Exploration of orally available calpain inhibitors 2: Peptidyl hemiacetal derivatives

Article abstract of DOI:10.1021/jm060157n

We previously reported a potent calpain inhibitor 1 (SJA6017, N-(4-fluorophenyl)-L-valyl-L-leucinal), which displayed relatively low oral bioavailability (BA). Replacing the metabolically labile aldehyde moiety of 1 with more chemically stable warheads, s

Full text of DOI:10.1021/jm060157n

3926
J. Med. Chem. 2006, 49, 3926-3932
Exploration of Orally Available Calpain Inhibitors 2: Peptidyl Hemiacetal Derivatives
Yoshihisa Shirasaki,* Masayuki Nakamura, Masazumi Yamaguchi, Hiroyuki Miyashita,† Osamu Sakai, and Jun Inoue
Senju Pharmaceutical Company, Ltd., 1-5-4 Murotani, Nishi-ku, Kobe, Hyogo 651-2241, Japan
ReceiVed February 11, 2006
We previously reported a potent calpain inhibitor 1 (SJA6017, N-(4-fluorophenyl)-L-valyl-L-leucinal), which
displayed relatively low oral bioavailability (BA). Replacing the metabolically labile aldehyde moiety of 1
with more chemically stable warheads, such as a cyclic hemiacetal, hydrazone, and R-ketoamide, provided
the inhibitors with improved in vitro metabolic stability. Cyclic hemiacetal 2 was the most stable of these
compounds. The optimization of 2 led to hemiacetal 8 (SNJ-1715) which exhibited high potency, good
aqueous solubility, excellent oral BA, and prolonged plasma half-life in rats. Furthermore, 8 showed
neuroprotective efficacy via oral administration in a rat retinal ischemia model.
Introduction
Calpains, which are activated by calcium ions, are nonlyso-
somal cytosolic cysteine endoproteases, and several isozymes
have been reported.1 Two major isoforms, µ-calpain (calpain
I) and m-calpain (calpain II), are ubiquitously found in mam-
malian cells. These enzymes are implicated in a variety of
biological processes and in numerous diseases, such as muscular
dystrophy, cardiac ischemia, cataract, stroke, Alzheimer’s
disease, central nervous system diseases, spinal cord injury, and
traumatic brain injury (TBI).2 Therefore, the development of
novel agents that target calpains could potentially offer a means
of treating these diseases.
Figure 1. Main metabolic pathway of 1.
Most calpain inhibitors consist of an active site (warhead)
solution in the eye cannot readily deliver drugs into the retina
and a di or tripeptide-based backbone that is structurally related
because several barriers lie between the retina and the surface
area of the eye.20 Oral administration of 1 showed efficacy in
this model, but a relatively high dosage (500 mg/kg × 2) is
required.21 Hence, this phenomenon would limit its potential
as a therapeutic agent for these diseases.
to the cleavage site of the substrates (Arg, Phe, Nle or Leu at
the P1 site and Leu or Val at the P2 site).3-5 Calpain inhibitors
are categorized into irreversible and reversible inhibitors by their
mechanism of action.3 Irreversible inhibitors have an epoxide,
R-haloketone, vinyl sulfone, or diazomethyl ketone moiety as
warheads. These react with the SH group in the catalytic center
of the enzymes and form irreversible covalent adducts.6
However, aldehydes, R-ketoesters, R-ketoacids, and R-ketoa-
mides act as reversible warheads and form a hemithioacetal or
ketal with the SH group.6 From the viewpoint of drug discovery,
previous studies of calpain inhibitors have focused on reversible
inhibitors because irreversible inhibitors may cause unexpected
adverse effects. A number of reversible calpain inhibitors have
been identified.3 Some, such as leupeptin,7 calpain inhibitor I,8
MDL 28170,9 AK295,10 A-705239,11 and SJA6017 (1),12 have
shown neuroprotective efficacy in animal ischemia models.13-18
Our group has previously reported that neuronal cell death
occurs because of the activation of calpains in a rat retinal
ischemia-reperfusion model. A potent calpain inhibitor, dipep-
tidyl aldehyde 1, showed neuroprotective efficacy in this model
when 3 mg/kg was administrated intravenously.19 Thus, 1 may
serve as a useful agent for the treatment of retinal neurological
disorders such as glaucoma and retinitis pigmentosa. Because
these retinal disorders are usually chronic diseases, the develop-
ment of an oral drug is desirable because intravenous admin-
istration (iv) would be inconvenient. Instillation of an ophthalmic
A pharmacokinetic (PK) study of 1 revealed that it showed
poor bioavailability and very high total clearance in rats. We
have reported that 1 possesses moderate Caco-2 membrane
permeability, whereas both its in vitro metabolic stability against
the human liver S9 fraction (S9) and its water solubility are
low.22 Because aldehyde 1 is rapidly metabolized to the
corresponding alcohol M1 and carboxylic acid M2 in human
S9 in vitro and rats in vivo (Figure 1), the aldehyde moiety
was a major metabolic soft spot. Assuming that metabolism is
the major route of clearance, the conversion of the aldehyde
moiety into more chemically stable warheads could lead to the
generation of compounds with improved pharmacokinetics.
In this article, we describe the design and synthesis of novel
compounds based on the modifications of the metabolic soft
spot of 1 into various alternative warheads and describe further
optimization studies to maximize potency. This article also
reports the results of pharmacokinetic studies and the neuro-
protective efficacy of a representative compound in the retinal
ischemia model in rats.
Chemistry
We have reported the synthesis of hemiacetal 2,23 hydrazone
derivative 3,22a and ketoamide 4.23 As shown in Scheme 1, nitrile
5 was synthesized from condensation of N-(4-fluorophenyl)-L-
valine succinimide ester (6) and L-leucine amide following
dehydration of the resulting dipeptidyl amide 7 with trifluoro-
* To whom correspondence should be addressed. Tel.: +81-78-997-
1010. Fax: +81-78-997-1016. E-mail: shirasaki@senju.co.jp.
† Current address: Faculty of Pharmaceutical Sciences, Kumamoto
University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
10.1021/jm060157n CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/26/2006

Orally AVailable Calpain Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3927
Scheme 1a
a Conditions: (a) L-Leu-NH2‚HCl, EtOAc; (b) (CF3CO2)2O, TEA, THF.
Table 1. Inhibitory Activities against Calpains, Solubility and
Metabolic Stability of Aldehyde 1 and Its Analogs 2-5
Table 2. Inhibitory Activities against Calpains, Solubility and
Metabolic Stability of Hemiacetal Derivatives
metab.
structure
R2
IC50 (µM)a
µ-calpain m-calpain (mg/mL)
solubilityb stab.c
compd P2
n
(%)
8d
9
Leu
Val
Ile
Leu
Leu 4-F
Leu 4-MeO
Leu 3-CN
Leu 4-CN
Leu 2-Me
Leu 3-Me
Leu 4-Me
H
H
H
H
0
0
0
1
0
0
0
0
0
0
0
0.086d
0.63
0.60
0.15
0.15
0.22
0.26
0.14
0.24
0.18
0.91
0.88
0.19d
1.9
1.7
1.5
NDe
NDe
0.60
3.3
2.4
1.5
2.2
1.7
100
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
NDe
79
10
11
12
13
14
15
16
17
18
2
0.24
0.19
0.20
0.17
0.12
0.25
0.34
0.17
1.2
1.3
1.1
1.5
a IC50 values are reported as the mean of two independent determinations;
errors are within (20%. b Aqueous solubility in pH 7 buffer at 25 °C (n )
1). c Metabolic stability represented in residual percent after the incubation
with human hepatic S9 fraction for 0.5 h at 37 °C (n ) 1). d Reference 23.
e Not determined.
a IC50 values are reported as the mean of two independent determinations;
errors are within (20%. b Aqueous-solubility in pH 7 buffer, at 25 °C (n
) 1). c Metabolic stability represented in residual percent after incubation
with human hepatic S9 fraction for 0.5 h at 37 °C (n ) 1). d Reference 23.
e Reference 22a. f Not determined. g Reference 12.
