Novel peptidyl α-keto amide inhibitors of calpains and other cysteine proteases

Article abstract of DOI:10.1021/jm950541c

A series of new dipeptidyl α-keto amides of the general structure R₁- L-Leu-D,L-AA-CONH-R₂ were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. They combine 10 different N-protecting groups (R₁), 3 amino acids residues in P1 (AA), and 44 distinct substituents on the α-keto amide nitrogen (R₂). In general, calpain II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors displaying dissociation constants (K(i)) in the 10-100 nM range. Calpain I was also effectively inhibited, but very low K(i) values were observed with a smaller number of inhibitors than with calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best inhibitors for calpain II were Z-Leu-Abu-CONH-CH₂-CHOH-C₆H₅ (K(i) = 15 nM), Z-Leu-Abu-CONH-CH₂-2-pyridyl (K(i) = 17 nM), and Z-Leu-Abu-CONH- CH₂-C₆H₃(3,5(OMe)₂) (K(i) = 22 nM). The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH₂-2-pyridyl (K(i) = 19 nM). The peptide α-keto amide Z-Leu-Abu-CONH-(CH₂)₂-3-indoly] was the best inhibitor for cathepsin B (K(i) = 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such inhibitors may be useful in elucidating the physiological and pathological events involving these proteases and may become possible therapeutic agents.

Full text of DOI:10.1021/jm950541c

J . Med. Chem. 1996, 39, 4089-4098
4089
Novel P ep tid yl r-Keto Am id e In h ibitor s of Ca lp a in s a n d Oth er Cystein e
P r otea ses
Zhaozhao Li,† Anne-Ce´cile Ortega-Vilain,† Girish S. Patil,† Der-Lun Chu,† J . E. Foreman,‡
David D. Eveleth,‡ and J ames C. Powers*,†
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, and
Cortex Pharmaceuticals, Inc., 15241 Barranca Parkway, Irvine, California 92618
Received J uly 24, 1995X
A series of new dipeptidyl R-keto amides of the general structure R1-L-Leu-D,L-AA-CONH-R2
were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II,
and cathepsin B. They combine 10 different N-protecting groups (R1), 3 amino acids residues
in P1 (AA), and 44 distinct substituents on the R-keto amide nitrogen (R2). In general, calpain
II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors
displaying dissociation constants (Ki) in the 10-100 nM range. Calpain I was also effectively
inhibited, but very low Ki values were observed with a smaller number of inhibitors than with
calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best
inhibitors for calpain II were Z-Leu-Abu-CONH-CH2-CHOH-C6H5 (Ki ) 15 nM), Z-Leu-Abu-
CONH-CH2-2-pyridyl (Ki ) 17 nM), and Z-Leu-Abu-CONH-CH2-C6H3(3,5(OMe)2) (Ki ) 22 nM).
The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH2-2-pyridyl (Ki ) 19 nM).
The peptide R-keto amide Z-Leu-Abu-CONH-(CH2)2-3-indolyl was the best inhibitor for cathepsin
B (Ki ) 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity
on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others
were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such
inhibitors may be useful in elucidating the physiological and pathological events involving these
proteases and may become possible therapeutic agents.
In tr od u ction
tones,12,13 R-keto acids,13,14 R-keto esters,12-14 and R-keto
amides,13-15 reversibly inhibit cysteine proteases as well
as serine proteases, possibly due to hemithioacetal
formation with the SH group of the active site cysteine,
in the case of cysteine proteases, or due to hemiacetal
formation with the OH group of the active site serine,
in the case of serine proteases.
Calpains and calcium-activated neutral cysteine pro-
teases widely distributed in animal cells and tissues.
Two types of calpains have been identified, calpain I (or
µ-calpain) and calpain II (or m-calpain), which require
micro- and millimolar concentrations of calcium, respec-
tively, for optimal enzyme activity in vitro. Proteolysis
by calpains is a vital regulatory mechanism mediating
biological responses elicited by the elevation of calcium.
Calpains are able to degrade cytoskeletal proteins and
hormone receptors as well as activate some kinases and
phosphatases (for a review on calpain, see ref 1). In-
creasing evidence suggests that calpain participates in
the initiation and/or expression of multiple degenerative
conditions. Enhanced calpain activity has been de-
scribed in conjunction with cell injury elicited by physi-
cal damage,2 hypoxia,3 and ischemia.4-7 Therefore,
specific calpain inhibitors may be useful therapeutic
agents in some degenerative disease states.
In a preceding article,14 we explored the inhibitory
potencies of dipeptidyl and tripeptidyl R-keto esters,
R-keto amides, and R-keto acids. The influence of the
type and number of amino acid residues, the nature of
the N-protecting group, and differences between the
ester or amide functional group were studied. Our
results showed that, in general, peptidyl R-keto acids
were more inhibitory toward both calpains than R-keto
amides which, in turn, were more effective than R-keto
esters in vitro. Lacking hydrophobicity, R-keto acids
had quite poor membrane penetration as illustrated in
a platelet membrane permeability assay. They were
also highly unstable in vivo due to their sensitivity to
esterases. After demonstrating that N-monosubstituted
peptide R-keto amides were much more potent inhibitors
than the corresponding N,N-disubstituted R-keto amides,
we hypothesized that the N-H on the R-keto amide
functional group formed a hydrogen bond with an amino
acid residue of the active site of calpain. Noteworthy
was the fact that R-keto amides with hydrophobic alkyl
groups or alkyl groups with an attached phenyl group
had the lowest Ki values, which suggest the existence
Several types of low molecular weight inhibitors have
been shown to effectively inhibit calpain. Acting by
affinity labeling, peptidyl diazomethyl ketones8 and
peptidyl fluoromethyl ketones9 irreversibly inhibit
calpain. E-64, a naturally occurring epoxide, and its
derivatives (E-64-c, E-64-d)10 irreversibly inhibit cys-
teine proteases, including calpain, by covalent bond
formation with the active site cysteine residue. Peptidyl
aldehydes11 and carbonyl derivatives, such as R-dike-
16
of a hydrophobic pocket on the S′ side of the active
site.
* Author to whom correspondence should be addressed. Tel: (404)
894-4038. Fax: (404) 894-7452. E-mail: james.powers@chemistry.
gatech.edu.
The protective effect of two dipeptide R-keto amides
against ischemic brain damage, using an animal model
of ischemia, has been reported.6,7 Focal ischemia was
induced using a variation of the middle cerebral artery
† Georgia Institute of Technology.
‡ Cortex Pharmaceuticals, Inc.
X Abstract published in Advance ACS Abstracts, September 1, 1996.
S0022-2623(95)00541-3 CCC: $12.00 © 1996 American Chemical Society

4090 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Li et al.
The appropriate aldehyde was treated with trimeth-
ylsilyl cyanide and followed by reduction with LiAlH4
to give the amine as a racemic mixture. 2-(Amino-
methyl)quinoline20 and 1-(aminomethyl)isoquinoline21
were prepared by palladium-catalyzed hydrogenation of
the corresponding nitrile derivatives. 1-(3-Aminopro-
pyl)-1,2,3,4-tetrahydroquinoline22 and 2-(3-aminopro-
pyl)-1,2,3,4-tetrahydroisoquinoline23 were prepared by
refluxing the corresponding unsubstituted tetrahydro
derivatives with acrylonitrile in acetic acid or toluene
followed by catalytic hydrogenation or lithium alumi-
num hydride reduction of the resulting N-cyanoethyl
derivatives.
8-(Aminomethyl)caffeine24 was synthesized in seven
steps according to the reported procedures. Three
peptidyl R-keto amides 94 were prepared from the
corresponding R-keto acids 93 by coupling using CDI
(compound 49) or DCC-HOBt (compounds 51 and 71)
and the appropriate amine in low yield. The starting
peptidyl R-keto acids Z-Leu-Abu-CO2H and Z-Leu-Phe-
CO2H (93) were prepared by alkaline hydrolysis of the
corresponding R-keto esters 91 in high yield as previ-
ously reported.14
F igu r e 1. Structure of a dipeptidyl R-keto amide (abbreviated
as R1-Leu-AA-CONH-R2).
occlusion method. One compound, Z-Leu-Abu-CONH-
Et (2), was perfused directly onto the infarcted cortical
surface.7 Alternately, Z-Leu-Abu-CONH-(CH2)3-4-mor-
pholinyl (42) was infused through the internal carotid
artery.6 After a delay, animals were sacrificed and the
infarct volume was quantified. With 2, a 75% reduction
in infarct volume was achieved, while the maximum
obtained with 42 was 32% using a different dosing
protocol. A dose-dependent neuroprotective effect could
be demonstrated in both cases. The morpholine com-
pound 42 (AK295) is also able to attenuate motor
dysfunction in rats following brain injury.17 The sig-
nificant improvement in behavioral outcome measure-
ments suggest that 42 could be used in the treatment
of brain injuries.
We have now investigated the inhibitory potencies of
R-keto amides toward cysteine proteases using a series
of new compounds. In addition, nine other R-keto
amides, whose syntheses and inhibitory potencies to-
ward cysteine proteases have been published else-
where,14 are presented here for comparison purposes.
