylpr opan e (14b). Com-
pound 13b was transformed into 14b as described for 14a in
98% yield: 1H NMR (CDCl3) δ 0.92 (d, 6H, J ) 6.0 Hz), 1.23-
1.29 (m, 3H), 3.02 (d, 2H, J ) 5.6 Hz), 3.06 (d, 2H, J ) 6.5
Hz), 3.69 (m, 1H), 4.04 (d, 1H, J ) 5.6 Hz), 4.28 (br, 1H), 4.69
(m, 1H), 5.17 (s, 2H), 6.95 (s, 1H), 7.20-7.65 (m, 15H); MS
(ESI) m/z 610.2 [M + Na]+, 588.1 [M + H]+.
2(R)-[3(S)-(N-Cbz-L-leu cyla m in o)-4-p h en yl-2-oxobu tyr -
yla m in o]-1-a m id in o-3-p h en ylp r op a n e (3a ). Compound 14a
(100 mg, 0.17 mmol) was dissolved in a 1:1 mixture of DMSO
and toluene (4 mL) and cooled on an ice bath, and pyridinium
trifluoroacetate (5 equiv) was added followed by the addition
of EDC (0.326 mg, 1.7 mmol). The mixture was stirred at 0 °C
for 10 min and the ice bath was removed. Stirring was
continued at room temperature until TLC [silica gel, EtOAc:
MeOH:NH4OH (6:3:1)] showed no starting material (ap-
proximately 2 h later). EtOAc and water were added, and the
organic phase was recovered and washed with saturated
3(S)-(N-Cbz-L-leu cyla m in o)-2(R,S)-h yd r oxy-4-p h en yl-
bu ta n oic Acid Hyd r a zid e (18). Compound 17 (2.1 g, 4.6
mmol) was dissolved in MeOH (15 mL), and hydrazine
monohydrate (0.576 g, 11.5 mmol) was added. The mixture
was stirred at room temperature for 36 h and the solvent was
evaporated in vacuo to give 18 as a white solid in 86% yield:
1H NMR (CDCl3) δ 0.85 (d, 6H, J ) 6.0 Hz), 1.36-1.55 (m,
3H), 3.05 (d, 1H, J ) 7.5 Hz), 3.10 (d, 1H, J ) 7.5 Hz), 3.71 (s,
1H), 4.10 (m, 1H), 4.21 (m, 1H), 4.98 (m, 1H), 5.10 (s, 2H),
6.49 (d, 1H, J ) 7 Hz), 7.10-7.47 (m, 10H), 7.93 (s, 2H); MS
(ESI) m/ z 479.3 [M + Na]+, 457 [M + H]+.
N′-Ben zyl-3(S)-(N-Cbz-L-leu cyla m in o)-2(R,S)-h yd r oxy-
4-p h en ylbu ta n oic Acid Hyd r a zon e (19). Compound 17 (408
mg, 0.83 mmol) and TEA (0.22 mL, 1.6 mmol) were dissolved
in absolute EtOH (10 mL), and benzaldehyde (106 mg, 1 mmol)

78 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Donkor et al.
was added. The mixture was stirred for 5 h at room temper-
ature and left to stand overnight. The precipitated solid was
recovered by filtration and the filtrate was concentrated to
afford more product. The solid was crystallized twice from 95%
EtOH and once from Et2O to give 19 as a white solid in 72%
yield: 1H NMR (CDCl3) δ 0.83 (d, 6H, J ) 6.0 Hz), 1.28-1.43
(m, 3H), 3.13 (d, 2H, J ) 7.2 Hz), 4.39 (s, 1H), 4.6 (s, 1H),
4.80-5.10 (m, 3H), 5.78 (d, 1H, J ) 4.8 Hz), 6.46 (d, 1H, J )
6 Hz), 7.05-7.77 (m, 15H), 8.12 (s, 1H), 9.86 (s, 1H); MS (ESI)
m/ z 567.3 [M + Na]+, 545.3 [M + H]+, 543.0 [M - H]-.
