Structural basis for the potent calpain inhibitory activity of peptidyl α-ketoacids

Article abstract of DOI:10.1021/jm800182c

A series of peptidyl α-ketoacids and α-ketoesters was synthesized and studied as μ-calpain inhibitors. Docking studies revealed that the μ-calpain inhibitory activity of the compounds is influenced by hydrogen bonding interactions and the potential for io

Full text of DOI:10.1021/jm800182c

4346
J. Med. Chem. 2008, 51, 4346–4350
Brief Articles
Structural Basis for the Potent Calpain Inhibitory Activity of Peptidyl r-Ketoacids
Isaac O. Donkor,* Haregewein Assefa,† and Jiuyu Liu
Department of Pharmaceutical Sciences, UniVersity of Tennessee Health Science Center, Memphis, Tennessee 38163
ReceiVed February 20, 2008
A series of peptidyl R-ketoacids and R-ketoesters was synthesized and studied as µ-calpain inhibitors. Docking
studies revealed that the µ-calpain inhibitory activity of the compounds is influenced by hydrogen bonding
interactions and the potential for ionic interaction with active site residues as well as placement of a planar
N-terminal capping group into the S3 pocket of the enzyme.
Table 1. Structure, Diastereomeric Ratio, and µ-Calpaina Inhibitory
Activity of Compounds 1a-e and 2a-e
Introduction
Calpains are a family of intracellular calcium-dependent
cysteine proteases with widespread expression in a variety of
cells and tissues.1 µ-Calpain (calpain I) and m-calpain (calpain
II) are the major calpain isoforms, which are ubiquitously
distributed in mammalian cells. These calpain isoforms catalyze
the partial hydrolysis of a wide variety of target proteins
including cytoskeletal proteins, receptors, and integral membrane
proteins.1–3 The enzymes have been implicated in several
physiological processes and a variety of degenerative diseases.1–4
The implication of calpain in human disease has aroused interest
in the search for calpain inhibitors as tools for studying the
enzyme and as potential therapeutic agents.5,6
The broad substrate specificity of the major calpains suggests
that the enzymes tolerate a wide variety of side chain residues
within their active site pockets.7 Recent crystallographic studies
have revealed the structure of calpain with and without bound
inhibitors and have made it possible to rationalize the structural
basis for ligand induced inhibition of the enzyme.8,9 We report
here the synthesis and structural basis for the potent calpain
inhibitory activity of a series of peptidyl R-ketoacids that
incorporate lipophilic residues as the P2 N-capping substituent
(Table 1).
Results and Discussion
Chemistry. Compounds 1a-e and 2a-e were synthesized
(Scheme 1) by employing general coupling procedures for the
solution phase synthesis of peptides. Thus, coupling the ap-
propriate carboxylic acid 4a-e with L-leucine methyl ester
hydrochloride (5) using EDC/HOBT as the coupling agent and
DMF/NMM mixture as solvent afforded pseudodipeptides 6a-e,
which were hydrolyzed with 1N NaOH in MeOH and coupled
with ꢀ-amino-R-hydroxy ester 7 to give 8a-e. Compound 7
was synthesized as previously reported.10 Dess-Martin oxida-
tion of 8a-e gave R-ketoesters 1a-e. 1H NMR analysis of the
crude products showed 1a-e to be diastereomerically pure.
a Porcine erythrocyte µ-calpain (Calbiochem). b Ki values were deter-
mined by Dixon plots using the average of triplicate assays and plotting
1/ν vs I (inhibitor concentration) to give intersecting lines with correlation
coefficient 0.95. c DR ) Diastereomeric ratio of the compounds as
determined by 1H NMR spectrometry in CDCl3. d MDL28170 (Calbiochem).
However, column chromatographic purification (silica gel) as
well as basic hydrolysis of the ester functionality led to
epimerization of the chiral center at P1 to yield 2a-e as pairs
of diastereomers. The diastereomeric ratios of the compounds
* To whom correspondence should be addressed. Phone: (901) 448-7736.
Fax: (901) 448-6828. E-mail: idonkor@utmem.edu.
as determined by 1H NMR spectrometry are shown in Table 1.
Racemization of the compounds is consistent with previous
†
Currently a Research Scientist at Paratek Pharmaceuticals, Boston, MA.
10.1021/jm800182c CCC: $40.75
2008 American Chemical Society
Published on Web 07/17/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4347
Scheme 1a
a Reagents: (a) EDC, HOBT, NMM, DMF; (b) 1N NaOH/CH3OH; (c) 7, EDC, HOBT, NMM, DMF; (d) Dess-Martin Reagent/CH2Cl2.