Table 3. In Vitro PK Properties of Hemiacetal 8 and Aldehyde 1
metab.
stab.a
(%)
Caco-2b
acetic anhydride and Et3N.24 The synthesis of hemiacetal 8 (SNJ-
c
Papp
compd
(10-6 cm‚s-1
)
log D7
d
23
1715) was also reported previously and hemiacetals 9-18
were prepared by the same method (Table 3, Scheme 2). Boc-
protected amino acids (Leu, Val, and Ile) were coupled to
homoserine lactone to give intermediates 19-21. The interme-
diates were deprotected with 4 M HCl/EtOAc, and the resulting
crude amine hydrochloride salts were reacted with various
isothiocyanates to give the thiourea intermediates. These were
reduced with DIBAL-H, and the resulting compounds were
purified by preparative HPLC to provide hemiacetals 9-18 as
a mixture of anomers.
8
1
100
3
14.3
2.6
0.70
1.7
a Metabolic stability represented in residual percent after the incubation
with human hepatic S9 fraction for 0.5 h at 37 °C (n ) 1). b The Caco-2
cell monolayer permeability assay (n ) 1) errors of standard compound
c
(propranolol, n ) 3) are within (5%. Papp is the apparent permeability
coefficient for the apical to basolateral flux in 10-6 cm/s. d Reference 23.
The corresponding analogues (2-5)22a,23 of aldehyde lead 1
were evaluated for their metabolic stability against human S9
(Table 2). Cyclic hemiacetal 2,23 hydrazone 3,22a and R-ketoa-
mide 423 demonstrated significantly higher metabolic stability
in human S9 than that in 1. Hemiacetal 2 was the most
metabolically stable inhibitor in this series and showed good
aqueous solubility. However, the activities were about 50-fold
lower than that of 1. The increase in metabolic stability and
the decrease in potency may result from a reduction in both the
electrophilicity of the R-carbonyl moiety and the lipophilicity.
The decrease in electrophilicity led to a suppression of the
nucleophilic attack from both calpains and metabolic enzymes.27
A reduced lipophilicity diminished the affinity of both enzymes
by a decrease in hydrophobic interactions.28 Hydrazone 3 also
exhibited good metabolic stability presumably due to a decrease
in electrophilicity and a high water solubility in acidic condi-
tions. However, it was found to rapidly decompose to aldehyde
1 in simulated gastric juice (pH 1.2). Ketoamide 4 showed
improved metabolic stability and very potent inhibitory activity,
but it had extremely low aqueous solubility. Nitrile 5 was also
Results and Discussion
The aldehyde functionality is an essential active site for
hemithioacetal formation by reaction with the SH group on the
Cys residue of calpains. The intrinsic instability of aliphatic
aldehydes renders them problematic as electrophilic warheads.
It is, therefore, desirable to replace the aldehyde with other
electrophilic warheads that can reversibly react with the SH
group but are more chemically stable than aldehydes. R-Ke-
toacids, R-ketoesters, R-ketoamides, hydrazones, nitriles, and
masked aldehydes such as hydroxyoxazolidines and cyclic
hemiacetals have been reported as reversible warheads for
calpain inhibitors.3,25 R-Ketoacids show very high potency, but
generally low membrane permeability.25a,26 Most R-ketoesters
would be rapidly metabolized to R-ketoacids by esterases in
the plasma and tissues.26 We, therefore, selected the cyclic
hemiacetal, hydrazone, nitrile, and R-ketoamide for further
development (Figure 2).

3928 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Shirasaki et al.
Scheme 2a
a Conditions: (a) (S)-R-amino-γ-butyrolactone, TEA, DMF; (b) 4 M HCl/EtOAc; (c) R-NCS, TEA, EtOAc; (d) DIBAL-H, CH2Cl2.
Figure 2. Warhead conversion for the improvement in metabolic stability.
unsuitable because it exhibited a complete loss of calpain
inhibitory activity. We focused our efforts accordingly on the
modification of the structure of 2 to improve its activity.
We conducted an optimization study of the P2 substituent
and P3 N-capping group to enhance inhibitory activity. The
activities and water solubility of the cyclic hemiacetals are
shown in Table 3. The substitution of the 4-F-phenylsulfonamide
moiety with the phenylthiourea moiety at P3 slightly increased
inhibition against µ-calpain (2 vs 9). The placement of a Leu
residue at P2 was the most effective in inhibiting calpains (8
vs 9 vs 10). Leu derivative 8 exhibited a 7-fold higher activity
than the Val and Ile derivatives. The P2 substituent SAR was
Figure 3. Plasma levels of 1 (po 500 mg/kg) and 8 (po 10 mg/kg) via
single oral administration in rats. Each value represents the mean (
SD (n ) 4-5, dose normalized to 10 mg/kg).
identical to that of N-Cbz-dipeptidyl aldehydes5 and cyclic
hemiacetals previously reported in the literatures.29 The incor-
poration of a methylene spacer into the phenylthiourea resulted
in a 2-fold reduction in potency and aqueous solubility (8 vs
11). The various substituents on the phenyl ring of 8 resulted
in the decreased inhibitory activity and no significant change
in the aqueous solubility (12-18). Therefore, the nonsubstituted
phenyl moiety was optimal. Hemiacetal 8 was the most potent
inhibitor in this series, and in addition, its metabolic stability
in human S9 was higher than that of parent hemiacetal 2 (Table
2). Furthermore, compound 8 showed good Caco-2 permeability
(Table 3).
Pharmacokinetics. Pharmacokinetic properties of hemiacetal
8 and aldehyde lead 1 were determined in rats, and the plasma
concentration time profiles after single oral administration are
shown in Figure 3. Hemiacetal 8 demonstrated a rapid absorp-
tion (Tmax ) 0.25 h) and an increased AUC (based on dose
normalized values) compared to those of 1. The pharmacokinetic
parameters of 8 and 1 after single oral and intravenous (iv)
administrations are summarized in Table 4. Hemiacetal 8
showed excellent oral bioavailability (F ) 66%) and an
improved iv terminal half-life (t1/2 ) 1.9 h). The plasma
clearance (55 mL/min/kg) and volume of distribution (2.3 L/kg)
of 8 were lower than those of 1 (89 mL/min/kg, 6.1 L/kg). We
considered that the improvement of in vitro hepatic stability
was associated with the decline in plasma total clearance and
first pass metabolism. Furthermore, hemiacetal 8 showed
adequate aqueous solubility and high Caco-2 permeability for
oral absorption (Table 3). The permeability of 8 was higher
Table 4. PK Parameters of Hemiacetal 8 and Aldehyde 1 in Rats
parametersa
8
1
route/dose (mg/kg)
number of animals
(n/time point)
iv/3
5
po/10
5
iv/3
4
po/500
4
AUCb (µM‚h)
2.6 ( 0.1
5.7 ( 0.4
5.6 ( 2.5
0.25
1.5 ( 0.3
45 ( 11
9.4 ( 5.4
1.0
C
max (µM)
Tmax (h) (median)
t1/2c (h)
1.9 ( 0.7
2.3 ( 0.3
55 ( 2.7
0.7 ( 0.8
6.1 ( 4.0
89 ( 86
Vssd (L/kg)
Cle (mL/min/kg)
Ff (%)
66 ( 7.3
18 ( 4.5
a The values are mean ( SD. b The area under the curves for 0 h to
infinity. c The terminal half-life. d The steady-state volume of distribution.