N′-Ben zyl-3(S)-(N-Cbz-L-leu cyla m in o)-2(R,S)-h yd r oxy-
4-p h en ylbu ta n oic Acid Hyd r a zid e (20). To a solution of 19
(330 mg, 0.6 mmol) in THF (15 mL) was added NaBH3CN (114
mg, 1.8 mmol) followed by 3 drops of HCl. The mixture was
heated to 60 °C and stirred overnight until TLC [silica gel,
hexane/EtOAc (2:3)] showed no starting material. The solvent
was evaporated and the residue was dissolved in EtOAc (120
mL) and washed successively with NaHCO3 solution, water,
and brine. It was dried (MgSO4) and concentrated to give 20
as a white solid in 88% yield:1H NMR (CDCl3) δ 0.88 (d, 6H,
J ) 5.7 Hz), 1.25-1.65 (m, 3H), 2.79 (m, 2H), 3.09 (d, 1H, J )
6.3 Hz), 3.72 (m, 2H), 3.91 (d, 1H, J ) 6.3 Hz), 4.09 (m, 2H),
5.02 (s, 1H), 7.00-7.65 (m, 15H); MS (ESI) m/ z 569.3 [M +
Na]+.
N′N′-(Ben zyl)(t er t -b u t yloxyca r b on yla m in o)-3(S)-(N-
Cbz-L-leu cylam in o)-2(R,S)-h ydr oxy-4-ph en ylbu tan oic Acid
Hyd r a zid e (21). Di-tert-butyl dicarbonate (158 mg, 0.72
mmol) was slowly added to a solution of 20 (360 mg, 0.66
mmol) in t-BuOH (10 mL) and 1 N NaOH (2 mL), and the
mixture was stirred at room temperature for 12 h. Following
this period the mixture was diluted with H2O (25 mL) and
extracted with pentane (3 × 50 mL). The aqueous phase was
cooled on an ice bath, the pH was adjusted to 3 with 0.5 N
HCl, and the phase was extracted with EtOAc. The combined
organic extracts were washed successively with water and
brine, dried over MgSO4, and concentrated in vacuo to give
21 as a white solid in 94% yield: 1H NMR (CDCl3) δ 0.88 (dd,
6H, J 1 ) 8 Hz, J 2 ) 8 Hz), 1.40 (s, 9H), 1.45-1.54 (m, 3H),
3.16 (m, 1H), 3.35 (m, 1H), 3.97 (m, 1H), 4.18 (dd, 1H, J 1 ) 3
Hz, J 2 ) 8 Hz), 4.29 (br s, 2H), 4.91 (m, 1H), 4.96 (d, 1H, J )
12 Hz), 5.09 (d, 1H, J ) 12 Hz), 5.99 (d, 1H, J ) 8 Hz), 6.27 (s,
1H), 7.05-7.47 (m, 15H), 8.44 (s, 1H); MS (ESI) m/ z 669.6 [M
+ Na]+.
N′,N′-(Ben zyl)(t er t -b u t yloxyca r b on yla m in o)-3(S)-(N-
Cbz-L-leu cyla m in o)-4-p h en yl-2-oxo-bu ta n oic Acid Hy-
d r a zid e (22). Dess-Martin reagent oxidation of 21 followed
by flash chromatographic purification [silica gel, hexane/EtOAc
(7:3)] of the crude product afforded 22 as a white solid in 93%
yield: 1H NMR (CDCl3) δ 0.89 (d, 6H, J ) 6.0 Hz), 1.24 (m,
1H), 1.42 (m, 2H), 1.47 (s, 9H), 3.08 (m, 1H), 3.30 (dd, 1H, J 1
) 5 Hz, J 2 ) 14.25 Hz), 4.10 (br s,1H), 4.69 (s, 2H), 5.08 (s,
2H), 6.51 (s, 1H), 7.01 (s, 2H), 7.17-7.45 (m, 13H), 8.20 (s,
1H); MS (ESI) m/ z 667.8 [M + Na]+; 643.0 [M - H]-.
of CaCl2, and the increase in fluorescence (λex ) 370 nm, λem
) 440 nm) was monitored at amibient temperature using a
SPECTRAmax Gemini fluorescence plate reader (Molecular
Devices). The Ki values were estimated from the semireciprocal
plot of the initial velocity versus the concentration of the
inhibitor according to the method of Dixon.30 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 triplicate determinations.