Figure 2. R-Ketoester 1e (the S,S-diastereomer) docked into the active
Figure 1. R-Ketoacid 2e (the S,S-diastereomer) docked into the active
site of µ-calpain. Hydrogen atoms are removed for clarity except those
involved in hydrogen bonding. The inhibitor is shown as ball and stick.
Hydrogen bonds are shown as black dashed lines; all protein residues
are colored orange except those involved in the hydrogen bonding.
site of µ-calpain. Hydrogen atoms are removed for clarity except those
involved in hydrogen bonding. The inhibitor is shown as ball and stick.
Hydrogen bonds are shown as black dashed lines; all protein residues
are colored orange except those involved in the hydrogen bonding.
hydrogen and H272.9 Compounds 1e and 2e also made hydrogen
bonding interactions with Q109, G208, G271, and H272 at the active
site of µ-calpain. Hydrogen bonding between the NH of the
P1-P2 amide bond of calpain inhibitors, and G271 has been
shown to be critical for inhibition of the enzyme. The absence
of such an interaction was shown to decrease calpain inhibition
by 250-fold.13 Compound 2e formed 12 hydrogen bonds with
active site residues, which included a network of eight hydrogen
bonds that were facilitated by two water molecules (Figure 1).
The carboxylate group of 2e was juxtapositioned to the
imidazole ring of H272 and formed a hydrogen bond with the
NH group of the imidazole ring, suggesting the possibility for
ionic interaction between the carboxylate of 2e and H272. On
the contrary, R-ketoester 1e formed nine hydrogen bonds with
active site residues (compared to 12 for R-ketoacid 2e) (Figure
2). Esterification of the carboxylate group eliminated the
possibility for ionic interaction between 1e and the enzyme. This
is supported by the absence of the hydrogen bond that was
observed between 2e and H272 and may account in part for the
150-fold difference in the µ-calpain inhibitory activity between
1e and 2e, the 30-fold difference in activity between 1d and
2d, the 10-fold difference in activity between 1c and 2c or 1b
and 2b, and the 20-fold difference between 1a and 2a.
reports, which indicate that R-keto carbonyl compounds are
prone to racemization in the presence of base.11
Calpain Inhibition and Docking Studies. The µ-calpain
inhibitory potency (Ki values) of 1a-e and 2a-e (as diaster-
eomeric mixtures) were determined employing a fluorometric
assay with Suc-Leu-Tyr-AMC as substrate and the peptidyl
aldehyde calpain inhibitor, MDL28170, as positive control. The
inhibition data (Table 1) showed that the R-ketoacids are better
inhibitors of µ-calpain compared to the corresponding R-ke-
toesters, suggesting that the carboxylic acid moiety is very
important for potent inhibition of the enzyme. For example,
R-ketoacid 2e (Ki ) 8 nM), which was the most potent member
of the series, was 150-fold more potent than its methyl ester
derivative 1e. This is consistent with the results of Li et al.,12
who reported that the calpain inhibitory activity of peptidyl
calpain inhibitors with R-keto carbonyl electrophilic centers
follows the order R-ketoacids > R-ketoamides > R-ketoesters.
To determine the structural basis for the potent calpain inhibitory
activity of peptidyl R-ketoacids compared to peptidyl R-ke-
toesters, we docked the S,S-diastereomers of 1e and 2e into the
active site of µ-calpain. This study revealed that water molecules
play an important role in the binding of the inhibitors to the
enzyme by serving as bridges to facilitate formation of a network
of hydrogen bonds between the inhibitors and active site residues
(Figures 1 and 2). This is consistent with the reported cocrystal
structure of mini-calpain in complex with an R-ketoamide
inhibitor, which showed a water bridge between the ketoamide
It has been reported that peptidyl aldehydes with hydrophobic
N-terminal capping groups are good inhibitors of calpain.4,5 To
further explore the structural requirements of the N-terminal
capping group that is important for potent inhibition of µ-calpain,
we investigated the effect of the molecular shape and molecular

4348 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Donkor et al.
added and stirred for 10 min. The mixture was extracted with
CH2Cl2, and the organic extract was washed with NaHCO3 solution,
water, and dried (MgSO4). The solvent was removed, and the crude
product was purified by column chromatography.
General Procedure for the Synthesis of r-Ketoacids 2a-e.
A solution of the appropriate ester 1a-e in MeOH was stirred with
1N NaOH solution until TLC showed completion of reaction (1-2
h). The MeOH was removed under vacuum, water was added, and
the solution was washed with CH2Cl2. The aqueous layer was
recovered and acidified with 2N HCl and extracted with EtOAc.
The combined EtOAc extracts were washed with water and dried
(MgSO4), followed by evaporation of the solvent to give the desired
acid.