e The total clearance. f Oral bioavailability.
than that of 1, even though 8 had lower lipophilicity (8, log D7
) 0.70; 1, log D7 ) 1.7).23 The reason for this may be attributed
to a reduced reactivity. The aldehyde group may react with
various membrane substances that contain nucleophiles such
as an NH or SH group.27 A decrease in the reactivity on
replacement of the aldehyde with the hemiacetal resulted in an
increase in membrane permeability. Therefore, the enhancement
of the oral BA of 8 is ascribed to an improvement in these
parameters. The prolongation of the terminal half-life of 8 was
caused by a reduced plasma clearance, which predominated over
a reduction in the volume of distribution. Thus, the replacement
of the aldehyde moiety with the cyclic hemiacetal significantly

Orally AVailable Calpain Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3929
carbon. The major and minor peaks of the same proton were
separately listed, in case each peak was identified. Specific rotations
were measured with a Horiba SEPA-2000 model. Matrix-assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-
TOF MS) were obtained on a Perceptive Voyager DE PRO using
R-cyano-4-hydroxycinnamic acid as a matrix. Analytical HPLC was
performed with a Shimadzu Class LC-10A system (LC-10AD, SPD-
10A, SIL-10A, CTO-10A, CBM-10A). Preparative HPLC was
performed using two Shimadzu LC-8A pumps with an SPD-6AV
UV-Vis detector set at 250 nm. Elemental analyses were performed
on an Elementar Vario EL analyzer.
N-(4-Fluorophenylsulfonyl)-L-valyl-L-leucinamide (7). N-(4-
Fluorophanylsulfonyl)-L-valine N-hydroxysuccinimide ester (6.0 g,
16 mmol) (6) was dissolved in EtOAc (200 mL), and l-leucinamide
HCl (3.2 g, 19 mmol) and Et3N (3.3 g, 32 mmol) were added
thereto. The mixture was stirred at room temperature for 18 h. After
the reaction occurred, 1 M HCl was added, and the precipitate was
dissolved in EtOAc. The organic layer was washed with 1 M HCl,
saturated NaHCO3, and saturated NaCl, dried over MgSO4, and
concentrated in vacuo. The solid was washed with hexane/EtOAc
(1:1) to give 7 (5.2 g, 83%) as colorless crystals. Mp 224.0-225.0
Figure 4. Cell density in the retinal ganglion cell layer seven days
after retinal ischemia. Each column represents the mean ( SEM for
eight rats. *:p < 0.05, significantly different from the control group
(student’s t-test).
1
°C. H NMR (300 MHz, DMSO-d6) δ 0.70 (d, 3H, J ) 5.7 Hz),
improved the BA and the terminal half-life through the
enhancement of metabolic stability, aqueous solubility, and
membrane permeability.
0.76 (d, 3H, J ) 6.9 Hz), 0.79 (d, 3H, J ) 5.7 Hz), 0.80 (d, 3H,
J ) 6.9 Hz), 1.16-1.27 (m, 3H), 1.82 (m, 1H), 3.55 (m, 1H), 3.94-
4.02 (m, 1H), 6.89 (s, 1H), 7.24 (s, 1H), 7.30-7.36 (m, 2H), 7.77-
7.83 (m, 3H), 7.87 (d, 1H, J ) 8.4 Hz).
Retinal Ischemia Model in Rats. We evaluated the neuro-
protective efficacy of hemiacetal 8 and aldehyde lead 1 in the
ischemia-reperfusion model in rats.19 As shown in Figure 4, 8
demonstrated a significant effect at po 100 mg/kg (the cell
density in the retinal ganglion cell layer: 8 vs control; 6.38 (
3.36 vs 3.69 ( 1.65, p < 0.05) and no effect at po 30 mg/kg
(4.41 ( 3.18 vs 3.69 ( 1.65, not statistically significant, student
t-test). Compound 8 is effective only at a 5-fold lower dose
compared to that of 1 (po 500 mg/kg). Hemiacetal 8 showed a
3-fold higher oral BA (F ) 66%) than that of lead 1 (F ) 18%),
although 8 displayed 4-fold less in vitro potency. Improvement
of oral BA led to an enhanced retinal efficacy.
N-(1-Cyano-3-methylbutyl)-2-((4-fluorophenyl)sulfonyl)-3-
methylbutanamide (5). Compound 7 (2.0 g, 5.2 mmol) was
dissolved in THF (50 mL), and Et3N (1.0 g, 10 mmol) and
trifluoroacetic anhydride (1.2 g, 5.7 mmol) were added under ice-
cold conditions. The mixture was stirred under the same conditions
for 2 h. After the reaction occurred, the mixture was evaporated,
and the residue was dissolved in EtOAc. The solution was washed
with 1 M HCl, saturated NaHCO3, and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The residue recrystallized from
EtOAc to give 5 (0.82 g, 43%) as colorless crystals. 1H NMR (300
MHz, DMSO-d6) δ 0.77 (d, 3H, J ) 6.3 Hz), 0.79-0.83 (m, 9H),
1.25-1.54 (m, 3H), 1.81 (m, 1H), 3.43 (dd, 1H, J ) 9.3, 7.9 Hz),
4.34 (dt, 1H, J ) 15.5, 7.5 Hz), 7.30-7.40 (m, 2H), 7.76-7.81
(m, 2H), 8.14 (d, 1H, J ) 9.3 Hz), 8.67 (d, 1H, J ) 7.5 Hz). Anal.
(C17H24FN3O3S) C, H, N.
Conclusion
On the basis of the assumption that the presence of the
aldehyde moiety on potent lead 1 decreased oral bioavailability
through its metabolic lability, we replaced the aldehyde moiety
with a more chemically stable warhead. The resulting inhibitors,
cyclic hemiacetal 2, hydrazone 3, and R-ketoamide 4, showed
higher in vitro metabolic stability compared to that of 1.
Hemiacetal 2 was the most metabolically stable compound with
only a modest loss of potency. Optimization of 2 at the P2/P3
sites led to hemiacetal 8 with an improved inhibitory activity.
In addition, 8 had excellent metabolic stability, oral BA (F )
66%), and a prolonged half-life. Furthermore, 8 was efficacious
in the rat retinal ischemia model at a 5-fold lower oral dose
than that of 1. Hemiacetal 8 is a useful tool for exploration of
orally active drugs for the treatment of retinal diseases. In future
work, it will be evaluated by the appropriate pharmacological
models that reflect clinical efficacy.
