Exploration of orally available calpain inhibitors: Peptidyl α-ketoamides containing an amphiphile at P3 site

Article abstract of DOI:10.1016/j.bmc.2005.04.059

A novel series of dipeptidyl α-ketoamide derivatives with amphiphile was designed and synthesized as water-soluble calpain inhibitors. The introduction of amphiphiles at the P3 site increased water solubility without loss of membrane permeability and prov

Full text of DOI:10.1016/j.bmc.2005.04.059

Bioorganic & Medicinal Chemistry 13 (2005) 4473–4484
Exploration of orally available calpain inhibitors: Peptidyl
a-ketoamides containing an amphiphile at P3 site
Yoshihisa Shirasaki,* Hiroyuki Miyashita, Masazumi Yamaguchi,
Jun Inoue and Masayuki Nakamura
Research Laboratory of Ocular Science, Senju Pharmaceutical Co., Ltd, 1-5-4 Murotani Nishi-ku Kobe, Hyogo 651-2241, Japan
Received 23 March 2005; revised 15 April 2005; accepted 15 April 2005
Available online 25 May 2005
Abstract—A novel series of dipeptidyl a-ketoamide derivatives with amphiphile was designed and synthesized as water-soluble cal-
pain inhibitors. The introduction of amphiphiles at the P3 site increased water solubility without loss of membrane permeability and
provided the oral available inhibitors. Extension of the ethylene glycol chain at the P3 site led to an improvement in persistence of
plasma levels. In particular, introduction of a combination of a diethylene glycol methyl ether moiety at the P3 site, a phenylalanine
residue at the P1 site and a cyclopropyl moiety at the P0 site was the most effective modification for an increase in plasma drug
exposure.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Several a-ketoamide inhibitors have demonstrated pro-
tective effects in in vivo experiments. For example, in
an animal model of stroke, it has been shown that an
original peptidyl a-ketoamide inhibitor, AK-275 (1),
has a protective effect against ischemic brain damage
(Fig. 1).4 Furthermore, its water-soluble derivative,
AK-295 (2), is also effective in an animal ischemia model
via internal carotid artery administration.5 Recently,
benzoylalanine-derived ketoamides have been reported
as non-peptidyl calpain inhibitors without an L-Leu or
L-Val moiety at the P2 site. The representative com-
pound showed neuroprotective efficacy in an experimen-
tal model of traumatic brain injury by intraperitoneal
administration.6
Calpains are nonlysosomal cysteine endoproteases and
the family of these enzymes has grown rapidly in recent
years.1 Two well-known calpains, l-calpain (calpain I)
and m-calpain (calpain II), are ubiquitously found in
mammalian cells and participate in a variety of biologi-
cal processes and numerous diseases such as stroke, Alz-
heimarꢀs disease, central nervous system (CNS) diseases,
spinal cord injury, brain trauma, cardiac ischemia, mus-
cular dystrophy, and cataracts.2 Therefore, calpains are
attractive targets for discovering novel therapeutic
agents for diseases.
Many of the known calpain inhibitors are composed of
a binding group and di- or tripeptide-based backbone,
which is structurally related to the cleavage site of sub-
strates of calpains. The binding groups are capable of
binding to the catalytic center of the enzymes in irrevers-
ible or reversible manners.3 Epoxide, haloketone, and
vinyl sulfone work as irreversible binding groups. Alde-
hyde, a-ketoacid, a-ketoester, and a-ketoamide act as
reversible binding groups, which form hemithioacetal
or ketal with the SH group of the cysteine residue in
the active site of calpains.
Previously, our group has reported a dipeptidyl alde-
hyde inhibitor SJA6017 (3), which has efficacy as an
anti-cataract agent in lens culture models.7 However,
in a rat retinal ischemia model,8 the high oral dosage
of 3 is needed for confirmation of the protective effect,
because of its poor oral bioavailability. It appears that
this poor availability is ascribed to the presence of an
aldehyde group that is easily metabolized.9 Further-
more, it is possible that the aldehyde group reacts with
an amino or thiol moiety of various biological sub-
stances and proteins through a Schiff base and this
may limit membrane permeation of the drugs.10 There-
fore, we examined whether introducing an a-ketoamide
moiety instead of the aldehyde moiety could improve
oral availability. The corresponding a-ketoamide
Keywords: Calpain inhibitor; Dipeptidyl a-ketoamides; Amphiphile;
Water-solubility; Pharmacokinetics; Ethylene glycol.
*
Corresponding author. Tel.: +81 78 997 1010; fax: +81 78 997
1016; e-mail: shirasaki@senju.co.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.059

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Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
O
O
O
O
O
H
N
H
N
H
N
H
N
N
O
N
H
O
N
H
HCl
O
O
O
O
O
2 (AK-295)
1 (AK-275)
O
O O
S
O
O O
S
H
N
H
N
H
N
N
H
N
H
H
O
O
F
F
4
3 (SJA6017)
Figure 1. Structure of known calpain inhibitors.
analogue 411 showed inhibitory activities equivalent to 3
and higher cellular permeability and higher metabolic
stability than 3. Unfortunately, it shows very low solu-
bility that can be disadvantageous to the bioavailability.
In fact, pharmacokinetic studies in monkey have dem-
onstrated that 4 displayed poor oral availability.
crease water solubility without decreasing the cellular
permeability. In this report, we describe the synthesis
of peptidyl a-ketoamides containing amphiphile, their
inhibitory activities, as well as the relationships between
the chemical structures, in vitro pharmacokinetic data
and in vivo pharmacokinetics in monkey.