N-(3-Cyclohexylpropionyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phe-
nylbutanoic Acid Methyl Ester (1a). Column chromatographic
purification of the crude product with EtOAc/hexanes (4:6) as
solvent gave a diastereomeric mixture of 1a as white solid in 80.3%
yield. 1H NMR (CDCl3, 300 MHz) Diastereomer 1: δ 7.22 (m,
5H), 6.73 (d, J ) 6.6 Hz, 0.60H), 5.75 (m, 1H), 5.33 (m, 1H), 4.42
(m, 1H), 3.84 (s, 1.8H), 3.23 (m, 1H), 3.00 (m, 1H), 2.16 (m, 2H),
1.49 (m, 10H), 1.19 (m, 4H), 0.89 (m, 8H). Diastereomer 2: δ 7.22
(m, 5H), 6.80 (d, J ) 6.9 Hz, 0.40H), 5.75 (m, 1H), 5.33 (m, 1H),
4.42 (m, 1H), 3.85 (s, 1.20H), 3.23 (m, 1H), 3.00 (m, 1H), 2.16
(m, 2H), 1.49 (m, 10H), 1.19 (m, 4H), 0.89 (m, 8H). ESI MS: m/z
513.6 (M + Na + CH3OH)+. Anal. (C26H38N2O5) C, H, N.
N-(1-Adamantylacetyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phenylbu-
tanoic Acid Methyl Ester (1b). The crude product was purified by
column chromatography with EtOAc/hexanes (2:3) as solvent to
give 0.71 g (94.6%) of 1b as a mixture of diastereomers. 1H NMR
(CDCl3, 300 MHz) Diastereomer 1: δ 7.23 (m, 5H), 6.77 (d, J )
6.9 Hz, 0.54H), 5.68 (d, J ) 8.1 Hz, 1H), 5.35 (m, 1H), 4.43 (m,
1H), 3.84 (s, 1.62H), 3.23 (m, 1H), 3.02 (m, 1H), 1.92 (m, 5H),
1.59 (m, 15H), 0.90 (m, 6H). Diastereomer 2: δ 7.23 (m, 5H), 6.88
(d, J ) 6.9 Hz, 0.46H), 5.68 (d, J ) 8.1 Hz, 1H), 5.35 (m, 1H),
4.43 (m, 1H), 3.85 (s, 1.38H), 3.23 (m, 1H), 3.02 (m, 1H), 1.92
(m, 5H), 1.59 (m, 15H), 0.90 (m, 6H). ESI MS: m/z 551.3 (M +
Na + CH3OH)+. Anal. (C29H40N2O5) C, H, N.
N-(2,2-Diphenylacetyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phenylbu-
tanoic Acid Methyl Ester (1c). Column chromatographic purification
of the crude product with EtOAc/hexanes (2:3) as solvent gave 1c
as white solid in 88% yield. 1H NMR (CDCl3, 300 MHz)
Diastereomer 1: δ 7.25 (m, 13H), 7.08 (m, 2H), 6.78 (d, J ) 6.6
Hz, 0.60H), 5.90 (m, 1H), 5.29 (m, 1H), 4.88 (s, 0.60H), 4.47 (m,
1H), 3.79 (s, 1.80H), 3.14 (m, 1H), 2.90 (m, 1H), 1.36 (m, 3H),
0.80 (m, 6H). Diastereomer 2: δ 7.25 (m, 13H), 7.08 (m, 2H), 6.92
(d, J ) 6.9 Hz, 0.40H), 5.90 (m, 1H), 5.29 (m, 1H), 4.94 (s, 0.40H),
4.47 (m, 1H), 3.87 (s, 1.20H), 3.14 (m, 1H), 2.90 (m, 1H), 1.36
(m, 3H), 0.80 (m, 6H). ESI MS: m/z 569.2 (M + Na + CH3OH)+.
Anal. (C31H34N2O5) C, H, N.
N-(3,3-Diphenylpropionyl)-L-leucyl-2-oxo-3(S)-amino-4-phenyl-
butanoic Acid Methyl Ester (1d). Compound 1d was obtained as
white solid in 89% yield after column purification with EtOAc/
hexanes (2:3) as solvent. 1H NMR (CDCl3, 300 MHz): δ 7.51 (m,
15H), 6.57 (d, J ) 6.9 Hz, 1H), 5.60 (d, J ) 8.1 Hz, 1H), 5.25 (m,
1H), 4.50 (m, 1H), 4.27 (m, 1H), 3.82 (s, 3H), 3.14 (m, 1H), 2.89
(m, 3H), 1.38 (m, 1H), 1.15 (m, 2H), 0.72 (m, 6H). ESI MS: m/z
551.3 (M + Na + CH3OH)+. Anal. (C32H36N2O5) C, H, N.