General Procedure for the Synthesis of Butyrolactone Com-
pounds 19-20. (2S)-2-((tert-Butoxy)carbonylamino)-3-methyl-
N-((3S)-tetrahydro-2-oxo-3-furanyl)butanamide (19). To a so-
lution of N-Boc-valine N-hydroxysuccinimide ester (17 g, 54 mmol)
in DMF (60 mL) was added (S)-(-)-alpha-aminobutyrolactone
hydrobromide (L-homoserine lactone hydrobromide) (15 g, 81
mmol) and Et3N (16 g, 162 mmol) under ice-cold conditions. The
mixture was stirred at room temperature for 18 h. The mixture was
diluted with EtOAc, and the solution was washed with 1 M HCl,
saturated NaHCO3, and saturated NaCl, dried over MgSO4, and
concentrated in vacuo. The residue was crystallized from hexane
to give 19 (13 g, 82%) as colorless crystals. Mp 113.9-114.9 °C.
1H NMR (300 MHz, DMSO-d6) δ 0.84 (d, 3H, J ) 6.9 Hz), 0.87
(d, 3H, J ) 6.9 Hz), 1.38 (s, 9H), 1.93 (m, 1H), 2.17 (m, 1H), 2.39
(m, 1H), 3.78 (dd, 1H, J ) 8.9 Hz, 7.2 Hz), 4.21 (m, 1H), 4.40 (m,
1H), 4.60 (m, 1H), 6.68 (d, 1H, J ) 8.9), 8.37 (d, 1H, J ) 7.8 Hz).
(2S)-2-((tert-Butoxy)carbonylamino)-3-methyl-N-((3S)-tet-
rahydro-2-oxo-3-furanyl)pentanamide (20). Colorless crystals.
Experimental Section
1
Mp 156.4-159.2 °C. H NMR (300 MHz, CDCl3) δ 0.90 (3H, t,
J ) 7.5 Hz), 0.95 (d, 3H, J ) 6.6 Hz), 1.15 (m, 1H), 1.43 (s, 9H),
1.50 (m, 1H), 1.88 (m, 1H), 2.21 (m, 1H), 2.73 (m, 1H), 4.01 (m,
1H), 4.26 (m, 1H), 4.46 (td, 1H, J ) 9.0 Hz, 1.2 Hz), 4.54 (m,
1H), 5.11 (d, 1H, J ) 8.7 Hz), 6.87 (m, 1H).
General Procedure for the Synthesis of Cyclic Hemiacetals
9-18. (2S)-3-Methyl-2-(((phenylamino)thioxomethyl)amino)-N-
((3S)-tetrahydro-2-hydroxy-3-furanyl)butanamide (9). To a solu-
tion of 19 (3.0 g, 10 mmol) in EtOAc (100 mL) was added 4 M
HCl in EtOAc (10 mL) under ice-cold conditions. The mixture was
stirred at room temperature for 18 h (oil-down was observed). The
solvent was removed in vacuo, and then EtOAc was added and
General. All reagents were of commercial grade and used
without further purification. Melting points were obtained using a
Yanaco micro melting point apparatus without correction. 1H NMR
and 13C NMR spectra were measured using a Varian Gemini-2000
Spectrometer. Chemical shifts were reported in parts per million
(δ value), and coupling constants (J) were reported in hertz.
Tetramethylsilane (δ 0) and DMSO (δ 39.7) were used as internal
standards (1H NMR and 13C NMR, respectively). Splitting patterns
are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; br, broad peak. The NMR spectra of cyclic hemiacetals
were extremely complicated because of the presence of anomeric

3930 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Shirasaki et al.
removed in vacuo (twice). The resulting oil was suspended with
EtOAc, and phenyl isothiocyanate (1.35 g, 10 mmol) and Et3N (4.8
g, 48 mmol) were added. The mixture was stirred at room
temperature for 3 h and washed with 1 M HCl, saturated NaHCO3
and saturated NaCl, dried over MgSO4, and evaporated in vacuo
to give the thiourea intermediate (2.9 g, 86%, 8.6 mmol) as a white
solid, which was used directly in the subsequent reaction without
further purification.
(2S)-2-((((4-Methoxyphenyl)amino)thioxomethyl)amino)-4-
methyl-N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (13).
Colorless crystals. Mp 67.9-68.8 °C. [R]25 +19.9° (c ) 0.2,
D
1
DMSO). H NMR (300 MHz, DMSO-d6) δ 0.88-0.91 (m, 6H),
1.47-1.87 (m, 4H), 2.14 (m, 1H), 3.69/3.94 (m/m, 1H), 3.74 (s,
3H), 3.86-3.90 (m, 2H), 4.89 (m, 1H), 4.99/5.11 (d/t, 1H, J )
4.8/4.4 Hz), 6.15/6.35 (d/d, 1H, J ) 4.8/4.2 Hz), 6.88-6.91 (m,
2H), 7.28-7.33 (m, 2H), 7.47/7.53 (d/d, 1H, J ) 8.1/6.9 Hz), 8.22/
7.81 (d/d, 1H, J ) 6.6/7.8 Hz), 9.49 (s, 1H), major/minor ) 1.0:
0.78. 13C NMR (75 MHz, DMSO-d6) δ 22.5/22.7, 23.1/23.2, 24.4/
24.5, 28.0/29.1, 41.7/42.0, 52.0/56.0, 55.4/55.9, 55.7, 64.1/65.3,
94.0/100.8, 114.0 (2C), 125.5 (2C), 132.1, 172.0, 180.5/180.7.
MALDI-TOF MS calcd for C18H27N3O3S [M + Na]+, 404.162;
found, 404.166.
To a solution of the resulting thiourea in CH2Cl2 (150 mL) was
added DIBAL-H (1.0 M solution in toluene) (26 mL, 26 mmol) at
-78 °C. The mixture was stirred below -70 °C for 3 h, and
saturated NH4Cl solution (3.0 mL) was added and stirred for 30
min. MgSO4 and EtOAc (150 mL) were added, and the inorganic
was filtered off (using Celite). After concentration in vacuo, the
residue was purified with HPLC system (column; YMC-Pack
ODS-A 250 × 20 mm, eluent CH3CN/H2O/TFA ) 30:70:0.1). The
main fractions were collected and extracted with EtOAc. The extract
was washed with saturated NaHCO3 and saturated NaCl, dried over
MgSO4, and concentrated in vacuo. The resulting residue was
crystallized from hexane to give 9 (1.3 g, 44%) as colorless crystals.
Mp 50.9-51.9 °C. [R]25D +39.8° (c ) 0.2, DMSO). 1H NMR (300
MHz, DMSO-d6) δ 0.86-0.92 (m, 6H), 1.80 (m, 1H), 2.03-2.26
(m, 2H), 3.69/3.89 (m/m, 1H), 3.93-3.97 (m, 2H), 4.92 (m, 1H),
5.02/5.13 (d/t, 1H, J ) 4.5/4.7 Hz), 6.16/6.31 (d/d, 1H, J ) 4.5/
4.8 Hz), 7.10 (m, 1H), 7.29-7.34 (m, 2H), 7.52-7.55 (m, 2H),
7.71 (m, 1H), 8.24/7.92 (d/d, 1H, J ) 6.6/7.5 Hz), 9.82 (s, 1H),
major/minor ) 1.0:0.83. 13C NMR (75 MHz, DMSO-d6) δ 18.3/
18.5, 18.9/19.0, 27.8/28.9, 31.4/31.6, 52.1/56.0, 61.2/61.3, 64.0/
65.3, 94.0/100.8, 122.9 (2C), 124.2, 128.7 (2C), 139.6, 170.6, 180.5/
180.6. MALDI-TOF MS calcd for C16H23N3O3S [M + Na]+,
360.136; found, 360.137.