To explore novel calpain inhibitors that have good oral
availability, we designed water-soluble a-ketoamide
derivatives based on known inhibitors such as 1, 2,
and 4. This study was focused on substitution of P3
N-capped moieties and P0 alkyl chain (Fig. 2). Although
an introduction of ionic moieties such as sulfonate, car-
boxylate, and guanidino groups increase the water-solu-
bility, the charged form of these molecules would show
low cellular permeability.9a,12 Thus, it is not easy to de-
sign the molecules with both suitable water solubility
and membrane permeability. Therefore, we decided to
introduce a non-ionic amphiphile as a water-soluble
moiety to the P3 site of dipeptidyl a-ketoamide on the
assumption that the introduction of amphiphile can in-
2. Results and discussion
2.1. Chemistry
The new peptidyl a-ketoamides were synthesized as re-
ported previously by Herbeson et al.4b The syntheses
of the P2/P3 units are shown in Scheme 1. The alcohols
5b–d as amphiphiles were coupled to L-leucine ethyl es-
ter (L-Leu-OEt) through carbamate linkage using di-(N-
succinimidyl) carbonate (CO(OSu)2) to prepare the N-
alkoxycarbonyl amino acid esters 6b–d.13 The esters
were hydrolyzed with NaOH to yield the corresponding
acids 8b–d. The carboxylic acid 8a was prepared by a di-
rect condensation of L-Leu-OH (7a) and 2-methoxyethyl
chloroformate. The carboxylic acids 8a–d were con-
verted to the corresponding N-succinimidyl esters 9a–d
as the P2/P3 units.
Amphiphile
O
The P1/P0 units were prepared from Boc-protected ami-
no alcohols 10 and 11 (Scheme 2). The alcohols 10 and
11 were oxidized by SO3–pyridine complex in DMSO to
afford the aldehydes 12 and 13.10 The aldehydes were
converted to the cyanohydrins, which were directly
hydrolyzed with HCl to give a-hydroxy-b-amino acids.
The a-hydroxy-b-amino acids were protected with the
H
N
H
N
R1
N
H
R2
O
R3
P1
O
P3
P2
P'
Figure 2. Strategy for oral available a-ketoamide.
a, b
O
O
O
c
e
O
R1OH
O
O
R1O
N
H
CO2Et
CO2H
R1O
N
H
CO2H
R1O
N
H
N
O
5b-d
6b-d
8a-d
9a-d
d
a: R1 = CH3OC2H4
b: R1 = 2-(S)-Tetrahydrofranyl
c: R1 = 4-Tetrahydropyranyl
d: R1 = CH3(OC2H4)2
H2N
7a
Scheme 1. Reagents: (a) CO(OSu)2, Et3N, MeCN; (b) L-leucine-OEtÆHCl, Et3N, CH2Cl2; (c) NaOH, aq EtOH; (d) MeOC2H4OCOCl, Et3N, CH2Cl2;
(e) N-hydroxysuccinimide, EDC, THF.

Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
4475
O
OH
CO2H
a
b, c, d
BocHN
BocHN
BocHN
OH
H
n
n
n
10 (n = 1)
11 (n = 2)
12 (n = 1)
13 (n = 2)
14 (n = 1)
15 (n = 2)
OH
CONHR2
O
O
H
N
BocHN
f, g, h
e
R1O
N
H
CONHR2
O
n
n
16F (R2 = CH2CF3, n = 1)
16G (R2 = 2-indanyl, n = 1)
16H (R2 = C2H4OMe, n = 1)
17A (R2 = Et, n = 2)
16A (R2 = Et, n = 1)
16B (R2 = c-Pr, n = 1)
16C (R2 = n-Pr, n = 1)
16D (R2 = c-Bu, n = 1)
16E (R2 = n-Bu, n = 1)
18Aa-c,18Ba-d,18Ca-Ha,18Ee (n = 1)
19Aa-b,19Ba-b (n = 2)
a: R1 = CH3OC2H4
b: R1 = 2-(S)-Tetrahydrofranyl
c: R1 = 4-Tetrahydropyranyl
d: R1 = CH3O(C2H4O)2
e: R1 = PhCH2
17B (R2 = c-Pr, n = 2)
Scheme 2. Reagents and conditions: (a) SO3/pyridine, DIPEA, DMSO/CH2Cl2; (b) NaHSO3, aq MeOH; then NaCN, EtOAc; (c) concentrated HCl/
dioxane (1:1), reflux; (d) Boc2O, aq NaOH, dioxane; (e) R2–NH2, EDC, HOBt, DMF; (f) 4 M HCl/dioxane; (g) 9a–d or Cbz-L-Leu-OSu, Et3N,
CH2Cl2; (h) Dess–Martin periodinane, CH2Cl2.
Boc group using di-tert-butyl dicarbonate (Boc2O) and
aqueous NaOH to afford the N-Boc-a-hydroxy-b-amino
acids 14 and 15 as mixtures of diastereomers.5b The
HOBt/EDC-mediated coupling between the acids and
the various amines afforded the a-hydroxyamides 16A–
H and 17A–B. The Boc group in 16 and 17 were
removed and the resulting amines were coupled to N-
succinimidyl esters 9a–d or Cbz-L-Leu-OSu to afford
the corresponding dipeptidyl a-hydroxyamide interme-
diates. Oxidation of the hydroxyl group of the interme-
diates with Dess–Martin periodinane gave the final
products 18 and 19 (Table 1).11
19Ba). Thus, SAR at P0 and P1 region for the novel a-
ketoamides were consistent with that of known a-ketoa-
mides.6,15–17
We recognized that the a-ketoamide derivatives contain-
ing amphiphile at the P3 site were water-soluble inhibi-
tors. For instance, compound 18Ea, which has an
ethylene glycol methyl ether (mEG) moiety, is 20-fold
more soluble in water than the corresponding Cbz deriv-
ative (18Ee). Decreasing of P0 alkyl chain length from n-
Bu to Et gradually increases water solubility (18Ea vs
18Ca vs 18Aa). On the other hand, c-Pr compound
(18Ba), which is more soluble than n-Pr compound
(18Ca), is as soluble as Et compound (18Aa). A similar
phenomenon was observed in other c-Pr derivatives
(18Ac vs 18Bc and 19Aa vs 19Ba). However, in the case
of THF derivative, replacement of Et with c-Pr leads to
14-fold less water-solubility (18Ab vs 18Bb). The exten-
sion of the ethylene glycol chain at the P3 site also re-
sulted in an unexpected decline in water-solubility
(18Ba vs 18Bd). Thus, it is not easy to predict the water
solubility from the combination of each building block
in the modification of a-ketoamides.