N-(9-Fluoreneacetyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phenylbu-
tanoic Acid Methyl Ester (1e). 1H NMR (CDCl3, 300 MHz)
Diastereomer 1: δ 7.73 (m, 2H), 7.31 (m, 11H), 6.74 (d, J ) 7.2
Hz, 0.55H), 5.62 (d, J ) 8.1 Hz, 0.55H), 5.29 (m, 1H), 4.41 (m,
2H), 3.84 (s, 1.65H), 3.22 (m, 1H), 2.99 (m, 1H), 2.77 (m, 1H),
2.59 (m, 1H), 1.34 (m, 3H), 0.81 (m, 6H). Diastereomer 2: δ 7.73
(m, 2H), 7.31 (m, 11H), 6.66 (d, J ) 6.9 Hz, 0.45H), 5.55 (d, J )
8.1 Hz, 0.45H), 5.29 (m, 1H), 4.41 (m, 2H), 3.82 (s, 1.35H), 3.22
(m, 1H), 2.99 (m, 1H), 2.77 (m, 1H), 2.59 (m, 1H), 1.34 (m, 3H),
0.81 (m, 6H). ESI MS: m/z 551.3 (M + Na + CH3OH)+. Anal.
(C32H34N2O5) C, H, N.
Figure 3. Compounds 2c (yellow) and 2d (purple) docked into the
active site of µ-calpain. Notice that the diphenyl group of 2c unlike
that of 2d does not effectively access the S3 pocket. The S,S-
diastereomers were studied.
size of the N-terminal group on µ-calpain inhibition. Our data
showed that large planar aromatic groups are preferred over
nonplanar saturated substituents (2b vs 2e). Compound 2e with
the planar fluorene moiety was over 5-fold more potent than
2a and 2b with cyclohexyl and adamantyl groups, respectively.
The docking studies also revealed that the N-terminal capping
group occupied a large pocket at the S3 subsite of the enzyme
(Figure 3). It was also noticed that the extent to which the
N-terminal capping substituent occupied this pocket (as deter-
mined by the linker length) influenced µ-calpain inhibition.
Thus, 2d with a diphenylpropionyl group was about twice as
potent as 2c with a diphenylacetyl group as the N-terminal
capping substituent (Figure 2).
In summary, our data suggest that the structural basis for the
potent calpain inhibitory activity of peptidyl R-ketoacids is
derived from a network of hydrogen bonding interactions and
the possibility for ionic interaction between the inhibitor and
active site residues of the enzyme. Esterification of the car-
boxylic acid group significantly reduced calpain inhibition due
in part to diminished potential for hydrogen bonding and ionic
interaction interactions with active site residues. Additionally,
large planar aromatic groups are preferred over nonplanar
saturated groups as the N-terminal capping substituent. Fur-
thermore, efficient positioning of the N-terminal capping group
into a large pocket at the S3 subsite of the enzyme potentiates
µ-calpain inhibition.
Experimental Section
General Procedure for the Synthesis of Pseudodipeptides
6a-e and r-Hydroxy Esters 8a-e. A solution of the appropriate
carboxylic acid (1 equiv) in DMF was cooled with an ice-bath and
EDC (1.15 equiv), HOBt (1.15 equiv), NMM (2.3 equiv), and the
appropriate amine hydrochloride (1 equiv) were added consecu-
tively. The reaction mixture was stirred overnight at RT. DMF was
removed in vacuo, water was added to the residue, and the mixture
was extracted with Et2O or EtOAc. The combined extracts were
washed successively with saturated NaHCO3 solution, 0.5N HCl,
and H2O, followed by drying (MgSO4). The solvent was removed,
and the crude product was purified by column chromatography.
General Procedure for the Synthesis of r-Ketoesters 1a-e.
Dess-Martin reagent (2-4 equiv) was added to a cooled (ice-bath)
solution of the appropriate R-hydroxy ester 8a-e in CH2Cl2 and
the mixture was stirred at RT for 5-6 h. Sodium thiosulfate
pentahydrate (14-28 equiv) in saturated NaHCO3 solution was
N-(3-Cyclohexylpropionyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phe-
nylbutanoic Acid (2a). 1H NMR (CDCl3, 300 MHz) Diastereomer
1: δ 7.22 (m, 6H), 6.47 (d, J ) 8.3 Hz, 0.60H), 5.36 (m, 0.6H),

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4349
4.53 (m, 1H), 3.33 (m, 1H), 3.00 (m, 1H), 2.21 (m, 2H), 1.51 (m,
10H), 1.15 (m, 4H), 0.85 (m, 8H). Diastereomer 2: δ 7.22 (m, 6H),
6.24 (d, J ) 8.4 Hz, 0.40H), 5.25 (m, 0.4H), 4.53 (m, 1H), 3.33
(m, 1H), 3.00 (m, 1H), 2.21 (m, 2H), 1.51 (m, 10H), 1.15 (m, 4H),
0.85 (m, 8H). ESI MS: m/z 442.9 (M - 1)+. Anal. (C25-
H36N2O5 ·0.25H2O) C, H, N.