All R-keto amides share the general formula R1-L-Leu-
Several ketoamides (75-80) were prepared from 2 by
deblocking the Z group and coupling HBr‚Leu-Abu-
CONHEt with the appropriate acid R1-OH (Scheme 2).
The synthesis of 81-89 involved coupling of R1-Leu-
OH with AA-OMe followed by hydrolysis to R1-Leu-AA-
OH which was then converted to the keto amide as
described in Scheme 1.14
18
D,L-AA-CONH-R2 (Figure 1). They combine 10 differ-
ent R1 groups, including heterocyclic (75-80) and
nonheterocylic aromatic (1-74, 81-89) groups, 3 dif-
ferent amino acids (AA) in the P1 position (Abu, Phe,
or Nva) and 44 different R2 groups including alkyl,
hydroxy, or alkoxy groups (1-10, 51-53), arylalkyl
groups (11-32, 54-65, 72), and heterocyclic derivatives
(33-50, 66-71, 73, 74).
Resu lts a n d Discu ssion
We have designed a series of new R-keto amides as
specific calpain inhibitors. Because our previous results
indicated that cysteine proteases possessed a hydrogen
bond donor in the S1′ subsite,14 we synthesized R-keto
amides having one or several heteroatoms in an effort
to further explore the H-bonding capacity of the S′
subsites. To take advantage of the hydrophobic pocket
we previously identified in the S′ region,14 we chose to
incorporate the heteroatoms into aromatic structures
(14-33, 35-40, 44-49, 56-69, 72, 73, 84, 85, 87, 88).
The hydrophobicity and H-bonding capacity of another
region of the active site, the S3 and S4 subsites, were
also explored using various heterocyclic (75-80) or
nonheterocyclic aromatic (1-74, 82-89) blocking groups
on the N-terminus of the dipeptide R-keto amide inhibi-
tors. The importance of the nature of the amino acid
in the P1 position was investigated using 29 compounds
which were analogs in all respects, except that they
contained either an R-aminobutyric acid (Abu), a phen-
ylalanine, or a norvaline (Nva) in P1.
The inhibitory potencies of the peptidyl R-keto amides
toward several cysteine proteases including calpain I,
calpain II, and cathepsin B were investigated, and the
results are presented in Tables 1 and 2. Data for the
serine proteases pancreatic elastase and R-chymotrypsin
and the cysteine protease papain are presented in the
Supporting Information. Standard errors on Ki are not
given individually but correspond to 5-15% of the given
values. Some compounds were tested on bovine cathe-
psin B and others on human cathepsin B, depending
on enzyme availability. After verification with several
inhibitors that the measured Ki values did not differ
significantly from one enzyme to the other, we chose to
use both indiscriminately for comparison purposes.
Ch em istr y
We have previously reported synthetic methods for
the preparation of peptidyl R-keto amides.14 The pro-
cedure (Scheme 1) involves a modified Dakin-West
reaction to prepare peptidyl R-enol esters 90, which are
then converted to R-keto esters 91 by reaction with al-
koxides. The R-carbonyl group of the R-keto ester is
then protected as a 1,3-dithiolane derivative (92) fol-
lowed by reaction with an excess amount of the appro-
priate amine (R2-NH2) to yield the R-keto amide 94. As
previously reported, the 1,3-dithiolane protection group
was unexpectedly lost during the reaction or workup
procedure. This method (91 f 92 f 94) was used with
compounds 1-48, 51-70, and 72-74 and as the final
steps in the preparation of 81-89. Many of the amines
used in the coupling reaction were commercially avail-
able. Others, including 2-amino-1-(4-methoxyphenyl)-
ethanol, 2-amino-1-(3,4,5-trimethoxyphenyl)ethanol,
2-amino-1-[4-(N,N-dimethylamino)phenyl]-ethanol,
2-amino-1-(pentafluorophenyl)ethanol, 2-amino-1-[3-
(trifluoromethyl)phenyl]ethanol, 2-amino-1-(3-phenoxy-
phenyl)ethanol, 2-amino-1-(4-phenoxyphenyl)ethanol,
2-amino-1-[4-(benzyloxy)phenyl]ethanol, 2-amino-1-[3-
[4-(trifluoromethyl)phenoxy]phenyl]ethanol, 2-amino-1-
[3-(3,4-dichlorophenoxy)phenyl]ethanol, 2-amino-1-[3,4-
bis(benzyloxy)phenyl]ethanol, 2-amino-1-(1-naphthyl)-
ethanol, and 2-amino-1-(2-naphthyl)ethanol, were pre-
pared using the trimethylsilyl cyanide method.19

R-Keto Amide Inhibitors of Calpains
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4091
Sch em e 1a
a
AA ) Abu, Nva, Phe; R ) Me, Et, Ph; R1, R2 ) see Tables 1 and 2.
Sch em e 2a
inhibitor (Ki ) 7.8 µM), while Abu (19) and Phe (56)
analogs have better inhibitory potencies, with Ki values
of 1.1 and 1.3 µM, respectively.
Ca lp a in II In h ibition . Among the various proteases
tested, calpain II was most potently inhibited by this
series of R-keto amides. A large number (27) of inhibi-
tors have Ki values in the 10-100 nM range, whereas
only five inhibitors have values this low with calpain I
and only six with cathepsin B. Among the former are
Z-Leu-Abu-CONH-CH2-CHOH-Ph (19, Ki ) 15 nM),
Z-Leu-Abu-CONH-(CH2)2-Ph (12, Ki ) 22 nM), and
Z-Leu-Phe-CONH-CH2-2-quinolinyl (67, Ki ) 23 nM).
a
R1 ) see Tables 1 and 2.
The introduction of heteroatoms into the amide
nitrogen substituent of the R-keto amide or into the
N-terminal blocking group of the inhibitors did not
produce a large increase in affinity. This indicates that
no additional significant H bonds are being formed
between either the S or S′ sites of the enzyme and the
inhibitor. Alternatively, the heteroatoms in the inhibi-
tors may be in the wrong location to interact with H
bond acceptors or donors in the enzyme. The existence
of a hydrophobic pocket in S1′ was confirmed since
Ca lp a in I In h ibition . Changing the substituent on
the nitrogen atom of the R-keto amide function resulted
in only a slight variation of inhibitory potency toward
calpain I (less than 5-fold change with most compounds).
The nitrogen substituents giving the best calpain I
inhibitors were a pyridyl group, Z-Leu-Nva-CONH-CH2-
2-pyridyl (73, Ki ) 19 nM), and phenyl-substituted
derivatives of CH2-CHOH-Ph (i.e., Z-Leu-Abu-CONH-
CH2-CHOH-C6F5, 23, Ki ) 50 nM; Z-Leu-Abu-CONH-
CH2-CHOH-C6H4-3-OC6H4(3-CF3), 28, Ki ) 70 nM).
In contrast, modification of the N-terminal blocking
group induced a large decrease in the inhibitory potency
(up to 32-fold). The N-terminal blocking groups which
gave the best calpain I inhibitors were the 1-naphthyl-
CO (83), 1-isoquinolinyl-CO (79), and 2-quinolinyl-CO
(78) derivatives which had inhibitory potencies in the
same order of magnitude as the benzyloxycarbonyl
analog 2 (Ki ) 0.30, 0.35, 0.50, and 0.21 µM, respec-
tively). Notably poor inhibition results were obtained
with the morpholinyl derivative 4-morpholinyl-CO-Leu-
Abu-CONHEt (80, Ki ) 7.9 µM), and the bulky branched
derivative Ph2CHCO-Leu-Abu-CONH-Et (82, Ki ) 5.0
µM).
The compounds possessing an Abu in the P1 position
had a modest advantage over those with Phe, while
compounds with a Nva residue in P1 were sometimes
favored and sometimes disfavored. For example, Z-Leu-
Nva-CONH-CH2-2-pyridyl (73) is the best calpain I
inhibitor presented in this paper with a Ki value of 19
nM, while the analogs with an Abu or a Phe residue in
place of the Nva in P1 are weak calpain I inhibitors (0.64
and 0.65 µM, respectively). Another compound, Z-Leu-
Nva-CONH-CH2-CHOH-Ph (72), is a poor calpain I
R-keto amides R1-L-Leu-D,L-AA-CONH-R2 with R2
)
CH2-CH2-Ph or CHOH-CH2-Ph (12, 19, 55, 56) were
excellent calpain II inhibitors, with Ki values ranging
from 15 to 50 nM. But when the phenyl rings of these
compounds were substituted (14-17, 20-32, 57-65),
the affinity for calpain II decreased and ranged from
46 nM for Z-Leu-Abu-CONH-CH2-CH2-Ph(4-OMe) (17)
to 670 nM for Z-Leu-Phe-CONH-CH2-CHOH-Ph(3,4-
(OCH2Ph)2) (65) indicating a very definite size for the
hydrophobic pocket. Heterocyclic derivatives such as
Z-Leu-Abu-CONH-CH2-2-furyl (33, Ki ) 33 nM), Z-Leu-
Abu-CONH-CH2-2-pyridyl (25, Ki ) 17 nM), or Z-Leu-
Phe-CONH-CH2-2-quinolinyl (67, Ki ) 23 nM) were also
good calpain II inhibitors.