2.2. Enzyme inhibition and water solubility
Table 1 shows the inhibitory activities against l- and m-
calpain and the water solubility of novel a-ketoamide
derivatives containing an amphiphile at the P3 site.
These compounds are less potent than the known inhib-
itor 18Ee14 that has a Cbz moiety at the P3 site,
although they are sufficiently potent inhibitors (l-cal-
pain IC50 0.086–0.45 lM). The conversion of alkyl chain
at the P0 site did not affect the inhibitory activities to-
ward calpains. For example, introduction of a 2-indanyl
substituent, the most bulky group, did not alter the
activities (18Ga vs 18Aa). In the case of an (S)-3-tetrahy-
drofuranyl (THF) moiety at the P3 site, replacement of
an Et group with a cyclopropyl (c-Pr) group resulted in a
2-fold increase in inhibitory activities (18Bb vs 18Ab).
Introduction of a trifluoroethyl (CH2CF3) moiety at
the P0 site slightly reduced the activities compared to
the isosteric Et analogue (18Fa vs 18Aa). The reduction
of activity may be due to an electrostatic repulsion be-
tween the CH2CF3 at the P0 site and the carboxylate
group at the S0 site of the calpain enzyme.15 Similar
activity reduction has been reported by Donkor et
al.16 On the other hand, replacement of phenylalanine
residue (Phe) with homophenylalanine (HomoPhe) at
the P1 site also did not affect the activities (19Aa and
Consequently, the introduction of amphiphiles enables
us to provide a-ketoamide derivatives with acceptable
water solubility, although it slightly decreased inhibitory
activities. However, if the enhancement in water solubil-
ity leads to improvement in plasma exposure of the
drug, it can compensate for the loss of activity.
2.3. Membrane permeability and metabolic stability
Membrane permeability and in vitro metabolic stability
of the compounds are presented in Table 2. Most inhib-
itors were evaluated by Caco-2 permeability assay.
These compounds showed generally acceptable perme-
ability (Papp (apical ! basolaterial) > 10ꢀ6 cm/s).18 Fur-
thermore, immobilized artificial membranes (IAM)

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Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
Table 1. Enzyme inhibitory activity and solubility of a-ketoamide derivatives
O
O
n
H
H
N
R1
N
R2
O
N
H
O
O
P3
P2
P1
P'
Compd
Structure
IC50 (lM)
Solubilityc (mg/mL)
R1
n
R2
l-Calpaina
m-Calpainb
18Aa
18Ab
18Ac
18Ba
18Bb
18Bc
18Bd
18Ca
18Da
18Ea
18Fa
18Ga
18Ha
19Aa
19Ba
18Ee
3
CH3OC2H4
THFd
1
Et
Et
0.17
0.15
0.25
0.11
0.086
0.12
0.17
0.099
0.17
0.10
0.45
0.17
0.18
0.30
0.18
0.038
0.021
0.021
0.11
0.11
1.2
1.0
1
THPe
1
Et
0.16
0.10
0.84
1.3
CH3OC2H4
THFd
THPe
1
c-Pr
c-Pr
c-Pr
c-Pr
n-Pr
c-Bu
n-Bu
1
0.047
0.13
0.074
0.76
0.65
1
CH3(OC2H4)2
CH3OC2H4
CH3OC2H4
CH3OC2H4
CH3OC2H4
CH3OC2H4
CH3OC2H4
CH3OC2H4
CH3OC2H4
PhCH2
1
0.099
0.070
0.080
0.14
1
0.33
0.066
0.11
1
1
1
–CH2CF3
2-Indanyl
–C2H4OCH3
0.34
0.12
0.071
<0.005
0.74
1
1
0.11
0.20
2
Et
0.22
0.27
2
c-Pr
n-Bu
—
0.14
1
0.020
0.021
0.045
<0.005
0.0053
0.1
—
—
—
—
4
—
a Human erythrocyte l-calpain.
b Porcine kidney m-calpain.
c Water-solubility in pH 7 buffer, at 25 ꢁC.
O
d THF:
e THP:
O
chromatography analysis was performed to determine
19
monkeys. The Cmax value and area under the plasma le-
vel concentration time curve (AUC0!4 h) after 10 mg/kg
oral administration are shown in Table 2. Compounds
18Ab, 18Ba, 18Bd, and 18Ca showed good PK proper-
ties. Compounds 18Bb, 18Da, 18Ea, and 18Ee displayed
no or very low plasma drug levels. The most effective
modification for the improvement in PK properties
was introduction of a combination of mEG at the P3
site, Phe at the P1 site, and c-Pr at the P0 site (18Ba
and 18Bd). Thus, introduction of amphiphiles to dipep-
tidyl a-ketoamide led to enhancement of water-solubil-
ity without loss of adequate membrane permeability
and improved the PK properties. In addition, extension
of the P3 ethylene glycol chain, namely conversion of
18Ba into 18Bd, led to enhanced 2 and 4 h plasma levels
with equal Cmax value (Fig. 3).
the capacity factors (k0IAM
)
of inhibitors as a parame-
ter of permeability. We confirmed that all compounds
had acceptable membrane permeability that was de-
duced from the results of Caco-2 permeability assay
and IAM chromatography analysis. Thus, introduction
of amphiphile at the P3 site provided water-soluble
inhibitors without loss of membrane permeability.
In vitro metabolic stability was determined by incuba-
tion of the inhibitors with human liver S9 fraction.
The inhibitors showed low to moderate metabolic stabil-
ity (17–64% of initial concentration). Increasing the al-
kyl chain length at the P0 and P1 sites reduced their
metabolic stability. For instance, metabolic stability
was reduced gradually by increasing the P0 alkyl chain
from Et (18Aa) to n-Bu (18Ea). We confirmed that the
increase of lipophilicity caused the reduction of meta-
bolic stability in this series of inhibitors.