N-(1-Adamantylacetyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phenylbu-
tanoic acid (2b). Column chromatographic purification with EtOAc/
hexanes (1:1) as solvent gave a diastereomeric mixture of 2b as
foamy white solid in 92% yield. 1H NMR (CDCl3, 300 MHz)
Diastereomer 1: δ 7.23 (m, 6H), 6.23 (d, J ) 8.4 Hz, 0.67H), 5.33
(m, 0.67H), 4.51 (m, 1H), 3.31 (m, 1H), 3.01 (m, 1H), 1.94 (m,
5H), 1.57 (m, 15H), 0.84 (m, 6H). Diastereomer 2: δ 7.23 (m, 6H),
6.02 (d, J ) 8.4 Hz, 0.33H), 5.22 (m, 0.33H), 4.51 (m, 1H), 3.31
(m, 1H), 3.01 (m, 1H), 1.94 (m, 5H), 1.57 (m, 15H), 0.84 (m, 6H).
ESI MS: m/z 480.9 (M - 1)+. Anal. (C28H38N2O5 ·0.25H2O) C,
H, N.
which was recrystallized from hexanes/EtOAc/MeOH to afforded
rod-like white solid: mp 109 °C. H NMR (CDCl3, 300 MHz): δ
1
7.35-7.23 (m, 10H), 6.01 (d, J ) 7.9 Hz, 1H), 5.00 (s, 1H), 4.62
(m, 1H), 1.67-1.43 (m, 3H), 0.87 (d, J ) 6.3 Hz, 3H) 0.86 (d, J
) 6.2 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 176.6, 172.7, 139.0,
138.9, 129.0, 128.9, 128.8, 127.5, 127.4, 58.9, 51.2, 40.8, 25.0,
22.8, 21.8.
N-(3,3-Diphenylpropionyl)-L-leucine (6d). Diphenylpropionic
acid (2.26 g, 10 mmol) was coupled with L-leucine methyl ester
hydrochloride (1.82 g, 10 mmol) to give N-(3,3-diphenyl-propio-
nyl)-L-leucine methyl ester. This was hydrolyzed to give 99% of
6d, which was recrystallization from EtOAc/hexanes to give a white
solid: mp 115-116 °C. 1H NMR (CDCl3, 300 MHz): δ 8.70 (brs,
1H), 7.36-7.15 (m, 10H), 6.00 (d, J ) 8.2 Hz, 1H), 4.59-4.47
(m, 2H), 3.02 (dd, J ) 14.1, 7.0 Hz, 1H), 2.96 (dd, J ) 14.1, 9.0
Hz, 1H), 1.52 (m, 1H), 1.33(m, 1H), 1.15 (m, 1H), 0.79 (d, J )
6.6 Hz, 3H) 0.76 (d, J ) 6.5 Hz, 3H). 13C NMR (CDCl3, 75 MHz):
δ 176.4, 171.8, 143.3, 143.2, 128.6, 127.9, 127.6, 126.7, 126.6,
50.6, 47.4, 43.0, 41.0, 24.3, 22.8, 21.6.
N-(9-Fluoreneacetyl)-L-leucine (6e). 9-Fluoreneacetic acid (1.45
g, 6.47 mmol) was coupled with L-leucine methyl ester hydrochlo-
ride (1.18 g, 6.47 mmol) to give N-(9-fluoreneacetyl)-L-leucine
methyl ester in 94% yield. This was hydrolyzed to give 6e (97%),
which was recrystallized from EtOAc/hexanes/MeOH as white
solid: mp 139-140 °C. 1H NMR (CDCl3, 300 MHz): δ 7.71 (d, J
) 7.3 Hz, 2H), 7.48 (t, J ) 6.8 Hz, 2H), 7.35 (m, 2H), 7.26 (m,
2H), 5.80 (d, J ) 8.0 Hz, 1H), 4.60 (m, 1H), 4.43 (t, J ) 6.9 Hz,
1H), 2.76 (dd, J ) 15.0, 6.9 Hz, 1H), 2.68 (dd, J ) 15.0, 7.0 Hz,
1H), 1.61 (m, 1H), 1.53-1.36 (m, 2H), 0.89 (d, J ) 3.6 Hz, 3H),
0.87 (d, J ) 3.6 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 176.4,
171.8, 145.9, 140.7, 127.5, 127.5, 127.3, 127.1, 124.4, 119.9, 50.8,
43.7, 40.8, 40.1, 24.6, 22.8, 21.5.