(2S,3S)-3-Methyl-2-(((phenylamino)thioxomethyl)amino)-N-
((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (10). Color-
less crystals. Mp 51.3-52.9 °C. [R]25D +33.6° (c ) 0.2, DMSO).
1H NMR (300 MHz, DMSO-d6) δ 0.81-0.87 (m, 6H), 1.03 (m,
1H), 1.48 (m, 1H), 1.68-1.86 (m, 2H), 2.11 (m, 1H), 3.48/3.86
(m/m, 1H), 3.91-3.96 (m, 2H), 4.88 (m, 1H), 4.99/5.10 (d/t, 1H,
J ) 4.5/4.5 Hz), 6.13/6.27 (d/d, 1H, J ) 4.5/4.2 Hz), 7.07 (m,
1H), 7.27-7.32 (m, 2H), 7.49-7.52 (m, 2H), 7.70/7.71 (d/d, 1H,
J ) 8.4/8.1 Hz), 7.85/8.21 (d/d, 1H, J ) 7.8/6.6 Hz), 9.76 (s, 1H),
major/minor ) 1.0:0.77. 13C NMR (75 MHz, DMSO-d6) δ 11.69,
15.3/15.4, 24.7/24.9, 27.8/28.9, 37.9/38.1, 52.1/56.0, 60.8/61.0, 64.0/
65.3, 94.0/100.8, 122.8 (2C), 124.2, 128.6 (2C), 139.6, 170.6, 180.4/
180.4. MALDI-TOF MS calcd for C17H25N3O3S [M + Na]+,
374.151; found, 374.185.
(2S)-2-((((3-Cyanophenyl)amino)thioxomethyl)amino)-4-methyl-
N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (14). Col-
orless crystals. Mp 54.1-55.1 °C. [R]25D +23.8° (c ) 0.2, DMSO).
1H NMR (300 MHz, DMSO-d6) δ 0.90-0.92 (m, 6H), 1.45-1.90
(m, 4H), 2.15 (m, 1H), 3.69/3.96 (m/m, 1H), 3.86-3.91 (m, 2H),
4.93 (m, 1H), 5.01/5.12 (d/t, 1H, J ) 4.8/4.5 Hz), 6.16/6.34 (d/d,
1H, J ) 4.8/4.5 Hz), 7.48-7.54 (m, 2H), 7.75 (m, 1H), 8.06-8.18
(m, 2H), 8.30/7.92 (d/d, 1H, J ) 6.9/7.5 Hz), 9.93 (s, 1H), major/
minor ) 1.0:0.75. 13C NMR (75 MHz, DMSO-d6) δ 22.5/22.6, 23.1/
23.2, 24.5/24.5, 27.9/29.2, 41.6/41.9, 52.1/56.0, 55.7, 64.1/65.3,
94.0/100.8, 111.2, 118.9, 125.1, 127.1, 130.0 (2C), 140.8, 171.7,
180.2/180.4. MALDI-TOF MS calcd for C18H24N3O3S [M + Na]+,
399.147; found, 399.167.
(2S)-2-((((4-Cyanophenyl)amino)thioxomethyl)amino)-4-methyl-
N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (15). Col-
orless crystals. Mp 80.0-82.9 °C. [R]25D +51.2° (c ) 0.2, DMSO).
1H NMR (300 MHz, DMSO-d6) δ 0.89-0.92 (m, 6H), 1.50-1.90
(m, 4H), 2.14 (m, 1H), 3.69/3.96 (m/m, 1H), 3.86-3.91 (m, 2H),
4.93 (m, 1H), 5.01/5.12 (d/t, 1H, J ) 5.1/4.5 Hz), 6.16/6.33 (d/d,
1H, J ) 4.5/4.2 Hz), 7.73-7.76 (m, 2H), 7.84-7.88 (m, 2H), 8.18
(m, 1H), 8.31/7.95 (d/d, 1H, J ) 6.6/7.8 Hz), 10.10 (s, 1H), major/
minor ) 1.0:0.75. 13C NMR (75 MHz, DMSO-d6) δ 22.4/22.5, 23.0/
23.1, 24.4/24.5, 27.9/29.2, 41.5/41.7, 52.0/56.1, 55.6, 64.0/65.2,
94.0/100.8, 104.9, 119.2, 121.2 (2C), 132.8 (2C), 144.4, 171.5,
179.7. MALDI-TOF MS calcd for C18H24N3O3S [M + Na]+,
399.147; found, 399.150.
(2S)-4-Methyl-2-((((2-methylphenyl)amino)thioxomethyl)-
amino)-N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (16).
Colorless crystals. Mp 57.9-58.9 °C. [R]25 +9.8° (c ) 0.2,
D
1
DMSO). H NMR (300 MHz, DMSO-d6) δ 0.88-0.91 (m, 6H),
1.48-1.86 (m, 4H), 2.16 (m, 1H), 2.17 (s, 3H), 3.69/3.94 (m/m,
1H), 3.86-3.90 (m, 2H), 4.91 (m, 1H), 4.98/5.11 (d/t, 1H, J )
4.5/4.5 Hz), 6.15/6.36 (d/d, 1H, J ) 4.8/3.9 Hz), 7.12-7.31 (m,
4H), 7.46/7.52 (d/d, 1H, J ) 8.4/9.9 Hz), 7.78/8.19 (d/d, 1H, J )
7.2/6.6 Hz), 9.20 (s, 1H), major/minor ) 1.0:0.89. 13C NMR (75
MHz, DMSO-d6) δ 17.8, 22.5/22.7, 23.1/23.2, 24.5/24.5, 28.1/29.1,
41.5/41.9, 51.9/56.0, 56.0, 64.1/65.2, 94.0/100.8, 126.3, 126.5,
127.9, 130.6, 134.5, 137.4, 171.9, 181.2/181.3. MALDI-TOF MS
calcd for C18H27N3O3S [M + Na]+, 388.167; found, 388.176.
(2S)-4-Methyl-2-((((3-methylphenyl)amino)thioxomethyl)-
amino)-N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (17).
(2S)-2-((Benzylamino)thioxomethyl)amino)-4-methyl-N-((3S)-
tetrahydro-2-hydroxy-3-furanyl)pentanamide (11). Colorless
1
crystals. Mp 62.2-64.4 °C. [R]25 +18.9° (c ) 0.2, DMSO). H
D
NMR (300 MHz, DMSO-d6) δ 0.84-0.89 (m, 6H), 1.44-1.82 (m,
4H), 2.14 (m, 1H), 3.68/4.03 (m/m, 1H), 3.85-3.97 (m, 2H), 4.66
(bs, 1H), 4.85 (m, 1H), 4.99/5.11 (d/t, 1H, J ) 4.5/4.4 Hz), 6.12/
6.34 (d/bs, 1H, J ) 4.8 Hz), 7.22-7.36 (m, 5H), 7.58 (m, 1H),
7.68/8.13 (brs, 1H), 7.96 (brs, 1H), major/minor ) 1.0:0.80. 13C
NMR (75 MHz, DMSO-d6) δ 22.4/22.5, 23.1/23.2, 24.4/24.5, 28.1/
29.2, 42.1, 47.2, 51.9/56.0, 55.8, 64.1/65.2, 94.0/100.8, 127.1/127.4,
127.4 (2C), 128.5 (2C), 139.3, 172.1, 182.8. MALDI-TOF MS calcd
for C18H27N3O3S [M + Na]+, 388.167; found, 388.162.