The compounds with good PK properties possess high
water-solubility and metabolic stability. Insoluble or
metabolically unstable compounds displayed poor oral
absorption. Therefore, in this series, the oral availabil-
ity is mainly restricted water solubility and metabolic
stability. However, since compound 18Ha, having high
solubility and metabolic stability, showed relatively low
plasma AUC0!4 h, some other minor factors such as
carrier-mediated active transport and non-hepatic
2.4. Pharmacokinetic analysis
The purpose of this study is to improve the pharmacoki-
netic (PK) properties of the peptidyl a-ketoamide deriv-
atives. The a-ketoamide derivatives were evaluated for
their PK properties by oral absorption experiments in

Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
Table 2. Solubility, permeability, and PK data of a-ketoamide derivatives
4477
Compd
Solubilitya (mg/mL)
Permeability
Metabolic stabf
Human S9 (%)
Pharmacokineticsg
Caco-2 Papp (cm/s) · 10ꢀ6
IAMd LogK0
Cmax (lM)
AUC0!4 h (lM h)
b
c
e
h
18Aa
18Ab
18Ba
18Bb
18Bc
18Bd
18Ca
18Da
18Ea
18Ha
19Ba
18Ee
3
1.2
1.0
3.1
NDi
2.7
1.13
1.22
1.20
1.55
1.36
1.27
1.28
1.53
1.70
1.13
1.36
>2.50
2.13
1.26
64
53
64
57
27
41
42
17
17
60
33
NDi
22
3
0.30
0.59
1.2
0.31
0.61
1.9
1.3
0.074
0.76
0.65
0.33
0.066
0.11
0.74
0.27
<0.005
0.0053
0.10
2.5
3.0
NDi
0.08
0.26
1.1
0.038
0.27
2.4
6.3
0.65
BDLj
BDLj
0.21
0.23
BDLj
0.12
NDi
0.87
BDLj
BDLj
0.16
0.25
BDLj
0.16
NDi
NDi
10.5
2.0
4.5
NDi
17.3
2.5
4
a Water-solubility in pH 7 buffer, at 25 ꢁC.
b Caco-2 cell monolayer permeability assay.
c Papp is the apparent permeability coefficient18 for apical to basolateral flux in 10ꢀ6 cm/s.
d Immobilized artificial membrane chromatography analysis.19
19
e Logarithm of the capacity factors k0
.
IAM
f Metabolic stability represented in residual percent after incubation with human S9 fraction for 0.5 h at 37 ꢁC.
g Cynomolgus monkey, p.o. 10 mg/kg, dosed as a suspension in 0.5% carboxymethylcellulose.
h The area under the curves 0–4 h.
i Not determined.
j Below detection limit.
the decrease in potency can be compensated by the
enhancement in oral availability. The most effective
modification for oral absorption is to introduce c-Pr at
the P0 site and an mEG moiety at the P3 site in the series
of a-ketoamides. Furthermore, the extension of the P3
ethylene glycol chain provided sustained plasma levels
(compound 18Bd). We will evaluate the efficacy of com-
pound 18Bd (SNJ-1945) in adequate in vivo pharmaco-
logical models to develop calpain inhibitors as orally
available anti-retinopathy agents.
1.4
1.2
1
18Ba
18Bd
0.8
0.6
0.4
0.2
0
0
1
2
3
4
Time (h)
4. Experimental
4.1. General
Figure 3. Mean plasma levels after oral administration (10 mg/kg) of
18Ba and 18Bd in cynomolgus monkeys (n = 2).
Optical rotations were measured with a Horiba SEPA-
200 polarimeter. Melting points were determined on a
Yanaco micromelting point apparatus without correc-
tion. 1H NMR spectra were recorded on a Varian Gem-
ini-2000 spectrometer. Chemical shifts were reported in
parts per million, and coupling constants (J) were re-
ported in hertz. Matrix-assisted laser desorption ioniza-
tion time-of-flight mass spectra (MALDI-TOF-MS)
were obtained on a Perceptive Voyager DE mass spec-
trometer, and the mass numbers were corrected with
an internal standard (a-cyano-4-hydroxycinnamic acid)
and displayed accurately.
metabolism, in addition to water solubility and meta-
bolic stability, would be partly attributed to the limita-
tion of oral availability. We will hereafter need to
investigate the involvement of these factors in
pharmacokinetics.
3. Conclusion
Based on the assumption that the poor PK properties of
dipeptidyl a-ketoamide 4 can be due to its insolubility,
we modified the dipeptidyl a-ketoamides to improve
their water solubility. Introduction of amphihiles at
the P3 site led to increased water-solubility without a
loss of membrane permeability, although potency was
lower than with the starting a-ketoamide 4. Since some
of those inhibitors showed remarkable improvement of
oral availability as compared with 4, it is expected that
4.2. General procedure for the preparation of compounds
6b–d
4.2.1. N-(((3S)-Tetrahydrofuran-3-yloxy)carbonyl)-L-leu-
cine ethyl ester (6b). To a stirred solution of (S)-3-
hydroxytetrahydrofuran (1.0 g, 11 mmol) in acetonitrile

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Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
(50 mL) were added N,N0-disuccinimidyl carbonate
(4.3 g, 17 mmol) and triethylamine (4.8 mL, 34 mmol)
at room temperature. The resulting mixture was stirred
at room temperature for 18 h and concentrated in va-
cuo. The residue was diluted into saturated NaHCO3
(100 mL) and extracted with EtOAc (200 mL). The
combined extracts were washed with saturated NaCl
(100 mL), dried over MgSO4, and concentrated in va-
cuo to give a brown oil. This mixed carbonate was
used directly for the next reaction. To a solution of
L-leucine ethyl ester hydrochloride (2.7 g, 14 mmol)
and triethylamine (2.9 g, 28 mmol, 4.0 mL) in CH2Cl2
(50 mL) was added a solution of the resulting mixed
carbonate (2.6 g, 11 mmol) in CH2Cl2 (20 mL). The
mixture was stirred at room temperature for 18 h,
and concentrated in vacuo. EtOAc (200 mL) was added
to the residue, and the solution was washed with 1 M
HCl (100 mL), saturated NaHCO3 (100 mL), and satu-
rated NaCl (100 mL), dried over MgSO4, and concen-
trated in vacuo. The residue was washed with hexane
to give 10b (3.1 g, 98%) as a colorless solid. 1H
NMR (300 MHz, DMSO-d6) d 0.80–0.92 (m, 6H),
1.18 (t, 3H, J = 7.2), 1.37–1.71 (m, 3H), 1.85 (m,
1H), 2.12 (m, 1H), 3.61–3.84 (m, 4H), 3.95–4.15 (m,
3H), 5.10 (m, 1H), 7.64 (d, 1H, J = 8.1). MALDI-
TOF-MS calcd for C13H23NO5 (M+Na)+, 296.1474,
found, 296.1444.