N-(2,2-Diphenylacetyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phenylbu-
tanoic Acid (2c). Compound 2c was obtained in 97% yield as
1
diastereomeric mixture of a white foamy solid. H NMR (CDCl3,
300 MHz) Diastereomer 1: δ 7.15 (m, 16H), 6.16 (d, J ) 8.1 Hz,
0.75H), 5.08 (m, 2H), 4.46 (m, 1H), 3.21 (m, 1H), 2.74 (m, 1H),
1.25 (m, 3H), 0.83 (m, 6H). Diastereomer 2: δ 7.15 (m, 16H), 6.06
(d, J ) 8.1 Hz, 0.25H), 5.08 (m, 2H), 4.46 (m, 1H), 3.21 (m, 1H),
2.74 (m, 1H), 1.25 (m, 3H), 0.83 (m, 6H). ESI MS: m/z 499.1
(M - 1)+. Anal. (C30H32N2O5 ·0.5H2O) C, H, N.
N-(3,3-Diphenylpropionyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phe-
nylbutanoic Acid (2d). Compound 2d was obtained in 99.7% yield
as diastereomeric mixture of a white foamy solid. 1H NMR (CDCl3,
300 MHz) Diastereomer 1: δ 7.15 (m, 16H), 6.36 (d, J ) 8.4 Hz,
0.69H), 5.27 (m, 0.69H), 4.41 (m, 2H), 3.23 (m, 1H), 2.92 (m,
3H), 1.04 (m, 3H), 0.62 (m, 6H). Diastereomer 2: δ 7.15 (m, 16H),
6.11 (d, J ) 8.4 Hz, 0.31H), 5.12 (m, 0.31H), 4.41 (m, 2H), 3.23
(m, 1H), 2.92 (m, 3H), 1.04 (m, 3H), 0.62 (m, 6H). ESI MS: m/z
512.9 (M - 1)+. Anal. (C31H34N2O5 ·0.5 H2O) C, H, N.
N-(3-Cyclohexylpropionyl)-L-leucyl-2-hydroxy-3(R,S)-amino-4-
phenylbutanoic Acid Methyl Ester (8a). N-(3-Cyclohexylpropio-
nyl)-L-leucine (6a) was coupled with (S)-methyl 3-amino-2-
hydroxy-4-phenylbutanoate hydrochloride (7)10 to give 8a as a
mixture of diastereomers in 80% yield after column chromato-
graphic purification with EtOAc/hexanes (3:7) as eluent. 1H NMR
(CDCl3, 300 MHz) Diastereomer 1: δ 7.21 (m, 5H), 6.53 (d, J )
8.7 Hz, 0.50H), 5.73 (d, J ) 8.2 Hz, 0.52H), 4.60 (m, 1H), 4.37
(m, 1H), 4.29 (m, 0.51H), 3.71 (s, 1.56H), 3.48 (d, J ) 5.6 Hz,
0.51H), 2.92 (d, J ) 7.9 Hz, 1H), 2.81 (d, J ) 7.4 Hz, 1H), 2.14
(m, 2H), 1.54 (m, 10H), 1.23 (m, 4H), 0.87 (m, 8H). Diastereomer
2: δ 7.21 (m, 5H), 6.46 (d, J ) 9.2 Hz, 0.50H), 5.64 (d, J ) 8.1
Hz, 0.48H), 4.60 (m, 1H), 4.37 (m, 1H), 4.10 (m, 0.49H), 3.61 (s,
1.44H), 3.38 (d, J ) 4.6 Hz, 0.49H), 2.92 (d, J ) 7.9 Hz, 1H),
2.81 (d, J ) 7.4 Hz, 1H), 2.14 (m, 2H), 1.54 (m, 10H), 1.23 (m,
4H), 0.87 (m, 8H).
N-(9-Fluoreneacetyl)-L-leucyl-2-oxo-3(R,S)-amino-4-phenylbu-
tanoic Acid (2e). Compound 2e was obtained in 78% yield as
1
diastereomeric mixture of a white foamy solid. H NMR (CDCl3,
300 MHz) Diastereomer 1: δ 7.68 (m, 2H), 7.26 (m, 12H), 6.09
(d, J ) 8.1 Hz, 0.53H), 5.25 (m, 0.53H), 4.37 (m, 2H), 3.27 (m,
1H), 2.84 (m, 3H), 1.34 (m, 3H), 0.75 (m, 6H). Diastereomer 2: δ
7.68 (m, 2H), 7.26 (m, 12H), 5.75 (d, J ) 7.8 Hz, 0.47H), 5.09
(m, 0.47H), 4.37 (m, 2H), 3.27 (m, 1H), 2.84 (m, 3H), 1.34 (m,
3H), 0.75 (m, 6H). ESI MS: m/z 510.9 (M - 1)+. Anal.