Variation of the N-terminal blocking group did not
produce a large variation in the inhibitory potencies of
the R-keto amides toward calpain II. None of the
derivatives tested gave better inhibitory results than
the parent benzyloxycarbonyl group. As observed with
calpain I, the morpholinyl derivative 4-morpholinyl-CO-
Leu-Abu-CONHEt (80, Ki ) 9.9 µM) and the bulky
branched derivative Ph2CHCO-Leu-Abu-CONH-Et (82,
Ki ) 1.2 µM) were quite poor inhibitors.

4092 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Li et al.
a
Ta ble 1. Inhibition of Cysteine Proteases and Platelet Membrane Permeability by Peptidyl R-Keto Amides R1-Leu-AA-CONH-R2
compound
Ki (mM)
Cal IIb
IC (µM) plat
no.
R1
AA
R2
Cal Ib
Cat Bc,d
memb permeabilitye
1
2
3
4
5
6
7
8
9
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Nva
Nva
Nva
Me
0.28
0.25
0.083
0.21
0.25
0.078
0.051
0.16
0.10
0.16
0.044
0.069
0.35
0.022
0.043
0.060
0.16
0.086
0.046
0.022
0.015
nldpf
0.22
0.41
0.20
0.18
0.59
0.29
0.12
0.28
0.12
0.10
0.17
0.11
0.033
0.066
0.017
0.12
0.11
0.47
0.076
0.068
0.16
0.041
0.27
0.050
0.60
0.15
0.20
0.19
4.6
0.28
0.15
0.039
0.050
0.046
0.024
0.050
0.31
0.35
0.29
0.34
0.17
0.24
0.45
0.12
0.67
0.27
0.023
0.26
0.20
0.33
0.16
11
0.33c
2.4d
Et14
100
70
78
54
58
n-Pr14
(CH2)2OH
(CH2)5OH
(CH2)2O(CH2)2OH
CH2CH(OCH3)2
CH2CH(OC2H5)2
CH2-C6H11
CH2-C6H7(1,3,3-(CH3)3-5-OH)
CH2C6H5
(CH2)2C6H5
8.0c
0.80
0.50
0.65
0.50
0.20
0.68
0.42
0.20
0.20
0.80
0.38
0.13
0.11
0.12
2.3
4.5d
0.28d
2.0d
0.19c
0.11c
14
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
0.89d
2.0d
103
30
50
100
23
14
1.3d
14
0.20d
nldpd,f
0.63d
0.31d
0.44d
1.8d
14
(CH2)3C6H5
(CH2)2C6H4(4-OH)
(CH2)2C6H4(2-OCH3)
(CH2)2C6H4(3-OCH3)
(CH2)2C6H4(4-OCH3)
CH2C6H3(3,5-(OCH3)2)
CH2CH(OH)Ph
CH2CH(OH)C6H4(4-OCH3)
CH2CH(OH)C6H2(3,4,5-(OCH3)3)
CH2CH(OH)C6H4(4-N(CH3)2)
CH2CH(OH)C6F5
CH2CH(OH)C6H4(3-CF3)
CH2CH(OH)C6H4(3-OPh)
CH2CH(OH)C6H4(4-OPh)
CH2CH(OH)C6H4(4-OCH2Ph)
CH2CH(OH)C6H4-3-OC6H4(3-CF3)
CH2CH(OH)C6H4-3-OC6H3(3,4-Cl2)
CH2CH(OH)C6H3(3,4-(OCH2Ph)2)
CH2CH(OH)-1-C10H7
CH2CH(OH)-2-C10H7
CH2-2-furyl
CH2-2-tetrahydrofuryl
CH2-2-pyridyl
CH2-3-pyridyl
CH2-4-pyridyl
(CH2)2-2-pyridyl
(CH2)2-2-(N-methylpyrrole)
(CH2-3-1-imidazolyl
(CH2)2-4-morpholinyl
(CH2)3-4-morpholinyl
(CH2)3-1-pyrrolidin-2-one
(CH2)2-3-indolyl
30
69
1.1
0.37d
0.12c
0.22c
0.072c
0.15c
0.11c
0.12c
0.21c
0.11c
0.30c
0.10c
0.32c
0.063c
0.063c
6.0d
0.24
0.38
0.33
0.050
0.35
0.90
0.10
0.080
0.070
0.27
0.23
0.12
0.35
0.80
0.33
0.64
58
22
110
>300
54
4.5d
3.0d
1.2d
1.1
0.41
0.16
0.29
1.0
0.14
1.2
0.30
0.13
0.25
0.37
0.31
32.0
0.65
0.35
0.20
0.35
6.4d
0.20d
1.2d
70
9.9d
2.5d
23
45
6.9d
2.0d
0.031c
0.45c
0.30d
0.094c
8.0d
180
CH2-2-quinolinyl
CH2-1-isoquinolinyl
(CH2)3-1-tetrahydroquinolinyl
(CH2)3-2-tetrahydroisoquinolinyl
CH2-8-caffeinyl
1.7c
(CH2)2NH-biotinyl
0.66c
0.57c
6.0d
Me
Et14
22
31
>300
100
44
n-Pr14
3.0d
CH2Ph14
4.6
(CH2)2Ph14
0.052
1.3
9.3
CH2CH(OH)Ph
CH2CH(OH)C6H4(4-N(CH3)2)
CH2CH(OH)C6F5
2.1d
0.62
0.70
0.46
0.60
0.20
0.20
0.18
0.59
0.48
0.65
0.11
0.38
0.22
0.12
0.22
7.8
0.31c
0.21c
0.14c
0.64c
2.1c
CH2CH(OH)C6H4(3-CF3)
CH2CH(OH)C6H4(3-OPh)
CH2CH(OH)C6H4(4-OPh)
CH2CH(OH)C6H4(4-OCH2Ph)
CH2CH(OH)C6H4-3-OC6H4(3-CF3)
CH2CH(OH)C6H4-3-OC6H3(3,4-Cl2)
CH2CH(OH)C6H3(3,4-(OCH2Ph)2)
CH2-2-pyridyl
0.38c
0.12c
0.64c
20c
0.80d
0.34d
0.14c
4.5d
CH2-2-quinolinyl
(CH2)3-1-tetrahydroquinolinyl
(CH2)3-2-tetrahydroisoquinolinyl
(CH2)3-4-morpholinyl
(CH2)2NH-biotinyl
CH2CH(OH)Ph
CH2-2-pyridyl
13d
54
18
0.23c
3.4c
0.019
0.25
0.12
0.10
0.75d
4.2d
(CH2)3-4-morpholinyl
a
All of the peptide R-keto amide inhibitors are diastereomeric at the R-carbon of the P1 residue; peptide R-keto amides 19-32, 56-65,
and 72 possess an additional chiral center (racemic mixture) on the P′ side. 25 mM Tris, pH 8.0, 10 mM CaCl2, 5% DMSO. c Bovine
b
d
cathepsin B, 20 mM sodium acetate, pH 5.2, 0.5 mM dithiothreitol, 2% DMSO. Human cathepsin B, 20 mM sodium acetate, pH 5.2, 0.5
mM dithiothreitol, 2% DMSO. e 14.7 mM HEPES, 0.4 mM sodium phosphate, 12 mM sodium bicarbonate, pH 7.35, 0.137 M NaCl, 3 mM
KCl, 1 mM MgCl2, 20 mM glucose, 2% DMSO. f nldp ) gives nonlinear Dixon plots.

R-Keto Amide Inhibitors of Calpains
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4093
a
Ta ble 2. Inhibition of Cysteine Proteases and Platelet Membrane Permeability by Peptidyl R-Keto Amides R1-Leu-AA-CONH-R2
compound
AA
Ki (µM)
Cal IIb
IC50 (µM) plat
no.
R1
R2
Cal Ib
Cat Bc,d
memb permeabilitye
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
2-furyl-CO
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Abu
Phe
Phe
Phe
Et
Et
Et
Et
Et
Et
Et
Et
Et
0.85
1.30
1.4
0.50
0.35
7.9
1.9
5.0
0.30
0.75
0.5
0.58
0.83
0.48
0.30
0.11
9.9
1.2c
3-pyridyl-CO
2-pyrazinyl-CO
2-quinolinyl-CO
1-isoquinolinyl-CO
4-morpholinyl-CO
Ph(CH2)2-CO
Ph2CH-CO
1-C10H7CH2-CO
Ph2CH-CO
Ph2CH-CO
0.88c
0.35c
0.096c
0.22c
4.3c
0.13
1.2
0.22c
0.55c
0.35d
0.17c
2.8d
0.25
0.20
0.09
0.11
0.73
0.36
0.074
CH2CH(OH)Ph
CH2-2-pyridyl
(CH2)3-morpholinyl
CH2CH(OH)Ph
CH2-2-pyridyl
180
88
82
Ph2CH-CO
Ph2CH-CO
Ph2CH-CO
Ph2CH-CO
0.8
10
1.1
0.76
2.3d
0.24c
2.2d
>300
(CH2)3-morpholinyl
3.8d
a
All of the peptide R-keto amide inhibitors are diastereomeric at the R-carbon of the P1 residue; peptide R-keto amides 84 and 87
possess an additional chiral center (racemic mixture) on the P′ side. 25 mM Tris, pH 8.0, 10 mM CaCl2, 5% DMSO. c Bovine cathepsin
b
d
B, 20 mM sodium acetate, pH 5.2, 0.5 mM dithiothreitol, 2% DMSO. Human cathepsin B, 20 mM sodium acetate, pH 5.2, 0.5 mM
dithiothreitol, 2% DMSO. e 14.7 mM HEPES, mM sodium phosphate, 12 mM sodium bicarbonate pH 7.35, 0.137 M NaCl, 3 mM KCl, 1
mM MgCl2, 20 mM glucose, 2% DMSO.