4.4. General procedure for the preparation of compounds
8b–d
4.4.1. N-(((3S)-Tetrahydrofuran-3-yloxy)carbonyl)-L-leu-
cine (8b). To a solution of 6b (2.9 g, 11 mmol) in EtOH
(100 mL) was added 1 M NaOH (33 mL). The mixture
was stirred under ice-cooled conditions for 3 h made
by the addition of HCl thereto (pH 3). The solution
was concentrated in vacuo and extracted with EtOAc
(100 mL). Then, the organic layer was separated, washed
with 1 M HCl and saturated NaCl, dried over MgSO4
and concentrated in vacuo. The residue was washed
with hexane and EtOAc to give 8b (2.6 g, 85%) as
colorless crystals. Mp 94.9–96.0 ꢁC. 1H NMR (300
MHz, DMSO-d6) d 0.85 (d, 3H, J = 6.3), 0.88 (d, 3H, J =
6.3), 1.38–1.73 (m, 3H), 1.85 (m, 1H), 2.10 (m, 1H), 3.61–
3.85 (m, 4H), 3.94 (m, 1H), 5.10 (m, 1H), 7.48 (d,
1H, J = 7.8), 12.49 (br s, 1H). MALDI-TOF-MS
calcd for C11H19NO5 (M+Na)+, 268.1161, found,
268.1181.
4.4.2. N-((Tetrahydro-4H-pyran-4-yloxy)carbonyl)-L-leu-
cine (8c). Colorless oil. H NMR (300 MHz, DMSO-d6)
1
d 0.83–0.90 (m, 6H), 1.38–1.73 (m, 5H), 1.84 (m, 2H),
3.41 (m, 2H), 3.84 (m, 2H), 3.95 (m, 1H), 4.68 (m,
1H), 7.38 (d, 1H, J = 8.1), 12.49 (br s, 1H). MALDI-
TOF-MS calcd for C12H21NO5 (M+Na)+, 282.1318,
found, 282.1354.
4.2.2. N-((Tetrahydro-4H-pyran-4-yloxy)carbonyl)-L-leu-
cine ethyl ester (6c). Brown oil. H NMR (300 MHz,
DMSO-d6) d 0.83–0.90 (m, 6H), 1.17 (t, 3H, J = 7.1),
1.38–1.71 (m, 5H), 1.83 (m, 2H), 3.41 (m, 2H), 3.81
(m, 2H), 3.93–4.16 (m, 3H), 4.68 (m, 1H), 7.55 (d, 1H,
J = 8.1).
1
4.4.3. N-((5-Methoxy-3-oxapentyloxy)carbonyl)-L-leucine
(8d). Colorless oil. H NMR (300 MHz, DMSO-d6) d
0.80–0.89 (m, 6H), 1.39–1.69 (m, 3H), 3.25 (s, 3H), 3.42–
3.46 (m, 2H), 3.51–3.58 (m, 4H), 3.94 (m, 1H), 4.02–
4.07 (m, 2H), 7.50 (d, 1H, J = 8.4), 12.45 (br s,
1H). MALDI-TOF-MS calcd for C12H23NO6 (M+
Na)+, 300.1423, found, 300.1470.
1
4.2.3. N-((5-Methoxy-3-oxapentyloxy)carbonyl)-L-leucine
ethyl ester (6d). Colorless oil. 1H NMR (300
MHz, DMSO-d6) d 0.80–0.90 (m, 6H), 1.18 (t, 3H,
J = 7.1), 1.38–1.69 (m, 3H), 3.25 (s, 3H), 3.42–3.46
(m, 2H), 3.51–3.58 (m, 4H), 3.99 (m, 1H), 4.04–4.12
(m, 4H), 7.64 (d, 1H, J = 7.8). MALDI-TOF-MS
calcd for C14H27NO6 (M+Na)+, 328.1736, found,
328.1775.
4.5. General procedure for the preparation of compounds
9a–d
4.5.1. N-((2-Methoxyethoxy)carbonyl)-L-leucine N-hy-
droxysuccinimide ester (9a). Compound 8a (20 g,
86 mmol) and N-hydroxysuccinimide (13 g, 0.11 mmol)
were dissolved in THF (200 mL), and a suspension of
4.3. N-((2-Methoxyethoxy)carbonyl)-L-leucine (8a)
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
HCl
(21 g, 0.11 mol) in CH2Cl2 (200 mL) was added thereto.
The mixture was stirred at room temperature for 18 h,
and concentrated in vacuo. The residue was dissolved
in EtOAc (300 mL), the solution was washed with 1 M
HCl (150 mL), saturated NaHCO3 (150 mL), and satu-
rated NaCl (150 mL), dried over MgSO4, and concen-
trated in vacuo to give 14a (27 g, 95%) as a colorless
oil. 1H NMR (300 MHz, DMSO-d6) d 0.89 (d, 3H,
J = 6.6), 0.93 (d, 3H, J = 6.6), 1.57–1.84 (m, 3H), 2.81
(s, 4H), 3.26 (s, 3H), 3.51 (t, 2H, J = 4.7), 4.10 (t, 2H,
J = 4.7), 4.40 (m, 1H), 8.04 (d, 1H, J = 8.1). MALDI-
TOF-MS calcd for C14H22N2O7 (M+Na)+, 353.1325,
found, 353.1301.