Colorless crystals. Mp 61.4-61.9 °C. [R]25 +4.8° (c ) 0.2,
D
1
DMSO). H NMR (300 MHz, DMSO-d6) δ 0.91 (d, 6H, J ) 6.3
Hz), 1.47-1.88 (m, 4H), 2.12 (m, 1H), 2.28 (s, 3H), 3.69/3.95 (m/
m, 1H), 3.86-3.91 (m, 2H), 4.92 (m, 1H), 5.00/5.11 (d/t, 1H, J )
4.8/4.5 Hz), 6.15/6.34 (d/d, 1H, J ) 4.8/4.5 Hz), 6.92 (m, 1H),
7.17-7.30 (m, 3H), 7.68/7.74 (d/d, 1H, J ) 8.4/7.8 Hz), 8.24/7.84
(d/d, 1H, J ) 6.6/7.5 Hz), 9.63 (s, 1H), major/minor ) 1.0:0.87.
13C NMR (75 MHz, DMSO-d6) δ 21.2, 22.5/22.6, 23.1/23.2, 24.5/
24.5, 28.0/29.1, 41.6/42.0, 52.0/56.0, 55.6/55.8, 64.1/65.3, 94.0/
100.8, 120.1, 123.4, 125.0, 128.5, 138.0, 139.4, 171.9, 180.1/180.3.
MALDI-TOF MS calcd for C18H27N3O3S [M + Na]+, 388.167;
found, 388.175.
(2S)-2-((((4-Fluorophenyl)amino)thioxomethyl)amino)-4-methyl-
N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (12). Col-
orless crystals. Mp 77.2-78.3 °C. [R]25D +20.1° (c ) 0.2, DMSO).
1H NMR (300 MHz, DMSO-d6) δ 0.90 (d, 6H, J ) 6.3 Hz), 1.48-
1.91 (m, 4H), 2.14 (m, 1H), 3.69/3.95 (m/m, 1H), 3.86-3.91 (m,
2H), 4.93 (m, 1H), 5.00/5.12 (d/t, 1H, J ) 5.1/4.5 Hz), 6.15/6.34
(d/d, 1H, J ) 4.8/3.9 Hz), 7.12-7.18 (m, 2H), 7.46-7.52 (m, 2H),
7.73/7.77 (d/d, 1H, J ) 8.1/7.8 Hz), 7.84/8.25 (d/d, 1H, J ) 7.8/
7.2 Hz), 9.64 (s, 1H), major/minor ) 1.0:0.85. 13C NMR (75 MHz,
DMSO-d6) δ 22.5/22.6, 23.1/23.2, 24.4/24.5, 28.1/29.1, 41.7/42.0,
52.0/56.0, 55.7, 64.1/65.3, 94.0/100.8, 115.1/115.4 (2C), 125.3 (2C),
135.9/136.0, 157.4/160.6, 171.9, 180.6. MALDI-TOF MS calcd for
C17H24FN3O3S [M + Na]+, 392.142; found, 392.141.
(2S)-4-Methyl-2-((((4-methylphenyl)amino)thioxomethyl)-
amino)-N-((3S)-tetrahydro-2-hydroxy-3-furanyl)pentanamide (18).
Colorless crystals. Mp 87.3-88.7 °C. [R]25 +23.9° (c ) 0.2,
D
1
DMSO). H NMR (300 MHz, DMSO-d6) δ 0.89 (d, 6H, J ) 6.6

Orally AVailable Calpain Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3931
Hz), 1.36-1.85 (m, 4H), 2.14 (m, 1H), 2.21 (s, 3H), 3.68/3.94 (m/
m, 1H), 3.85-3.90 (m, 2H), 4.28 (m, 1H), 4.98/5.10 (d/t, 1H, J )
4.8/4.7 Hz), 6.14/6.33 (d/d, 1H, J ) 4.5/3.6 Hz), 6.24 (m, 1H),
7.01-7.03 (m, 2H), 7.23-7.26 (m, 2H), 8.19/7.84 (d/d, 1H, J )
7.2/7.8 Hz), 8.46 (s, 1H), major/minor ) 1.0:0.46. 13C NMR (75
MHz, DMSO-d6) δ 20.4, 22.1/22.3, 23.2/23.4, 24.4/24.5, 27.9/29.1,
42.5/42.8, 51.1/51.2, 51.9/55.9, 64.1/65.3, 94.0/100.9, 117.7 (2C),
129.3 (2C), 130.0, 138.0, 154.9, 172.8. MALDI-TOF MS calcd
for C18H27N3O3S [M + Na]+, 388.167; found, 388.165.
Inhibition Assays for Calpains. The inhibition assays were
performed as described in the literature22,30 using commercial
µ-calpain (human erythrocyte, Calbiochem) and m-calpain (porcine
kidney, Calbiochem). An assay solution containing 0.5 mg/mL of
casein, 20 mM dithiothreitol, 50 mM Tris-HCl (pH 7.4), and 1.0
nmol of enzyme was used. The assay solution (200 µL) and DMSO
(2.5 µL) containing inhibitors of different concentrations were
placed in each well. The reaction was started by the addition of 20
mM CaCl2 (50 µL) in a test well and 1 mM EDTA (50 µL) in a
blank well. After incubation for 60 min at 30 °C, the mixture (100
µL) was transferred to another plate, and H2O (100 µL) and Bio-
Rad protein assay dye reagent (50 µL) were placed in each well.
After incubation at room temperature for 15 min, the OD of the
mixture at 595 nm was recorded on a plate reader (Multiscan
Multisoft, Labsystems). The percent inhibition was calculated from
the differences in the ODs between when the compound was present
and when it was absent.
Metabolic Stability. Human hepatic S9 incubations were
performed in the presence of an NADPH-generating system
composed of 3 mM MgCl2, 1 mM NADP+, 5 mM glucose-6-
phosphate, and 1 Unit/mL of glucose-6-phosphate dehydrogenase
in an 50 mM potassium phosphate buffer (pH 7.4). The human S9
prepared by XenoTech LLC (Lenexa, CS) was purchased from the
Nosan Corporation (Yokohama, Japan). All concentrations are
relative to the final incubation volume (5 mL). The compounds
were added in acetonitrile to a final concentration of 5 µM. The
inncubations were conducted at 37 °C. After 30 min, 1 mL of the
incubations were sampled, and the reaction was terminated by the
addition of 4 mL of acetonitrile. The precipitated proteins were
removed by centrifugation, the supernatants were evaporated, and
the residue was dissolved in mobile phase. The solution was
analyzed by LC-UV or LC-MS/MS.
Solubility Determination. A suspension of test compound in
2.0 mL of buffer solution (pH 7.0) or simulated gastric juice (pH
1.2) was shaken at 25 °C for 5 h. The suspension was filtered
through CHROMATODISK (0.45 µm, GL Sciences), and the
filtrate (1.0 mL) was made exactly 10 mL by the addition of the
mobile phase as a sample solution. The measurement was performed
with 20 µL each of the sample solution and the standard solution
under analytical HPLC according to the following conditions:
wavelength, UV 250 nm; column, YMC-Pack ODS A-303 (250 ×
4.6 mm, 5 µm); column temperature, 45 °C; mobile phase, CH3-
CN/H2O/TFA ) 40:60:0.1; and flow rate, 1.0 mL/min. The
concentration of the test compound in the sample solution was
calculated from the ratio of the peak area in the sample solution
and the standard solution.