We observed that Abu in P1 is slightly favored over
the other two amino acids in our study. In the large
majority of cases (13), replacement of the Abu residue
by a Phe did not produce a significant change in
inhibitory potency (<2-fold). However, with seven pairs
of inhibitors, replacement of the Abu by Phe resulted
in a decrease in affinity, while in two cases, it resulted
in an increase in affinity.
bridges with the substrate (Figure 3).25 The poor inhi-
bitory results obtained with our P′ positively charged
inhibitors can be explained by an electrostatic repulsion
effect between the inhibitors and these two histidine res-
idues. Interestingly, this repulsive interaction is not ob-
served with calpains I and II. Therefore, placement of
positively charged residues in P2′ may be a useful tech-
nique for increasing the specificity of calpain inhibitors.
Cath epsin B In h ibition . The peptidyl R-keto amides
presented here displayed a lower affinity toward cathe-
psin B, since they were designed primarily to target
calpains. However, the hypothetical H bond donor or
acceptor that we have been probing for in the active site
of the cysteine proteases is likely to be present in the
S′ sites of cathepsin B. While alkyl substituents on the
nitrogen (R2) were poor inhibitors, hydroxy or alkoxy
analogs (5, 7, 8) were more potent, with Ki values
ranging from 51 to 160 nM. Similarly, unsubstituted
phenyl derivatives (11, 12, 19, 55, 56) in R2 showed poor
inhibitory potencies (i.e., Z-Leu-Abu-CONH-CH2-CH2-
Ph, 12, Ki ) 1.3 µM; Z-Leu-Phe-CONH-CH2-CHOH-Ph,
56, Ki ) 2.1 µM) compared to analogs having the phenyl
ring substituted with a group carrying halogens or
heteroatoms (15-18) (i.e., Z-Leu-Abu-CONH-CH2-CH2-
Ph(3-OMe), 16, Ki ) 0.31 µM; Z-Leu-Phe-CONH-CH2-
CHOH-Ph(3-CF3), 59, Ki ) 0.14 µM). Almost all com-
pounds having R2 ) CH2CHOHPh (19-32, 56-65, 72)
were good cathepsin B inhibitors with Ki values as low
as 63 nM (Z-Leu-Abu-CONH-CH2-CHOH-1-naphthyl,
31, or 2-naphthyl, 32 or 72 nM (Z-Leu-Abu-CONH-CH2-
CHOH-Ph(4-NMe2), 22). Conversely, heterocycles in P′
(33-50, 66-71, 73, 74) resulted in a decreased inhibi-
tory potency, although this was not observed with the
pyridyl (28, Ki ) 200 nM), indolyl (44, Ki ) 31 nM), or
tetrahydroquinolinyl (47, Ki ) 94 nM) derivatives.
Derivatives of imidazole, (CH2)n-4-morpholinyl, and
tetrahydroquinoline should be positively charged at the
pH of the inhibitory assay (pH 6). Extremely poor
inhibitory results were obtained with all positively
charged compounds in the P′ region (40-42, 48, 69, 70,
74), with Ki values as high as 13 µM.
Modification of the N-terminal blocking group of the
dipeptidyl R-keto amide chain led to rather small
increases (up to 3.4-fold) or decreases (up to 3.6-fold) in
the inhibitory potency of the R-keto amides. The best
result was obtained with 2-quinolinyl-CO-Leu-Abu-
CONH-Et (78, Ki ) 96 nM). As observed with calpain
II, no real preference for an Abu, Phe, or Nva residue
in the P1 position was observed.
Selectivity. Considerable specificity for calpain was
obtained in the dipeptide R-keto amides with Leu-Abu,
Leu-Phe, or Leu-Nva sequences in the P2-P1 position.
Serine proteases, such as R-chymotrypsin and pancre-
atic elastase, and cysteine proteases, papain and cathe-
psin B, are, in general, poorly inhibited. The specificity
of serine proteases is determined primarily by the P1
residue, with chymotrypsin preferring aromatic amino
acid residues and elastase, small hydrophobic residues.
Peptidyl R-keto amides having an Abu residue in P1
position (1-50, 75-86) are not inhibitors for R-chymo-
trypsin even at a high inhibitor concentration (150 µM).
Pancreatic elastase is inhibited by such compounds but
with poor Ki values, ranging from 10 µM for Z-Leu-Abu-
CONH-CH2-CHOH-Ph (19) up to 190 µM for Z-Leu-Abu-
CONH-CH2-C6H3(3,5-(OMe)2) (18). Peptidyl R-keto
amides with a Phe residue in the P1 position (51-72,
87-89) are not pancreatic elastase inhibitors at con-
centrations as high as 100 µM. Only a few of these P1
Phe derivatives have been tested with R-chymotrypsin,
and some are effective inhibitors with rather low Ki
values. For example, Z-Leu-Phe-CONH-CH2-CH2-Ph
(55) inhibits chymotrypsin with a Ki value of 0.8 µM.
Despite its high affinity toward R-chymotrypsin, com-
pound 55 remains principally, a calpain inhibitor, being
15- and 33-fold more active on calpains I and II,
respectively. In general, the lack of inhibitory potency
of dipeptidyl R-keto amides toward serine proteases can
be explained by the small size of the inhibitors, with
Cathepsin B has dipeptidyl carboxypeptidase activity
and interacts specifically with the C-terminal carboxyl
group at the P2′ subsite of such substrates. This dipep-
tidyl carboxypeptidase activity is due to the presence
of S2′ His-110 and His-111 residues which can form salt

4094 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Li et al.
pancreatic elastase in particular preferring longer pep-
tide chains.
The cysteine proteases cathepsin B and papain pref-
erentially cleave peptide substrates having a bulky
residue such as Phe in the P2 position and a small
hydrophobic residue in P1. Papain was, in general, very
poorly inhibited by dipeptide R-keto amides having a
Leu residue in P2 and a Phe or an Abu residue in P1,
with affinity values greater than 38 µM. Cathepsin B
was far more sensitive to their inhibitory potency, with
one Ki value as low as 31 nM (44). However, the main
target was calpains. Calpains preferentially cleave
peptide substrates having a bulky residue such as Phe,
Arg, Met, Abu, or Nva in the P1 position. Val or Leu
residues are favored in the P2.
F igu r e 2. Proposed interaction of a dipeptidyl R-keto amide
(Z-Leu-Abu-CONH-R2) with the active site of calpain.
Specificity for calpain I, calpain II, or both calpains
was obtained in various dipeptide R-keto amides having
a Leu residue in P2 and a Phe, Abu, or Nva residue in
P1. Among the R-keto amides specific for calpain I is
Z-Leu-Nva-CONH-CH2-2-pyridyl (73), having a Ki value
of 19 nM toward calpain I and being 6.3- and 40-fold
poorer with calpain II and cathepsin B, respectively.
Other derivatives are specific for calpain II. For ex-
ample, Z-Leu-Abu-CONH-CH2-CHOH-Ph (19), Z-Leu-
Abu-CONH-CH2-2-pyridyl (35), and Z-Leu-Abu-CONH-
CH2-C6H3 (3,5-(OMe)2) (18) have Ki values of 15, 17, and
22 nM toward calpain II and are 73-, and 38-, and 100-
fold more potent on calpain II than on calpain I,
respectively. They have respectively 25-, 180-, and 82-
fold poorer affinity for cathepsin B. Some R-keto
amides, such as Z-Leu-Abu-CONH-(CH2)3-2-tetrahy-
droisoquinolinyl (48) and Z-Leu-Phe-CONH-CH2-CHOH-
C6H3(3,4-(OCH2Ph)2) (65), are highly specific for both
calpains without discriminating between calpains I
and II.
F igu r e 3. Proposed interaction of a substrate with the active
site of cathepsin B.
morpholinyl (74), Z-Leu-Abu-CONH-(CH2)2-4-morpholi-
nyl (41), Z-Leu-Abu-CONH-CH2-2-tetrahydrofuryl (34),
and Z-Leu-Abu-CONH-(CH2)2-C6H4(4-OH) (14) dis-
played the highest membrane penetrance in this assay,
with IC50 values of 18-23 µM.