L-Leucine (7a) (25 g, 0.19 mol) was dissolved in 2 M
NaOH (0.12 L). To this solution were slowly added
chloroformic acid 2-methoxyethyl ester (30 g, 0.22 mol)
and 1 M NaOH (0.22 L) at the same time under ice-
cooled conditions. The mixture was stirred at room tem-
perature for 18 h, diluted into water (600 mL), and
washed with ethyl ether (2 · 200 mL). The aqueous
phase was chilled in an ice bath and acidified to pH 3
with 6 M HCl. This mixture was extracted with EtOAc
(5 · 150 mL). The organic phase was dried over MgSO4
and concentrated in vacuo to yield 13a (41 g, 92%) as a
1
colorless oil. H NMR (300 MHz, DMSO-d6) d 0.85 (d,
3H, J = 6.6), 0.88 (d, 3H, J = 6.6), 1.47 (m, 2H), 1.63 (m,
1H), 3.26 (s, 3H), 3.49 (t, 2H, J = 4.7), 3.92 (m, 1H), 4.06
(t, 2H, J = 4.5), 7.50 (d, 1H, J = 8.4), 12.50 (br s, 1H).
MALDI-TOF-MS calcd for C10H19NO5 (M+Na)+,
256.1161, found, 256.1241.
4.5.2. N-(((3S)-Tetrahydrofuran-3-yloxy)carbonyl)-L-leu-
cine N-hydroxysuccinimide ester (9b). Colorless viscous
oil. 1H NMR (300 MHz, DMSO-d6) d 0.89 (d, 3H,
J = 6.0), 0.92 (d, 3H, J = 6.3), 1.55–1.82 (m, 3H), 1.88

Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
4479
(m, 1H), 2.12 (m, 1H), 2.81 (s, 4H), 3.64–3.84 (m, 4H),
4.39 (m, 1H), 5.15 (m, 1H), 8.04 (d, 1H, J = 7.8).
MgSO4, and concentrated in vacuo to yield crude cya-
nohydrin as a colorless viscous oil. This crude cyanohy-
drin was dissolved in 1,4-dioxane (250 mL),
concentrated HCl (250 mL), and anisole (10 mL). The
solution was gently refluxed and stirred overnight. The
hydrolysis reaction was allowed to cool and then con-
centrated in vacuo to give a brown oil. The residue
was dissolved in water (100 mL) and washed with ethyl
ether (3 · 50 mL). The aqueous phase was then placed
on a Dowex 50X8 (100–200 mesh) column (H+ form;
25 · 2 cm). The column was washed with water until
pH 5.5 and eluted with 2 M ammonium hydroxide (ca.
1.5 L). The eluent was evaporated in vacuo to yield
crude a-hydroxy-b-amino acid as a white solid. The
resulting a-hydroxy-b-amino acid was dissolved in 1 M
NaOH (70 mL). To this solution was added a solution
of di-tert-butyl dicarbonate (12 g, 56.9 mmol) in dioxane
(70 mL). The reaction was stirred at room temperature
while the pH was maintained at pH 11 with 1 M NaOH.
The mixture was stirred at room temperature for 18 h,
diluted into water (600 mL) and washed with ethyl ether
(2 · 200 mL). The aqueous phase was chilled in an ice
bath and acidified to pH 2 with 1 M HCl. This mixture
was extracted with EtOAc (3 · 250 mL). The organic
phase was dried with MgSO4 and concentrated in vacuo
to yield a mixture of diastereomers (ca. 55:45) of 14
4.5.3. N-((Tetrahydro-4H-pyran-4-yloxy)carbonyl)-L-leu-
cine N-hydroxysuccinimide ester (9c). Colorless viscous
oil. 1H NMR (300 MHz, DMSO-d6) d 0.89 (d, 3H,
J = 6.0), 0.92 (d, 3H, J = 6.3), 1.43–1.93 (m, 7H), 2.80
(s, 4H), 3.42 (m, 2H), 3.78–3.82 (m, 2H), 4.39 (m, 1H),
4.72 (m, 1H), 7.94 (d, 1H, J = 7.8). MALDI-TOF-MS
calcd for C16H24N2O7 (M+Na)+, 379.1428, found,
379.0930.
4.5.4. N-((5-Methoxy-3-oxapentyloxy)carbonyl)-L-leucine
N-hydroxysuccinimide ester (9d). Colorless oil. 1H
NMR (300 MHz, DMSO-d6) d 0.90 (dd, 6H, J =
9.5, 6.5), 1.56–1.80 (m, 3H), 2.80 (s, 4H), 3.24 (s, 3H),
3.41–3.46 (m, 2H), 3.50–3.54 (m, 2H), 3.56–3.60
(m, 2H), 4.08–4.11 (m, 2H), 4.39 (m, 1H), 8.05 (d, 1H,
J = 7.8).
4.6. General procedure for the preparation of compounds
12 and 13
4.6.1. N-(tert-Butoxycarbonyl)-L-phenylalaninal (12). N-
Boc-L-phenylalaninol (10) (69 g, 0.28 mol) was dissolved
in DMSO (280 mL) and CH2Cl2 (140 mL), and cooled
on an ice bath. N,N-Diisopropylethylamine (110 g,
0.82 mol) and a suspension of purified sulfur trioxide
pyridine complex (130 g, 0.82 mol) in DMSO (100 mL)
were added thereto. The mixture was stirred for 1 h un-
der the same conditions. The reaction mixture was di-
luted with EtOAc (1.5 L), and the solution was washed
with 1 M HCl (1 L), saturated NaHCO3 (0.8 L), and satu-
rated NaCl (0.8 L), dried over MgSO4, and concen-
trated in vacuo. The residue was crystallized from a
mixture of hexane and EtOAc to give 5a (53 g, 77%)
as colorless crystals. Mp 42.5–43.6 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 1.34 (s, 9H), 2.73 (m, 1H),
3.10 (dd, 1H, J = 14.0, 4.7), 4.08 (m, 1H), 7.15–7.34
(m, 6H), 9.53 (s, 1H). MALDI-TOF-MS calcd for
C14H19NO3 (M+H)+, 250.1143, found, 250.1172.
1
(12 g, 72%) as a white solid. (Major diastereomer) H
NMR (300 MHz, DMSO-d6) d 1.31 (s, 9 H), 2.75 (m,
2H), 3.87 (m, 1H), 4.00 (m, 1H), 6.39 (d, 1H, J = 9.6),
7.15–7.34 (m, 6H). (Minor diastereomer) 1H NMR
(300 MHz, DMSO-d6) d 1.27 (s, 9H), 2.68 (m, 2H),
3.83–4.05 (m, 2H), 6.70 (d, 1H, J = 9.0), 7.12–7.33 (m,
6H), 12.55 (br s, 1H). MALDI-TOF-MS calcd for
C15H21NO5 (M+Na)+, 318.1318, found, 318.1312.