Molecu la r Mod elin g Stu d ies. The 2.1 Å resolution X-ray
crystal structure of the calcium-bound protease core of calpain
I (PDB accession number 1KXR)22 was retrieved from the
Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The
Ser115 residue was mutated to Cys115 to mimic the wild-type
enzyme. The structures of the inhibitors were constructed
using Sybyl 6.831 and energetically minimized using Tripos
force field with Gasteiger-Hu¨ckel charges.32 The Gold 1.2
program34 was applied for flexibly docking the inhibitors into
the active site of the enzyme. Cys115 and all residue within
10 Å of it defined the active site pocket for the docking studies.
The complexes of the inhibitors with calpain I obtained from
the molecular docking were further minimized using the Tripos
force field in Sybyl 6.8 and the Powell method for 5000 steps
or until the gradient was lower than 0.01 kcal mol-1 Å -1. All
simulations were performed with the molecular dynamics
module (Sybyl 6.8) in a vacuum with a dielectric constant ꢀ )
1 using the Tripos force field. The inhibitors were allowed to
be fully flexible, while in the enzyme, only residues within 10
Å radius of the inhibitor were made fully flexible, the other
residues being kept fixed.
Ack n ow led gm en t. The study was supported in part
by grant 1 R 15 HL68675-01 from the U.S. Public
Health Service and grant 0255066B from the South-
eastern Affiliate of the American Heart Association. We
thank Dr. Shantaram Kamath for his helpful sugges-
tions with the molecular modeling studies.
Refer en ces
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N′-Ben zyl-3(S)-(N-Cbz-L-leu cyla m in o)-4-p h en yl-2-oxo-
bu ta n oic Acid Hyd r a zid e (4b). Compound 22 (150 mg, 3.98
mmol) was dissolved in a mixture of 2 N HCl (2 mL) and
dioxane (8 mL) and stirred at room temperature for 1 h. The
solvent was evaporated in vacuo to give 4b as light brown solid
in 90% yield: 1H NMR (CDCl3) δ 0.89 (d, 6H, J ) 6.0 Hz),
1.27-1.45 (m, 3H), 3.03 (m, 2H), 4.06 (s, 1H), 4.25 (m, 2H),
4.86 (s, 1H), 5.06 (d, 1H, J ) 12 Hz), 5.10 (d, 1H, J ) 12 Hz),
6.44 (s, 1H), 6.70 (s, 1H), 7.05-7.60 (m, 15H), 8.21 (s, 1H); 13
C
NMR (CDCl3) δ 20.5, 21.7, 34.6, 43.1, 55.2, 56.3, 56.8, 70.3,
126.1, 126.6, 127.5, 127.8, 127.9, 128.0, 128.3, 128.4, 128.7,
139.2, 141.0, 155.7, 160.8, 178.9, 196.7; MS (ESI) m/ z 567.4
[M + Na]+. Anal. (C31H36N4O5), C, H, N.
Ca lp a in I In h ibition Assa y. Calpain activity was moni-
tored 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 (Calbiochem), 1
mg porcine erythrocyte calpain I (Calbiochem), varying con-
centrations of inhibitor dissolved in DMSO (2% total concen-
tration), and 5 mM CaCl2 in a final volume of 250 mL in a
polystyrene microtiter plate. Assays were initiated by addition

Novel R-Ketoamides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 79
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