Con clu sion
A series of dipeptide R-keto amides with the general
structure R1-L-Leu-D,L-AA-CONH-R2 are effective in-
hibitors of cysteine proteases. The majority of the
inhibitors have inhibitory potency toward the cysteine
proteases tested in the order: calpain II > calpain I >
cathepsin B. In general, replacing the benzyloxycarbo-
nyl blocking group at the N-terminus did not increase
the potency of these inhibitors. None of the P1 amino
acid residues (Abu, Nva, Phe) examined was clearly
favored with calpain II or cathepsin B, although Nva
appeared to be the best choice for calpain I. Introduc-
tion of a hydrophobic group (i.e., phenyl ring) on the P′
side improved inhibitory potency toward calpain II
indicating a hydrophobic pocket at the S1′ site of the
enzyme. Introduction of heteroatoms on the P′ side of
the inhibitors increased potency toward cathepsin B but
had less affect with the calpains.
Stroke ranks third in the number of deaths in the
United States and is a major cause of disability. Pos-
tocclusion administration of the calpain keto amide
inhibitor Z-Leu-Abu-CONH-(CH2)3-4-morpholinyl (42)
gives a dose-dependent neuroprotective effect after focal
brain ischemia in rats. This suggests that calpain
proteolysis plays an important role in brain degenera-
tion associated with cerebral ischemic events. Selective
calpain inhibitors may provide a rational, novel, and
viable means of treating such neurodegenerative dis-
eases.
In h ibitor y Mech a n ism . The mechanism of inhibi-
tion of cysteine proteases by R-keto amides involves the
formation of a reversible enzyme-inhibitor complex
prior to the attack of the active site cysteine residue on
the keto carbonyl group of the R-keto amides. This leads
to the formation of a stable but reversible tetrahedral
hemithioketal adduct (Figure 2). In addition to interac-
tions with the S3-S1 subsites of the enzyme, we have
proposed that the active site histidine is hydrogen-
bonded to the carboxamide group.14 In the vicinity of
this hydrogen-bonding group, the S′ subsites contains
a hydrophobic region or regions which are interacting
with hydrophobic P′ nitrogen substituents in our inhibi-
tors. It is also likely that there are additional hydrogen-
bonding sites (donors or acceptors) in the S′ subsites,
since many of the more potent calpain inhibitors have
either hydrogen bond donor or acceptor groups (2-
pyridyl, CH2CHOHPh, etc.). However, this structural
feature is not required since many derivatives lacking
a hydrogen-bonding group are also potent calpain
inhibitors.
P la telet Mem br a n e P er m ea bility Assa y. The
membrane penetrance of a number of dipeptide R-keto
amide inhibitors was investigated. Treatment of rat
platelets with a calcium ionophore results in the eleva-
tion of intracellular calcium concentration, activation
of calpain, and calpain-mediated cleavage of the cyto-
skeletal proteins including spectrin. Using spectrin
cleavage as an indicator or calpain inhibition, the
membrane penetrance of the inhibitors can be inferred.
Z-Leu-Phe-CONH-Et (52), Z-Leu-Nva-CONH-(CH2)3-4-
Exp er im en ta l Section
Ch em istr y. Unless otherwise noted, materials were ob-
tained from commercial suppliers and used without further
purification. The purity of each compound was checked by
TLC, mp, 1H NMR, and MS. TLC was performed on Baker
Si250F silica gel plates. Melting points were obtained on a
Bu¨chi capillary apparatus and are not corrected. 1H NMR
spectra were determined on a Varian Gemini 300 spectrom-

R-Keto Amide Inhibitors of Calpains
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4095
Ta ble 3. Physical Properties of R-Keto Amides
no.
mp (°C)
% yielda (solvent)c
TLC Rf (solvent)d
formula
FAB MSb
anal.
1
4
5
6
7
125-127
151-154
122-123
103-105
99-102
100-103
59-61
151-153
101-103
99-100
152-155
153-155
152-154
128-129
170-172
104 dec
167-171
(ss)e
37 (B)
40 (C)
42 (C)
34 (C)
25 (B)
36 (B)
51 (C)
60 (C)
71 (A)
56 (A)
50 (A)
45 (A)
50 (C)
26 (F)
29 (C)
23 (E)
66 (C)
72 (C)
67 (C)
48 (B)
39 (B)
57 (C)
55 (B)
60 (C)
15 (H)
17 (F)
43 (B)
35 (B)
50 (C)
35 (C)
45 (C)
53 (C)
16 (A)
27 (C)
55 (C)
42 (C)
33 (C)
18 (A)
16 (H)
12 (H)
40 (A)
20 (B)
30 (B)
42 (D)
58 (B)
46 (C)
22 (C)
47 (B)
42 (B)
50 (B)
30 (A)
49 (B)
52 (B)
41 (B)
45 (B)
41 (B)
33 (H)
40 (A)
51 (B)
40 (C)
35 (D)
54 (C)
50 (C)
37 (C)
39 (A)
49 (D)
18 (C)
45 (H)
46 (H)
33 (C)
72 (A)
24 (C)
67 (A)
30 (A)
9 (J )
0.39 (B)
0.42 (C)
0.54 (C)
0.42 (C)
0.47 (B)
0.37 (B)
0.55 (C)
0.56 (C)
0.47 (A)
0.46 (A)
0.46 (A)
0.44 (A)
0.48 (C)
0.56 (H)
0.54 (C)
0.41 (E)
0.28 (C)
0.48 (C)
0.54 (C)
0.22 (B)
0.40 (B)
0.40 (C)
0.28 (B)
0.48 (C)
0.48 (H)
0.39 (H)
0.68 (C)
0.59 (B)
0.50 (C)
0.54 (C)
0.55 (C)
0.60 (C)
0.34 (A)
0.33 (C)
0.49 (C)
0.50 (C)
0.51 (C)
0.47 (A)
0.27 (H)
0.34 (H)
0.26 (B)
0.51 (B)
0.35 (C)
0.41 (D)
0.37 (B)
0.72 (C)
0.68 (C)
0.45 (B)
0.48 (C)
0.25 (B)
0.20 (A)
0.45 (B)
0.23 (B)
0.40 (B)
0.42 (B)
0.40 (B)
0.30 (H)
0.58 (B)
0.62 (C)
0.55 (C)
0.42 (D)
0.56 (C)
0.55 (C)
0.23 (C)
0.51 (C)
0.56 (C)
0.33 (C)
0.48 (H)
0.47 (H)
0.45 (C)
0.23 (A)
0.40 (C)
0.47 (A)
0.40 (H)
0.30 (C)
0.25 (C)
0.41 (L)
0.33 (L)
0.45 (L)
C
20H29N3O5
392
422
464
466
466
494
532
498
512
512
512
528
498
528
588
541
588
566
590.3378
590
604
658
658.2764
710
548
548
458
462
469
469
469
483
485
486
491
505
503
521.2745
519
519
551
551
584
647
454
560
603
650
628
652
652
666
720
721
772
531
581
613
613
567
709
512.3378
483.2656
519.3208
366
377
378
427
427
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
ndf
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
nd
C21H31N3O6
C24H37N3O6
C23H35N3O7
C23H35N3O7
C25H39N3O7
C29H45N3O6
C27H35N3O6
C28H37N3O6
C28H37N3O6
C28H37N3O6
C28H37N3O7
C27H35N3O6
C28H37N3O7
C30H41N3O9
C29H40N4O6
C27H30N3O6F5
C28H34N3O6F3
C33H39N3O7
C33H39N3O7
C34H41N3O7
C34H38N3O7F3
C33H37N3O7Cl2
C41H47N3O8
C31H37N3O6
C31H37N3O6
C24H31N3O6
C24H35N3O6
C25H32N4O5
C25H32N4O5
C25H32N4O5
C26H34N4O5
C26H36N4O5
C25H35N5O5
C25H38N4O6
C26H40N4O6
C26H38N4O6
C29H36N4O5
C29H34N4O5
C29H34N4O5
C31H42N4O5
C31H42N4O5
C28H37N7O7
C31H46N6O7S
C25H31N3O5
C32H37N3O6
C34H42N4O6
C32H32N3O6F5
C33H36N3O6F3
C38H41N3O7
C38H41N3O7
C39H43N3O7
C39H40N3O7F3
C38H39N3O7Cl2
C46H49N3O8
C30H34N4O5
C34H36N4O5
C36H44N4O5
C36H44N4O5
C31H42N4O6
C36H48N6O7S
C28H37N3O6
C26H34N4O5
C27H42N4O6
C18H27N3O5
C19H28N4O4
C18H27N5O4
C23H30N4O4
C23H30N4O4
C18H32N4O5
C22H33N3O4
C27H35N3O4
C25H33N3O4
C33H39N3O5
C31H36N4O4
C32H44N4O5
C38H41N3O5
C36H38N4O4
C37H46N4O5
8
10
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
(oil)
55-60
59-62
97-101
63-67
101-104
63-71
137-140
138-139
126-128
117-119
122-123
124-126
128-130
120-123
52-55
124-126
125-126
(ss)
(ss)
135-138
121-125
(oil)
(ss)
171-177 dec
188-192 dec
151-153
164-166
130 dec
191-192
(ss)
(ss)
146-149
133-134
142-143
136-137
149-152
144-146
131-135
115-120
107-111
155-156
204-206 dec
75-77
65-70
nd
nd
108-110
58-59
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
nd
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
57-61
51-56
56-59
104-106
(oil)
385.0209
404
467
440
558
529
565
620
591
134-136
78-83
201-203
178-180
161-163
170-174
192-196
160-162
158-160
25 (J )
16 (M)
9 (K)
20 (M)
627
a
b
Yields are for the conversion of the peptide keto ester to keto amide. Compounds which are oils or semisolids have high-resolution
mass spectral data (FAB), while the solids have C, H, and N analyses. c Solvent in the purification of the product by column chromatography.