Retinal Ischemia-Reperfusion Model in Rats.19 Male Spra-
gue-Dawley rats (150-250 g) were used, and the compounds
formulated as a suspension in 0.5% CMC solution were orally
administrated to rats 15 min before ischemia (1, 500 mg/kg; 8, 30
and 100 mg/kg). The control group received the vehicle solution.
The rats were anesthetized with an intraperitoneal injection of
sodium pentobarbital (60 mg/kg). The body temperature was
maintained at 37 °C. Retinal ischemia on the left eye was produced
by the occlusion of the central retina and posterior ciliary arteries
with a clip. After retinal ischemia was produced for 55 min, the
clip was removed. The retinal arteries were allowed to reperfuse.
A sham operation was performed on the right eye without ligation.
After reperfusion, the same dose of compounds was orally
administrated. The efficacy was evaluated by a measurement of
cell density in the retinal ganglion cell layer 1-2 mm from the
optic disk. The cells were stained with hematoxylin and eosin (HE
staining). The number of cell nuclei in the ganglion cell layers was
counted at a width of 250 µm at three sites.
Acknowledgment. We thank Dr. Wataru Takayama, Yusuke
Sakai, and Noriko Nada for supporting our laboratory work. In
addition, we thank Dr. Yukuo Yoshida and Akiko Shirasaki
for the critical reading of this manuscript. Also, we thank Drs.
Yutaka Kawamatsu and Mitsuyoshi Azuma for useful discus-
sions.
Pharmacokinetic Studies in Rats. The in vivo pharmacokinetic
results were determined after oral or intravenous (po 10 or 500
mg/kg or iv 3 mg/kg) single agent dosing to male Sprague-Dawley
rats (n ) 4 or 5/time point, weight 200-250 g). Each compound
(1 or 8) was formulated as a suspension in 0.5% carboxymethyl
cellulose solution (CMC) for oral administration. In the case of iv
administration, each compound was dissolved in a vehicle consisting
of 60% poly(ethylene glycol) and 40% water at a concentration of
10 mg/mL. The animals were anesthetized with isoflurane at
predetermined times (0.1 (iv only), 0.25, 0.5, 1, 2, 4, and 8 h), and
the blood was sampled from the abdominal aorta into heparinized
syringes. The blood was centrifuged to obtain plasma. All plasma
samples were frozen and stored at -30 °C until analysis. The frozen
solution was fused at room temperature, and test sample solutions
were prepared by a solid-phase extraction procedure using OASIS
HLB (60 mg) cartridges by Waters. The plasma concentration of
each test compound was determined by turboionspray on an Applied
Biosystems/MDS-Sciex API 4000 triple-quadrupole mass spec-
trometer equipped with a turbo ion spray source using multiple
reaction monitoring (MRM). The samples were chromatographed
on a NANOSPACE SI-2 (Shiseido) HPLC system with Shiseido
Capcell pak C18 MG-II column (75 × 1.5 mm, 5 µm) at 40 °C
using a mobile phase (MeOH/H2O/HCO2H ) 40:60:0.1, for
compound 1) or Shiseido Capcell pak C18 ACR (150 × 1.5 mm,
3 µm) at 40 °C using a mobile phase (MeCN/H2O ) 70:30, for
compound 8) at a flow rate of 0.20 mL/min. Data reduction was
performed using MDS-Sciex Analyst ver. 1.3 software. The
noncompartmental model was used to calculate pharmacokinetic
parameters from the resulting plasma concentration vs time profiles
using WinNonLin (Scientific Consulting, Inc.).
Supporting Information Available: Elemental analyses for
compound 5. HPLC analysis data for compounds 9-18. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) (a) Ono, Y.; Sorimachi, H.; Suzuki, K. Pharmacology and toxicology
of calcium-dependent protease. In Calpain; Wang, K. K. W., Yuen,
P.-W., Eds.; Taylor & Francis: Philadelphia, 1999; pp1-3. (b)
Huang, Y.; Wang, K. K. The calpain family and human disease.
Trends Mol. Med. 2001, 7, 355-362.
(2) (a) Wang, K. K. W.; Yuen, P.-W. Calpain inhibition: an overview
of its therapeutic potential. Trends Pharmacol. Sci. 1994, 15, 412-
419. (b) Stracher, A. Calpain inhibitors as therapeutic agents in nerve
and muscle degeneration. Ann. N.Y. Acad. Sci. 1999, 884, 52-59.
(c) Yuen, P.-W.; Wang, K. K. W. Therapeutic potential of calpain
inhibitors in neurodegenerative disorders. Expert Opin. InVest. Drugs
1996, 5, 1291-1304. (d) Zatz, M.; Starling, A. Calpains and disease.
N. Engl. J. Med. 2005, 352, 2413-2423.
(3) (a) Donkor, I. O. A survey of calpain inhibitors. Curr. Med. Chem.
2000, 7, 1171-1188. (b) Wells G. J.; Bihovsky, R. Calpain inhibitors
as potential treatment for stroke and other neurodegenerative
disease: recent trends and developments. Expert Opin. Ther. Pat.
1998, 8, 1707-1727 (c) Leung, D.; Abbenante, G.; Fairlie, D. P.
Protease inhibitors: current status and future prospects. J. Med. Chem.
2000, 43, 305-341.
(4) (a) Sasaki, T.; Kikuchi, T.; Yumoto, N.; Yoshimura, N.; Murachi,
T. Comparative specificity and kinetic studies on porcine calpain I
and calpain II with naturally occurring peptides and synthetic
fluorogenic substrates. J. Biol. Chem. 1984, 259, 12489-12494. (b)
Tompa, P.; Buzder-Lantos, P.; Tantos, A.; Farkas, A.; Szila´gyi, A.;
Ba´no´czi, Z.; Hudecz, F.; Friedrich, P.; On the sequential determinants
of calpain cleavage. J. Biol. Chem. 2004, 279, 20775-20785.

3932 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Shirasaki et al.
(5) Iqbal, M.; Messina, P. A.; Freed, B.; Das, M.; Chatterjee, S.; Tripathy,
R.; Tao, M.; Josef, K. A.; Dembofsky, B.; Dunn, D.; Griffith, E.;
Siman, R.; Senadhi, S. E.; Biazzo, W.; Bozyczko-Coyne, D.; Meyer,
S. L.; Ator, M. A.; Bihovsky, R. Subsite requirements for peptide
aldehyde inhibitors of human calpain I. Bioorg. Med. Chem. Lett.
1997, 7, 539-544.
(6) Moldoveanu, T.; Campbell, R. L.; Currier, D.; Davis, P. L. Crystal
Structures of calpain-E64 and -leupeptin inhibitor complexes reveal
mobile loops gating the active site. J. Mol. Biol. 2004, 343, 1313-
1326.
(7) Aoyagi, T.; Takeuchi, T.; Matsuzaki, A.; Kawamura, K.; Kondo, S.
Leupeptines, new protease inhibitors from Actinomycetes. J. Antibiot.
1969, 22, 283-286.
(8) Sasaki, T.; Kishi, M.; Saito, M.; Tanaka, T.; Higuchi, N.; Kominami,
N.; Katunuma, N.; Murachi, T. Inhibitory effect of di- and tripeptidyl
aldehydes on calpains and cathepsins. J. Enzyme Inhib. 1990, 3, 195-
201.
(9) Mehdi, S. Cell-penetrating inhibitors of calpain. Trends Biochem.
Sci. 1991, 16, 150-153.