4.7.2. (3S)-3-(tert-Butoxycarbonylamino)-2-hydroxy-5-phen-
ylpentanoic acid (15). Colorless oil as a mixture of
diastereomers. 1H NMR (300 MHz, DMSO-d6) d
1.31–1.38 (m, 9H), 1.57–1.76 (m, 2H), 2.40–2.60 (m,
2H), 3.66–3.78 (m, 1H), 3.96 (m, 1H), 6.32 (d, 0.5H,
J = 9.6), 6.68 (d, 0.5H, J = 8.7), 7.12–7.27 (m, 5H).
MALDI-TOF-MS calcd for C16H23NO5 (M+Na)+,
332.1474, found, 332.1469.
4.6.2. N-(tert-Butoxycarbonyl)-L-homophenylalaninal (13).
1
Colorless oil. H NMR (300 MHz, DMSO-d6) d 1.40–
1.42 (m, 9H), 1.72 (m, 1H), 1.96 (m, 1H), 2.58–2.73
(m, 2H), 3.77–3.84 (m, 1H), 7.16–7.31 (m, 5H), 7.43
(d, 1H, J = 7.2), 9.45 (s, 1H). MALDI-TOF-MS calcd
for C15H21NO3 (M+H)+, 264.1599, found, 264.1686.
4.8. General procedure for the preparation of compounds
16A–H, 17A–B
4.8.1. ((1S)-1-Benzyl-3-ethylamino-2-hydroxy-3-oxopro-
pyl)carbamic acid 1,1-dimethylethyl ester (16A). Com-
pound 14 (6.3 g, 21 mmol) and HOBt (3.0 g, 22.4
mmol) were dissolved in DMF (45 mL) and cooled on
an ice bath. EDC (4.6 g, 24 mmol) was added followed
by 70% aqueous ethylamine solution (3.0 mL). The solu-
tion was stirred for 18 h at room temperature. The solu-
tion was diluted into EtOAc (200 mL) and washed with
1 M HCl (150 mL), saturated NaHCO3 (150 mL), and
saturated NaCl (150 mL). The organic layer was dried
over MgSO4 and concentrated in vacuo to yield 16A
4.7. General procedure for the preparation of compounds
14 and 15
4.7.1. (3S)-3-(tert-Butoxycarbonylamino)-2-hydroxy-4-phen-
ylbutanoic acid (14). Compound 12 (17 g, 67 mmol) was
dissolved in MeOH (100 mL) and chilled to 5 ꢁC.
Sodium bisulfite (7.0 g, 67 mmol) was dissolved in water
(150 mL) and chilled to 5 ꢁC before addition to the alde-
hyde solution. The solution was added to the aldehyde
solution and the mixture was stirred overnight at 5 ꢁC.
NaCN (4.0 g) was dissolved in water (150 mL) and
added with EtOAc (300 mL) to the above mixture.
The reaction was allowed to stir for 5 h at room temper-
ature. The organic layer was separated, dried with
1
(5.8 g, 84%) as a white solid. Mp 122.7–123.8 ꢁC. H
NMR (300 MHz, DMSO-d6) d 1.02 (m, 3H), 1.28 (s,
9H), 2.54–2.67 (m, 2H), 3.11 (m, 2H), 3.94 (m, 1H),
4.01 (m, 1H), 5.65–5.85 (m, 1H), 6.55 (d, 1H, J = 8.7),
7.08–7.29 (m, 5H), 7.83 (m, 1H). MALDI-TOF-MS

4480
Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
calcd for C17H26N2O4 (M+Na)+, 345.1791, found,
345.1762.
3.83 (m, 0.5H), 3.93–4.07 (m, 1.5H), 4.52 (m, 1H), 5.56
(d, 0.5H, J = 6.6), 5.75 (d, 0.5H, J = 5.4), 6.23 (d,
0.5H, J = 5.4), 6.56 (d, 0.5H, J = 9.3), 7.12–7.30 (m,
9H), 7.96 (m, 1H). MALDI-TOF-MS calcd for
C24H30N2O4 (M+Na)+, 433.2104, found, 433.2213.
4.8.2. ((1S)-1-Benzyl-3-cyclopropylamino-2-hydroxy-3-oxo-
propyl)carbamic acid 1,1-dimethylethyl ester (16B).
1
White solid. Mp 103.5–104.0 ꢁC. H NMR (300 MHz,
DMSO-d6) d 0.47 (m, 2H), 0.58 (m, 2H), 1.28 (s,
4.5H), 1.21 (s, 4.5H), 2.53–2.68 (m, 2.5H), 2.78 (m,
0.5H), 3.77 (dd, 0.5H, J = 6.6, 2.7), 3.97 (m, 1.5H),
5.50 (d, 0.5H, J = 6.3), 5.67 (d, 0.5H, J = 5.7), 6.14 (d,
0.5H, J = 9.3), 6.50 (d, 0.5H, J = 9.0), 7.14–7.32 (m,
5H), 7.75 (m, 1H). MALDI-TOF-MS calcd for
C18H26N2O4 (M+Na)+, 357.1791, found, 357.1803.
4.8.8. ((1S)-1-Benzyl-2-hydroxy-3-(2-methoxyethylamino)-
3-oxopropyl)carbamic acid 1,1-dimethylethyl ester (16H).
1
White solid. Mp 113.5–116.5 ꢁC. H NMR (300 MHz,
DMSO-d6) d 1.28 (s, 4.5H), 1.29 (s, 4.5H), 2.53–2.67
(m, 1.5H), 2.79 (m, 0.5H), 3.23 (s, 1.5H), 3.23 (s,
1.5H), 3.29–3.39 (m, 4H), 3.81 (m, 0.5H), 3.91–4.08
(m, 1.5H), 5.76 (d, 0.5H, J = 6.0), 5.82 (d, 0.5H,
J = 5.4), 6.11 (d, 0.5H, J = 9.6), 6.53 (d, 0.5H, J = 8.7),
7.14–7.30 (m, 5H), 7.74 (m, 1H). MALDI-TOF-MS
calcd for C18H28N2O5 (M+Na)+, 375.1896, found,
375.1937.