Solvent used in the TLC. e ss ) semisolid. f nd ) not determined.
d

4096 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Ta ble 4. Proton NMR Data (CDCl3) for Selected Compoundsa
Li et al.
C6H5CH2OCO-NHb-CHc(R1)-CO-NHd-CHe(R2)-CO-CO-NHf-R3
R1 ) -CH2CH(CH3)2; R2 ) -C2H5 or -CH2C6H5
no.
Hb
Hc
Hd
He
Hf
other major signals
7
6.85, brd
5.26, m
5.36 brs
4.26, m
7.11, brs
5.12 s, 2H (OCH2Ph); 4.52, t, 1H (CH(OEt)2); 3.72, m, 2H;
3.54, m, 2H; 3.44, q, 2H; 1.22, t, 6H
16
19
22
23f
26
27
28
31
35
42
45
50
6.77, brs
6.30, brs
6.70, d
5.20, brs
5.17, m
5.18, m
5.05, m
5.15, m
5.10, m
5.10, m
5.45, m
5.15, m
5.15, m
5.22, brs
4.91, m
5.56, brd
5.25, m
5.18, m
5.96, m
5.25, m
5.20, m
5.20, m
5.68, m
5.29, m
5.25, brs
5.36, brs
7.66, d
4.20, m
4.22, m
4.20, m
4.11, m
4.22, m
4.22, m
4.21, m
4.28, m
4.22, m
4.21, m
4.26, m
4.12, m
7.07, brs
7.19, brs
6.70, d
5.09, s, 2H (OCH2Ph); 3.75, s, 3H (OCH3); 3.53, m, 2H (CH2N);
2.81, m, 2H( CH2CH2N)
5.11, s, 2H (OCH2Ph); 4.86, m, 1H (CHOH); 3.73 & 3.41,
d of m, 2H (CH2CHOH); 2.95, brs, 1H (OH)
5.10, s, 2H (OCH2Ph); 4.75, brs, 1H (CHOH); 3.65 & 3.38,
d of m, 2H (CH2CHOH); 2.95, s, 6H (N(CH3)2)
5.01, s, 2H (OCH2Ph); 4.86, brs, 1H (CHOH); 3.44 & 3.25,
d of m, 2H (CH2CHOH)
5.11, s, 2H (OCH2Ph); 4.86, brs, 1H (CHOH); 3.67 & 3.41,
d of m, 2H (CH2CHOH)
5.11, s, 2H (OCH2Ph); 5.06, s, 4H (OCH2Ph); 4.84, brs,
1H (CHOH); 3.70 & 3.39, d of m, 2H (CH2CHOH)
5.11, s, 2H (OCH2Ph); 4.88, brs, 1H (CHOH); 3.70 & 3.40,
d of m, 2H (CH2CHOH)
5.25, brs, 1H (CHOH); 5.07, s, 2H (OCH2Ph); 3.96 & 3.36,
d of m, 2H (CH2CHOH)
8.57, d, 1H; 8.02, s, 1H; 7.68, t, 1H; 7.34, m, 6H; 5.11, s,
2H (OCH2Ph); 4.59, d, 2H (CH2N)
5.12, s, 2H (OCH2Ph); 3.77, t, 4H (CH2O); 3.42, m,
2H (CH2N); 2.48, t, 6H (CH2N)
8.18, brs
6.77, brs
6.94, brd
6.72, brs
6.99, brs
6.68, brd
6.62, brd
6.77, brd
7.96, m
8.69, brs
7.12, s
7.04, s
7.09, s
7.05, brs
7.21, brs
8.71, brs
8.47, brs
8.14, m, 2H; 7.81, d, 1H; 7.74, t, 1H; 7.56, t, 1H; 7.34, m, 6H;
5.13, s, 2H (OCH2Ph); 4.78, d, 2H (CH2N)
8.68, d
8.26, m
6.43, d, 1H; 6.36, s, 1H; 4.29, t, 1H; 4.12, m, 1H; 3.07, m, 1H;
2.82, d of d, 1H; 2.56, s, 1H (H of the ring of biotin);
3.20-3.07, m, 4H (NH(CH2)2NH); 2.09, t, 2H (NHCOCH2)
5.09, s, 2H (OCH2Ph); 4.91, brs, 1H (CHOH); 3.73 & 3.44,
d of m, 2H (CH2CHOH); 3.31 & 3.10, d of m, 2H (Phe)
5.35, s, 2H (OCH2Ph); 5.11, brs, 1H (CHOH); 3.98 & 3.68,
d of m, 2H (CH2CHOH); 3.56 & 3.33, d of m, 2H (Phe)
7.68, s, 1H; 7.58, t, 2H; 7.50, m, 1H; 7.36, s, 5H; 5.08, s,
2H (OCH2Ph); 3.75 & 3.35, d of m, 2H (CH2CHOH);
3.28 & 3.08, d of m, 2H (Phe)
56
61
63
6.54, brs
6.88, brs
6.58, brs
5.04, brs
5.40, m
5.35, m
5.50, m
5.74, m
5.45, m
4.15, m
4.40, m
4.12, m
7.09, brs
7.26, brs
7.11, brs
67
6.75, brs
5.22, m
5.64, m
4.20, m
8.47, brs
8.13, d of d, 2H; 7.81, d, 1H; 7.73, t, 1H; 7.56, t, 1H; 7.40-7.15, m,
10H; 7.10, d, 1H; 4.78, d, 2H (CH2-C9H6N); 3.36 & 3.13,
d of m, 2H (Phe)
70
71
6.51, brs
7.87, m
5.03, brs
5.22, m
5.56, m
5.05, m
4.16, m
4.10, m
8.78, brs
3.72, t, 4H (CH2O); 3.44, q, 2H (CH2N); 3.37 & 3.13, d of m,
2H (Phe); 2.46, m, 6H (CH2N)
6.41, s, 1H; 6.35, s, 1H; 4.28, t, 1H; 8.32, d of d; 4.10, m, 1H;
3.07, m, 1H; 2.78, d of d, 1H; 2.55, s, 1H (H of the ring of biotin);
3.20, m, 4H (NH(CH2)2NH); 2.06, t, 2H (NHCOCH2)
9.40, d, 1H; 8.78, s, 1H; 8.57, s, 1H
9.52, d, 1H; 8.66, t, 1H; 8.46, d, 1H; 7.81, m, 1H; 7.66, m, 2H
3.32, q, 2H (NCH2); 2.92, t, 2H (CH2CO); 2.50, t, 2H (CH2Ph);
1.20, t, 3H (NCH2CH3)
8.73, d of d
77
79
81
7.11, d
7.37, m
6.78, brs
5.30, m
5.33, m
5.22, m
7.11, d
7.12, brd
5.92, brs
4.81, m
4.87, m
4.48, m
8.23, brs
7.81, m
6.90, brs
a
Solvent was DMSO-d6.
eter. Chemical shifts are expressed in ppm relative to internal
tetramethylsilane. Mass spectra were obtained on a Varian
MAT 112S spectrometer. Column chromatography was per-
formed on silica gel (32-63 µm) using the following solvent
systems: A, CHCl3:CH3OH ) 30:1; B, CHCl3:CH3OH ) 20:1;
C, CHCl3:CH3OH ) 10:1; D, CHCl3:CH3OH ) 5:1; E, AcOEt:
hexane ) 6:1; F, AcOEt:hexane ) 3:2; G, AcOEt:hexane ) 2:1;
H, AcOEt:hexane ) 1:1; I, butanal:AcOH:H2O ) 4:1:1; J ,
CHCl3:AcOEt ) 7:3; K, CHCl3:AcOEt ) 9:1; L, AcOEt:CH3-
OH ) 9:1; M, AcOEt.
Abu sequence.14 The synthesis of 92 was described earlier.14
Thus, after a mixture of 92 (1,3-dithiolane derivative of Z-Leu-
Abu-CO2Et; 1 g, 2.07 mmol) and the amine (6.7 g, 61.9 mmol)
in ethanol (5 mL) was stirred overnight at room temperature,
the solvent was evaporated and AcOEt (50 mL) was added.
This mixture was filtered to remove a white precipitate. The
filtrate was washed with water (4 × 20 mL) and saturated
NaCl (2 × 20 mL) and dried over MgSO4. Chromatography
on a silica gel column using solvent C followed by precipitation
from AcOEt/hexane afforded a yellow solid.
The physical properties of the peptide R-keto amides inhibi-
tors are given in Table 3. Proton NMR data for the inhibitors
are given in Table 4.
Z-Leu -Abu -CONH-CH2-2-p yr id yl (35): Gen er a l P r oce-
d u r e for th e Syn th esis of Keto Am id es fr om Keto Ester s.