(10) Li, Z.; Ortega-Vilain, A.; Patil, G. S.; Chu, D.; Foreman, J. E. Novel
peptidyl R-keto amide inhibitors of calpains and other cysteine
proteases. J. Med. Chem. 1996, 39, 4089-4098.
(11) Lubisch, W.; Beckenbach, E.; Bopp, S.; Hofmann, H. P.; Kartal, A.;
Ka¨stel, C.; Lindner, T.; Metz-Garrecht, M.; Reeb, J.; Regner, F.;
Vierling, M.; Mo¨ller, A. Benzoylalanine-derived ketoamides carrying
vinylbenzul amino residues: Discovery of potent water-soluble
calpain inhibitors with oral bioavailability J. Med. Chem. 2003, 46,
2404-2412.
(12) (a) Fukiage, C.; Azuma, M.; Nakamura, Y.; Tamada, Y.; Nakamura,
M.; Shearer, T. R. SJA6017, A newly synthesized peptide aldehyde
inhibitor of calpain: amelioration of cataract in cultured rat lenses.
Biochim. Biophys. Acta 1997, 1361, 304-312. (b) Inoue, J.;
Nakamura, M.; Cui, Y.; Sakai, Y.; Sakai, O.; Hill, J. R.; Wang, K.
K. W.; Yuen, P.-W. Structure-activity relationship study and drug
profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017)
as a potent calpain inhibitor. J. Med. Chem. 2003, 46, 868-871.
(13) Lee, K. S.; Franks, S.; Vanderklish, P.; Arai, A.; Lynch, G. Inhibition
of proteolysis protects hippocampal neurons from ischemia. Proc.
Natl. Acad. Sci. U.S.A. 1991, 57, 7233-7237.
(14) Rami, A.; Krieglstein, J. Protective effects of calpain inhibitors against
neuronal damage caused by cytotoxic hypoxia and ischemia in ViVo.
Brain Res. 1993, 609, 67-70.
(15) (a) Hong, S.-C.; Goto, Y.; Lanzino, G.; Saleau, S.; Kassel N. F.;
Lee, K. S. Neuroprotection with a calpain inhibitor in a model of
focal cerebral ischemia. Stroke 1994, 25, 663-669. (b) Markgraf,
C. G.; Velayo, N. L.; Johnson, M. P.; McCarty, D. R.; Medhi, S.;
Koehl, J. R.; Chmielewski, P. A.; Linnik, M. D. Six-hour window
of opportunity for calpain inhibition in focal cerebral ischemia in
rats. Stroke 1998, 29, 152-158.
(18) Nath, R.; Davis, M.; Probert, A. W.; Kupina, N. C.; Ren, X., Schielke
G. P.; Wang, K. K. Processing of cdk5 activator p35 to its truncated
form (p25) by calpain in acutely injured neuronal cells. Biochem.
Biophys. Res. Commun. 2000, 274, 16-21.
(19) Sakamoto, Y.; Nakajima, T.; Fukiage, C.; Sakai, O.; Yoshida, Y.;
Azuma, M.; Shearer, T. R. Involvement of calpain isoforms in
ischemia-reperfusion injury in rat retina. Curr. Eye. Res. 2000, 21,
571-580.
(20) Sasaki, H.; Yamamura, K.; Mukai, T.; Nishida, K.; Nakamura, J.;
Nakashima, M.; Ichikawa, M. Enhancement of ocular drug penetra-
tion. Crit. ReV. Ther. Drug Carrier Syst. 1999, 16, 85-146.
(21) Sakamoto, Y. Unpublished results.
(22) (a) Nakamura, M.; Inoue, J. Exploration of peptidyl hydrazones as
water-soluble calpain inhibitors. Bioorg. Med. Chem. Lett. 2002, 12,
1603-1606. (b) Shirasaki, Y.; Miyashita, H.; Yamaguchi, M.; Inoue,
J.; Nakamura, M. Exploration of orally available calpain inhibitors:
peptidyl R-ketoamides containing an amphiphile at P3 site. Bioorg.
Med. Chem. 2005, 13, 4473-4484.
(23) Nakamura, M.; Yamaguchi, M.; Sakai, O.; Inoue, J. Exploration of
cornea permeable calpain inhibitors as anticataract agents. Bioorg.
Med. Chem. 2003, 11, 1371-1379.
(24) Altmann, E.; Betschart, C.; Gohda, K.; Horiuchi, M.; Lattmann, R.;
Missbach, M.; Sakaki, J.; Takai, M.; Teno, N.; Cowen, S. D.;
Greenspan, P. D.; McQuire, L. W.; Tommasi, R. A.; Van D., John
H. PCT Int. Appl. WO9924460, 1999.
(25) (a) Angelastro, M. R.; Mehdi, S.; Burkhart, J. P.; Peet, N. P.; Bey,
P. R-Diketone 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. (b) Mehdi, S. Synthetic and naturally
occurring protease inhibitors containing an electrophilic carbonyl
group. Bioorg. Chem. 1993, 21, 249-259. (c) Otto, H.-H.; Schirmeis-
ter, T. Cysteine protease and their inhibitors. Chem. ReV. 1997, 97,
133-171. (d) Peet N. P.; Kim, H. O.; Marquart, A. L.; Angelastro,
M. R.; Nieduzak, T. R.; White, J. N.; Friedrich, D.; Flynn, G. A.;
Webster, M. E.; Vaz, R. J.; Linnik, M. D.; Koehl, J. R.; Mehdi, S.;
Bey, P.; Emary, B.; Hwang K. K. Hydroxyoxazolidines as alpha-
aminoacetaldehyde equivalents: novel inhibitors of calpain. Bioorg.
Med. Chem. Lett. 1999, 9, 2365-2370.
(26) Li, 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-keto
ester, peptide R-keto amide and peptide R-keto acid inhibitors of
calpains and other cysteine proteases. J. Med. Chem. 1993, 36, 3472-
3480.
(27) Nakamura, M.; Miyasita, H.; Yamaguchi, M.; Shirasaki, Y.; Naka-
mura, Y.; Inoue, J. Novel 6-hydroxy-3-morpholinones as cornea
permeable inhibitors. Bioorg. Med. Chem. 2003, 11, 5449-5460.
(28) van de Waterbeemed, H.; Smith, D. A.; Beaumont, K.; Walker, D.
K. Property-based design: optimization of drug absorption and
pharmacokinetics. J. Med. Chem. 2001, 44, 1313-1334.
(29) Ando, R.; Sasaki, T.; Masuda, H.; Inakoshi, N.; Jikihara, T.; Fujimura,
Y.; Niwa, T.; Yoshii, N.; Tabata, R. PCT Int. Appl. WO9625408,
1998.
(16) Bartus, R. T.; Hayward, 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. Calpain inhibitor AK-295 protects neurons from focal
brain damage. Stroke 1994, 25, 2265-2270.
(17) Neuhof, W.; Go¨tte, O.; Trumbeckaite, S.; Attenberger, M.; Kuzkaya,
N.; Gellerish, F.; Mo¨ller, A.; Lubisch, W.; Speth, M.; Tillmanns,
H.; Neuhof, H.; A novel water-soluble and cell-permeable calpain
inhibitor protects myocardial and mitochondrial function in postis-
chemic reperfusion. Biol. Chem. 2003, 384, 1597-1603.
(30) Buroker-Kilgore, M.; Wang, K. K. W. A coomassie brilliant blue
G-250-based colorimetric assay for measuring activity of calpain and
other proteases. Anal. Biochem. 1993, 208, 387-392.
JM060157N