(C31H32N2O5 ·0.5 H2O) C, H, N.
N-(3-Cyclohexylpropionyl)-L-leucine (6a). 3-Cyclohexylpropionic
acid (0.688 g, 2.8 mmol) was coupled with L-leucine methyl ester
hydrochloride (0.755 g, 2.8 mmol) to give yellow viscous oil, which
upon recrystallization from hexanes at freezer temperature afforded
N-(3-cyclohexylpropionyl)-L-leucine methyl ester as short rod-like
crystals in 98% yield. This was hydrolyzed to give a white solid,
which upon recrystallization from EtOAc/hexanes gave 6a as rods
N-(1-Adamantylacetyl)-L-leucyl-2-hydroxy-3(R,S)-amino-4-phe-
nylbutanoic Acid Methyl Ester (8b). N-(1-Adamantylacetyl)-L-
leucine (6b) was coupled with 7 to give 0.95 g (63.6%) of 8b as a
1
1
mixture of diastereomers. H NMR (CDCl3, 300 MHz) Diastere-
in 97% yield: mp 175-176 °C. H NMR (CDCl3, 300 MHz): δ
omer 1: δ 7.24 (m, 5H), 6.64 (br, 1H), 5.73 (d, J ) 8.4 Hz, 0.57H),
4.56 (m, 1H), 4.38 (m, 1H), 4.10 (m, 0.59H), 3.70 (s, 1.89H), 3.52
(d, J ) 4.5 Hz, 0.56H), 2.88 (m, 2H), 1.91 (m, 5H), 1.54 (m, 15H),
0.92 (m, 6H). Diastereomer 2: δ 7.24 (m, 5H), 6.64 (br, 1H), 5.65
(d, J ) 7.8 Hz, 0.43H), 4.56 (m, 1H), 4.38 (m, 1H), 4.28 (m, 0.41H),
3.59 (d, J ) 5.7 Hz, 0.44H), 3.57 (s, 1.11H), 2.88 (m, 2H), 1.91
(m, 5H), 1.54 (m, 15H), 0.92 (m, 6H).
5.86 (d, J ) 8.1 Hz, 1H), 4.62 (m, 1H), 2.28 (m, 2H), 1.81-1.52
(m, 10H), 1.27-1.04 (m, 5H), 0.99 (d, J ) 3.2 Hz, 3H), 0.97 (d,
J ) 3.2 Hz, 3H), 0.90 (m, 1H). 13C NMR (CD3OD, 75 MHz): δ
176.6, 176.2, 51.9, 41.6, 38.5, 34.4, 34.4, 34.3, 34.2, 26.5, 27.7,
27.4, 26.1, 23.4, 21.7.
N-(1-Adamantylacetyl)-L-leucine (6b). 1-Adamantaneacetic acid
(1.94 g, 10 mmol) was coupled with L-leucine methyl ester
hydrochloride (1.817 g, 10 mmol) to give N-(1-adamantylacetyl)-
L-leucine methyl ester as light-yellow solid in 98% yield. This was
hydrolyzed to give 6b as white solid in 94% yield: mp 204-205
N-(2,2-Diphenylacetyl)-L-leucyl-2-hydroxy-3(R,S)-amino-4-phe-
nylbutanoic Acid Methyl Ester (8c). N-(2,2-Diphenylacetyl)-L-
leucine (6c) (1.0 g, 3.07 mmol) was coupled with 7 (0.75 g, 3.07
mmol) to give 1.23 g (77.6%) yield of 8c as a diastereomeric
mixture after column chromatographic purification of the crude
product. 1H NMR (CDCl3, 300 MHz) Diastereomer 1: δ 7.23
(m, 15H), 6.62 (br, 1H), 6.03 (d, J ) 8.1 Hz, 0.56H), 4.89 (d, J )
4.28 Hz, 1H), 4.49 (m, 2H), 4.07 (m, 0.57H), 3.66 (s, 1.71H), 3.48
(d, J ) 4.8 Hz, 0.54H), 2.81 (m, 2H), 1.41 (m, 3H), 0.84 (d, J )
6.3 Hz, 6H). Diastereomer 2: δ 7.23 (m, 15H), 6.62 (br, 1H), 5.93
(d, J ) 7.8 Hz, 0.44H), 4.89 (d, J ) 4.28 Hz, 1H), 4.49 (m, 2H),
1
°C. H NMR (CD3OD, 300 MHz): δ 4.41 (m, 1H), 2.00 (s, 2H),
1.95 (m, 3H), 1.76-1.59 (m, 15H), 0.97 (d, J ) 6.5 Hz, 3H), 0.93
(d, J ) 6.5 Hz, 3H). 13C NMR (CD3OD, 75 MHz): δ 174.7, 172.5,
50.5, 50.1, 42.3, 40.1, 36.5, 32.5, 28.8, 24.6, 21.9, 20.2.