This procedure was used for the synthesis of compounds 1-48,
51-70, and 72-74 using the route shown in Scheme 1 (91 f
92 f 94). The procedure was also used as the final steps in
the synthesis of 81-89.
Z-Leu-Abu-CONH-CH2-2-pyridyl was synthesized from the
1,3-dithiolane derivative of Z-Leu-Abu-COOEt (92, R ) Me,
R1 ) Z) and 2-(aminomethyl)pyridine as described earlier for
the synthesis of other keto amide derivatives with the Z-Leu-
Z-Leu -Abu -CONH-CH2-8-ca ffein yl (49) via th e Keto
Acid Usin g CDI. Z-Leu-Abu-CONH-CH2-8-caffeinyl was
prepared from Z-Leu-Abu-COOH (93, R ) Me, R1 ) Z)14 and
8-(aminomethyl)caffeine (Scheme 1, 93 f 94).23 Thus, CDI
(0.32 g, 1.98 mmol) was added to a solution of Z-Leu-Abu-
COOH (0.5 g, 1.32 mmol) in DMF (5 mL), and the mixture
was stirred for 2 h at room temperature. The reaction mixture
was then added to a stirred suspension of 8-(aminomethyl)-
caffeine (0.36 g, 1.58 mmol) in DMF (20 mL) and the reaction
mixture stirred for 3 days at room temperature. After
evaporating DMF, AcOEt (30 mL) and water (10 mL) were
added, and the AcOEt layer was washed with H2O (5 × 20
mL) and saturated NaCl (2 × 20 mL), dried over MgSO4, and

R-Keto Amide Inhibitors of Calpains
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20 4097
concentrated. Chromatography on a silica gel column using
solvent B followed by precipitation from AcOEt/hexane af-
forded a yellow solid.
(AA ) Abu; 4.62 g, 30 mmol) in the presence of TEA (4.22 mL,
30 mmol), HOBt (3.4 g, 25 mmol), and DCC (6.7 g, 32.5 mmol)
in DMF for 16.5 h at room temperature to give the dipeptidyl
methyl ester R1-Leu-AA-OMe (1-C10H7CH2-CO-Leu-Abu-
OMe): 1H NMR (CDCl3) 0.65-0.89 (m, 9H), 1.10-1.90 (m, 5H),
3.72 (s, 3H), 3.95-4.14 (m, 2H), 4.43 (dd, 2H), 5.70 (d, 1H),
6.59 (dd, 1H), 7.39-7.55 (m, 4H), 7.79-7.98 (m, 3H).
The dipeptidyl acid (R ) Me, R1 ) 1-C10H7CH2-CO) was
quantitatively obtained by hydrolysis of the corresponding
dipeptidyl methyl ester with NaOH in methanol followed by
the Dakin-West procedure to give the dipeptidyl enol ester
90.14 Subsequent hydrolysis by NaOEt and protection of the
R-keto group with 1,2-ethanedithiol gave the corresponding
R-keto ester 91 and its 1,3-dithiolane derivative 92.14
The final product 83 (1.13 g, 2.59 mmol, 67% yield) was
prepared by reacting 92 (2 g, 3.87 mmol) with ethylamine (3.5
g, 77 mmol) under the reaction conditions described earlier
for 35.
Bioch em istr y. The materials, enzymes, enzyme kinetic
assays, and platelet membrane permeability assay have been
described previously.14 With calpain, initial velocities (t ) 30-
60 s) of Suc-Leu-AMC hydrolysis were determined at room
temperature after a delay of 30 s. Five or more concentrations
of inhibitor (not exceeding 2 × Kobs) and two fixed concentra-
tions of substrate were utilized. Ki values were determined
by Dixon plots. The average of triplicate assays, plotted as
1/v versus I, gave intersecting lines with a correlation coef-
ficient g0.95. No other attempt was made to correct for slow
binding or autolysis. The intra-assay variation in the platelet
membrane assay is approximately 5%. An internal standard
was always run to control for inter-assay variation. The total
variance in this assay is less than 15%.
Z-Leu -Abu -CONH-(CH2)2NH-biotin yl (50) via th e Keto
Acid Usin g DCC/HOBt: P r oced u r e for Com p ou n d s 50
a n d 71. Biotin (1 g, 4.1 mmol) was dissolved in 20 mL of DMF
at 70 °C and then cooled to 40 °C, CDI (0.97 g, 6 mmol) in 3
mL of DMF was added, and a white precipitate appeared. After
the mixture was stirred at room temperature for 2 h, ethyl-
enediamine (1.34 mL, 20 mmol) in 10 mL of DMF was added
and stirring continued for another 3 h. After evaporating the
DMF, the semisolid residue was dissolved in 50 mL of refluxing
methanol and the unreacted biotin was removed by filtration.
The solution was evaporated to dryness. The residue was
washed with CHCl3 to remove the imidazole and then dissolved
in 6 mL of water, acidified to pH 3.0 with 1 N HCl, and
evaporated to dryness. The crude biotinylethylenediamine
hydrochloride was crystallized from methanol to give 1.04 g
of biotinylethylenediamine hydrochloride (81% yield): long
spot on TLC, Rf ) 0.21 (I); mp 241-242 °C; 1H NMR (DMSO-
d6) 8.05 (brs, 1H), 7.86 (brs, 3H), 6.42 (brd, 2H), 4.31 (t, 1H),
4.12 (t, 1H), 3.25 (m, 2H), 3.09 (m, 1H), 2.83 (m, 3H), 2.56 (d,
1H), 2.05 (t, 2H), 1.53-1.28 (m, 6H).
To a stirred solution of Z-Leu-Abu-CO2H (93, R ) Me, R1 )
Z; 0.6 g, 1.58 mmol)14 in DMF (15 mL) were added HOBt (0.22
g, 1.58 mmol) and DCC (0.49 g, 2.38 mmol), and stirring was
continued for 2 h at room temperature. TEA (0.28 mL, 2.03
mmol) was added to a stirred solution of biotinylethylenedi-
amine hydrochloride (0.6 g, 1.85 mmol) in DMF (10 mL) at
0-5 °C and stirred for 2 h at room temperature. This solution
was then added to the DCC/HOBt reaction mixture and stirred
for 3 days. After filtration, the filtrate was evaporated to get
a semisolid which was washed with H2O (30 mL), 1 M HCl
(30 mL), and H2O (30 mL) and dried under vacuum. Chro-
matography on a silica gel column using solvent D afforded a
yellow solid.
Su p p or tin g In for m a tion Ava ila ble: Inhibition studies
of peptidyl R-keto amides on porcine pancreatic elastase,
bovine R-chymotrypsin, and papaya latex papain (3 pages).
Ordering information is given on any current masthead page.
2-F u r yl-CO-Leu -Abu -CONHEt (75): Gen er a l P r oced u r e
for Com p ou n d s 75-80. A solution of hydrogen bromide in
acetic acid (30 wt %, 1.52 mL, 7.40 mmol) was added to Z-Leu-
Abu-CONHEt (2; 1 g, 2.47 mmol) at room temperature. The
mixture was vigorously stirred for 1 h, during which all the
keto amide dissolved in the acetic acid. The reaction was
quenched with Et2O (30 mL), and then the mixture was
filtered. The semisolid product was triturated and washed
successively with Et2O (5 × 30 mL). After removal of solvent,
the Leu-Abu-CONHEt‚HBr was dried under vacuum, leaving
a very hygroscopic solid (70% yield). 1H NMR (CDCl3) showed
the loss of the Z group.
DCC (0.44 g, 2.13 mmol) and HOBt (0.29 g, 2.13 mmol) were
added to a stirred solution of 2-furoic acid (0.24 g, 2.13 mmol)
in DMF (10 mL), and the mixture was stirred for 2 h at room
temperature. TEA (0.2 mL, 1.42 mmol) was added to a stirred
solution of Leu-Abu-CONHEt‚HBr (0.5 g, 1.42 mmol) in DMF
(5 mL) at 0-5 °C and stirred for 3 min. This solution was
then added to the DCC/HOBt reaction mixture at 0-5 °C and
stirred overnight at room temperature. After evaporating
DMF and adding AcOEt (40 mL), the precipitate was filtered,
and the filtrate was washed with 0.25 M HCl (10 mL), H2O
(20 mL), 10% Na2CO3 (3 × 20 mL), H2O (20 mL), and saturated
NaCl (2 × 20 mL), dried over MgSO4, and concentrated.
Chromatography on a silica gel column with solvent C afforded
a yellow solid.
1-C10H7CH2-CO-Leu -Abu -CONHEt (83): Gen er a l P r o-
ced u r e for Com p ou n d s 81-89. Leucine methyl ester
hydrochloride (3.9 g, 26.8 mmol) was acylated with R1-OH (R1
) 1-C10H7CH2-CO; 5 g, 26.8 mmol; R1 ) Ph2CHCO or PhCH2-
CH2CO for 81, 82, 84-89) in the presence of TEA (3 g, 29.7
mmol) and CDI (4.8 g, 29.5 mmol) by stirring the mixture in
DMF (20 mL) for 2 hours at room temperature to form R1-
Leu-OMe (7.5 g, 23.96 mmol, 89% yield): 1H NMR (CDCl3)
0.70 (d, 3H), 0.76 (d, 3H), 1.26 (m, 2H), 1.46 (m, 1H), 3.61 (s,
3H), 4.04 (m, 2H), 4.59 (m, 1H), 5.61 (d, 1H), 7.46-7.53 (m,
4H), 7.86-7.96 (m, 3H).