N-(2,2-Diphenylacetyl)-L-leucine (6c). Diphenylacetic acid (2.12
g, 10 mmol) was coupled with L-leucine methyl ester hydrochloride
(1.82 g, 10 mmol) to give N-(2,2-diphenylacetyl)-L-leucine methyl
ester in 93% yield. This was hydrolyzed to give 6c (98% yield),

4350 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Donkor et al.
4.26 (m, 0.43H), 3.59 (d, J ) 5.4 Hz, 0.46H), 3.55 (s, 1.29H),
2.81 (m, 2H), 1.41 (m, 3H), 0.84 (d, J ) 6.3 Hz, 6H).
only residues within 10 Å radius of the inhibitor were made fully
flexible, the other residues being kept fixed.
N-(3,3-Diphenylpropionyl)-L-leucyl-2-hydroxy-3(R,S)-amino-4-
phenylbutanoic Acid Methyl Ester (8d). N-(3,3-Diphenylpropio-
nyl)-L-leucine (6d) (1.02 g, 3 mmol) was coupled with 7 (0.737 g,
3 mmol), followed by column chromatographic purification using
EtOAc/hexanes (3:7) as solvent to give 1.2 g (75.4%) of a
diastereomeric mixture of 8d as white solid. 1H NMR (CDCl3, 300
MHz) Diastereomer 1: δ 7.23 (m, 15H), 6.43 (d, J ) 9.3 Hz,
0.64H), 5.70 (d, J ) 8.4 Hz, 0.64H), 4.51 (m, 2H), 4.17 (m, 2H),
3.69 (s, 1.92H), 3.43 (d, J ) 4.8 Hz, 0.59H), 2.86 (m, 4H), 1.22
(m, 3H), 0.71 (m, 6H). Diastereomer 2: δ 7.23 (m, 15H), 6.49 (d,
J ) 8.7 Hz, 0.36H), 5.59 (d, J ) 7.8 Hz, 0.36H), 4.51 (m, 2H),
4.17 (m, 2H), 3.59 (s, 1.08H), 3.55 (d, J ) 5.7 Hz, 0.41H), 2.86
(m, 4H), 1.22 (m, 3H), 0.71 (m, 6H).
N-(9-Fluoreneacetyl)-L-leucyl-2-hydroxy-3(R,S)-amino-4-phe-
nylbutanoic Acid Methyl Ester (8e). N-(9-Fluoreneacetyl)-L-leucine
(6e) (1.012 g, 3 mmol) was coupled with 7 (0.736 g, 3 mmol) and
the crude product was purified by column chromatography using
EtOAc/hexanes (4:6) as solvent to give 1.12 g (70.7%) of 8e as a
mixture of diastereoisomers. 1H NMR (CDCl3, 300 MHz) Diaste-
reomer 1: δ 7.74 (d, J ) 7.2 Hz, 2H), 7.47 (d, J ) 7.5 Hz, 2H),
7.27 (m, 9H), 6.53 (d, J ) 9.3 Hz, 1H), 5.67 (d, J ) 7.8 Hz, 0.61H),
4.48 (m, 3H), 4.12 (m, 0.61H), 3.69 (s, 1.80H), 3.56 (d, J ) 4.8
Hz, 0.61H), 2.84 (m, 3H), 2.56 (m, 1H), 1.38 (m, 3H), 0.86 (d, J
) 3.2 Hz, 6H). Diastereomer 2: δ 7.74 (d, J ) 7.2 Hz, 2H), 7.47
(d, J ) 7.5 Hz, 2H), 7.27 (m, 9H), 6.53 (d, J ) 9.3 Hz, 1H), 5.52
(d, J ) 8.1 Hz, 0.39H), 4.48 (m, 3H), 4.29 (m, 0.39H), 3.61 (d, J
) 5.4 Hz, 0.39H) 3.59 (s, 1.20H), 2.84 (m, 3H), 2.56 (m, 1H),
1.38 (m, 3H), 0.86 (d, J ) 3.2 Hz, 6H).
Acknowledgment. The study was supported by NIH grant
R03CA125775 to IOD.
Supporting Information Available: Elemental analysis data for
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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JM800182C