Refer en ces
(1) Saido, T. C.; Sorimachi, H.; Suzuki, K. Calpain: New Perspec-
tives In Molecular Diversity and Physiological-Pathological
Involvement. FASEB J . 1994, 8, 814-822.
(2) Seubert, P.; Ivy, G.; Larson, G.; Lee, J .; Stahi, K.; Baudry, M.;
Lynch, G. Lesions of Entorhinal Cortex Produce a Calpain-
Mediated Degradation of Brain Spectrin in Dentate Gyrus. I.
Biochemical Studies. Brain Res. 1988, 459, 226-232.
(3) Arai, A.; Vanderklish, P.; Kessler, M.; Lee, K.; Lynch, G. A. Brief
Period of Hypoxia Causes Proteolysis of Cytoskeletal Proteins
in Hippocampal Slices. Brain Res. 1991, 555, 276-280.
(4) Wang, K. K. W.; Yuen, P.-W. Calpain Inhibition: An Overview
of Its Therapeutic Potential. Trends Pharmacol. Sci. 1994, 15,
412-419.
(5) Hong, S.-C.; Goto, Y.; Lanzino, G.; Soleau, S.; Kassel, N. F.; Lee,
K. S. Neuroprotection with a Calpain Inhibitor in a Model of
Focal Ischemia. Stroke 1994, 25, 663-669.
(6) Bartus, R. T.; Haywood, N. J .; Elliott, P. J .; Sawyer, S. D.; Baker,
K. L.; Dean, R. L.; Akiyama, A.; Straub, J . A.; Harbeson, S. L.;
Li, Z.; Powers, J . C. Calpain Inhibitor AK295 Protects Neurons
From Focal Brain Ischemia. Stroke 1994, 25, 2265-2270.
(7) Bartus, R. T.; Baker, K. L.; Heiser, A. D.; Sawyer, S. D.; Dean,
R. L.; Elliott, P. J .; Straub, J . A. Postischemic Administration
of AK275, A Calpain Inhibitor, Provides Substantial Protection
Against Focal Ischemic Brain Damage. J . Cereb. Blood Flow
Metab. 1994, 14, 537-544.
(8) Wikstrom, P.; Anagli, J .; Angliker, H.; Shaw, E. Additional
Peptidyl Diazomethyl Ketones, Including Biotinyl Derivatives,
Which Affinity-Label Calpain and Related Cysteinyl Proteinases.
J . Enzyme Inhib. 1993, 6, 259-269.
(9) Angliker, H.; Anagli, J .; Shaw, E. Inactivation of Calpain by
Peptidyl Fluoromethyl Ketones. J . Med. Chem. 1992, 35, 216-
220.
(10) Huang, Z.; McGowan, E. B.; Detwiler, T. C. Ester and Amide
Derivatives of E64c as Inhibitors of Platelet Calpains. J . Med.
Chem. 1992, 35, 2048-2054.
(11) Sasaki, T.; Kishi, M.; Saito, M.; Tanaka, T.; Higushi, N.;
Kominami, E.; Katunuma, N.; Murachi, T. Inhibitory Effect of
Di- and Tripeptidyl Aldehydes on Calpains and Cathepsins. J .
Enzyme Inhib. 1990, 3, 195-201.
(12) Angelastro, M. R.; Mehdi, S.; Burkhart, J . P.; Peet, N. P.; Bey,
P. R-Diketones and R-Keto Ester Derivatives of N-Protected
Amino Acids and Peptides as Novel Inhibitors of Cysteine and
Serine Proteinases. J . Med. Chem. 1990, 33, 11-13.
(13) Hu, L.-Y.; Abeles, R. H. Inhibition of Cathepsin B and Papain
by Peptidyl R-Keto Esters, R-Keto Acids, R-Diketones, and
R-Keto Amides. Arch. Biochem. Biophys. 1990, 2, 271-274.
The methyl ester was then hydrolyzed with 1.2 equiv of
NaOH in methanol (30 mL) for 3 h at room temperature to
give a quantitative yield of R1-Leu-OH (R1 ) 1-C10H7CH2-CO;
7.16 g, 23.96 mmol) followed by coupling with AA-OMe‚HCl

4098 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Li et al.
(14) Li. Z. Z.; Patil, G. S.; Golubski, Z. E.; Hori, H.; Tehrani, K.;
Foreman, J . E.; Eveleth, D. D.; Bartus, R. T.; Powers, J . C.
Peptide R-Ketoesters, R-Ketoamides and R-Ketoacids Inhibitors
of Calpains and Other Cysteine Proteases. J . Med. Chem. 1993,
36, 3472-3480.
(15) Harbeson, S. L.; Abelleira, S. M.; Akiyama, A.; Barret, R., III;
Carroll, R. M.; Straub, J . A.; Tkacz, J . N.; Wu, C.; Musso, G. F.
Stereospecific Synthesis of Peptidyl R-Keto Amides as Inhibitors
of Calpain. J . Med. Chem. 1994, 37, 2918-2929.
(16) We have used the subsite nomenclature of Schechter and Berger
(Schechter, I.; Berger, A. On the Size of the Active Site in
Protease. 1. Papain. Biochim. Biophys. Res. Commun. 1967,
27, 157-162) to describe the individual residues of the inhibitors
and the corresponding subsites of the enzyme.
(17) Saatman, K. E.; Murai, H.; Bartus, R. T.; Smith, D. H.; Hayward,
N. J .; Perri, B. R.; McIntosh, T. K. Calpain inhibitor AK295
attenuates motor and cognitive deficits following experimental
brain injury in the rat. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
3428-3433.
(18) Abbreviations: Abu, R-aminobutyric acid; AcOEt, ethyl acetate;
n-Bu, n-butyl; Bzl, benzyl; -C6H7 (1,3,3-(CH3)3-5-OH), 1,3,3-
trimethyl-5-hydroxylcyclohexyl; 8-caffeinyl, 8-substituted deriva-
tive of caffeine or 8-substituted 1,3,7-trimethylxanthine; Cal I,
calpain I; Cal II, calpain II; Cat B, cathepsin B; CDI, 1,1-
carbonyldiimidazole; DCC, dicyclohexylcarbodiimide; HOBt,
1-hydroxybenzotriazole; Pap, papain; Ph, phenyl; TEA, triethyl-
amine; TLC, thin layer chromatography; Z, benzyloxycarbo-
nyl. The peptidyl R-keto amides RCO-L-Leu-D,L-NHCH(R′)-
CO-CONHR′′ are abbreviated as R1-L-Leu-D,L-AA-CONHR2 or
simply as R1-Leu-AA-CONHR2.
(19) Evans, D. A.; Carroll, G. L.; Truesdale, L. K. Synthetic Applica-
tions of Trimethylsilyl Cyanide. An Efficient Synthesis of
â-Aminomethyl Alcohols. J . Org. Chem. 1974, 39, 914.
(20) Nagata, M.; Yamazaki, T. Synthesis in Azabenzo[1,2-a]quinoline.
Yakugaku Zasshi 1963, 83, 679-682.
(21) Zimmer, H.; Glasgow, D. G.; McClanahan, M.; Novinson, T. Imid-
azo[5,1-a]isochinoline. Tetrahedron Lett. 1968, 24, 2805-2807.
(22) Aroyan, A. A.; Azaryan, A. S.; Iradyan, M. A. Quinoline deriva-
tives. IV. Some reactions of 1,2,3,4-Tetrahydroquinoline. Izv.
Akad. Nauk Arm. SSR, Khim. Nauki 1965, 18 (1), 76-82.
(23) Yonan, P. 3-Substituted 1-(1,2,3,4-Tetrahydro-2-isoquinolyl)al-
kyl-2-thioureas. U.S. Patent 3,245,997 (Cl. 260-288), April 12,
1966.
(24) (a) Rybar, A.; Antos, K. 1,3,7-Trisubstituted 8-Isothiocyanatom-
ethylxanthine. Collect. Czech. Chem. Commun. 1970, 35, 1415-
1433. (b) Golovchinskava, E. S.; Chaman, E. S. The 8-Substi-
tution Derivatives of Methylated Xanthines. J . Gen. Chem.
1952, 22, 599-603. (c) Nantka-Namirski, P.; J arymowicz, B.;
Wojciechowski, J . Syntheza 7-Podstawionych Pochodnych 8-Ami-
nometyl-1,3-dwumetyloksantyny. Acta Pol. Pharm. 1974, 1,
5-12.
(25) Musil, D.; Zucic, D.; Turk, D.; Engh, R. A.; Mayr, I.; Huber, R.;
Popovic, T.; Turk, V.; Towatari, T.; Katunuma, N.; Bode, W. The
refined 2.15 Å X-ray structure of human liver cathepsin B: The
structural basis of specificity. EMBO J . 1991, 10, 2321-2330.
J M950541C