4.8.3. ((1S)-1-Benzyl-2-hydroxy-3-oxo-3-(propylamino)-
propyl)carbamic acid 1,1-dimethylethyl ester (16C).
1
White solid. Mp 104.8–112.5 ꢁC. H NMR (300 MHz,
DMSO-d6) d 0.82 (m, 3H), 1.28 (s, 4.5H), 1.29 (s,
4.5H), 1.42 (m, 2H), 2.53–2.72 (m, 1.5H), 2.80 (m,
0.5H), 3.05 (m, 2H), 3.79 (m, 0.5H), 3.97 (m, 1.5H),
5.70 (d, 0.5H, J = 6.3), 5.76 (d, 0.5H, J = 5.7), 6.10 (d,
0.5H, J = 9.3), 6.54 (d, 0.5H, J = 8.7), 7.14–7.30 (m,
5H), 7.75–7.83 (m, 1H). MALDI-TOF-MS calcd for
C18H28N2O4 (M+Na)+, 359.1947, found, 359.2062.
4.8.9. ((1S)-3-Ethylamino-2-hydroxy-3-oxo-1-(phenylethyl)-
propyl)carbamic acid 1,1-dimethylethyl ester (17A).
White solid. Mp 134.4–134.9 ꢁC. H NMR (300 MHz,
DMSO-d6) d 0.95–1.03 (m, 3H), 1.38–1.41 (m, 9H),
1.59–1.81 (m, 2H), 2.50–2.66 (m, 2H), 3.03–3.13 (m,
2H), 3.70–3.80 (m, 1H), 3.88 (m, 1H), 5.59 (m, 1H),
6.14 (d, 0.5H, J = 9.3), 6.58 (d, 0.5H, J = 9.3), 7.13–
7.30 (m, 5H), 7.77 (m, 1H). MALDI-TOF-MS
calcd for C18H28N2O4 (M+Na)+, 359.1947, found,
359.2041.
1
4.8.4. ((1S)-1-Benzyl-3-cyclobutylamino-2-hydroxy-3-oxo-
propyl)carbamic acid 1,1-dimethylethyl ester (16D).
White solid. Mp 94.7–96.5 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 1.28 (s, 4.5H), 1.29 (s, 4.5H), 1.60 (m,
2H), 1.99 (m, 2H), 2.09 (m, 2H), 2.54–2.67 (m, 1.5H),
2.79 (dd, 0.5H, J = 13.4, 6.8), 3.76 (dd, 0.5H, J = 6.3,
2.7), 3.89–4.04 (m, 1.5H), 4.20 (m, 1H), 5.57 (d, 0.5H,
J = 6.3), 5.71 (d, 0.5H, J = 5.7), 6.15 (d, 0.5H, J = 9.3),
6.52 (d, 0.5H, J = 9.0), 7.13–7.30 (m, 5H), 7.92 (d, 1H,
J = 8.4). MALDI-TOF-MS calcd for C19H28N2O4
(M+Na)+, 371.1947, found, 371.1936.
4.8.10. ((1S)-3-Cyclopropylamino-2-hydroxy-3-oxo-1-(phen-
ylethyl)propyl)carbamic acid 1,1-dimethylethyl ester
(17B). White solid. Mp 142.1–143.6 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 0.42–0.60 (m, 4H), 1.38–1.40
(m, 9H), 1.56–1.77 (m, 2H), 2.35–2.65 (m, 3H), 3.67–
3.80 (m, 1H), 3.82–3.88 (m, 1H), 5.41 (d, 0.5H,
J = 6.3), 5.54 (d, 0.5H, J = 6.0), 6.16 (d, 0.5H, J = 9.3),
6.56 (d, 0.5H, J = 9.0), 7.14–7.30 (m, 5H), 7.69 (d,
0.5H, J = 4.8), 7.74 (d, 0.5H, J = 4.2). MALDI-TOF-
MS calcd for C19H28N2O4 (M+Na)+, 371.1947, found,
371.1930.
4.8.5. ((1S)-1-Benzyl-3-butylamino-2-hydroxy-3-oxopro-
pyl)carbamic acid 1,1-dimethylethyl ester (16E). White
solid. Mp 114.9–116.5 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 0.85 (m, 3H), 1.15–1.44 (m, 4H), 1.28 (s,
4.5H), 1.29 (s, 4.5H), 2.48–2.68 (m, 1.5H), 2.80 (m,
0.5H), 3.07 (m, 2H), 3.79 (m, 0.5H), 3.97 (m, 1.5H),
5.68 (d, 0.5H, J = 6.3), 5.75 (d, 0.5H, J = 5.7), 6.10 (d,
0.5H, J = 9.3), 6.54 (d, 0.5H, J = 8.7), 7.11–7.30 (m,
5H), 7.77 (m, 1H). MALDI-TOF-MS calcd for
C19H30N2O4 (M+Na)+, 373.2104, found, 373.2164.
4.9. General procedure for the preparation of compounds
18Aa–Ac, 18Ba–Bd, 18Ca–Ha, 18Ee, 19Aa–Ab, 19Ba–Bb
4.9.1. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(ethylamino)-
propyl)amino)carbonyl)-3-methylbutyl)carbamic acid 2-
methoxyethyl ester (18Aa). Compound 16A (5.5 g,
17 mmol) was dissolved in 4 N HCl/dioxane (65 mL)
and stirred for 3 h at room temperature. The reaction
mixture was evaporated to yield the deprotected amine
hydrochloride (4.4 g, quant) as a white solid. To a solu-
tion of the deprotected amine hydrochloride (1.0 g,
4.0 mmol) and 9a (1.2 g, 3.6 mmol) in DMF (10 mL)
was added triethylamine (1.1 g, 11 mmol). The mixture
was stirred at room temperature for 18 h and concen-
trated in vacuo. The residue was dissolved in EtOAc
(50 mL) and the solution was washed with 1 M HCl
(50 mL), saturated NaHCO3 (50 mL), and saturated
NaCl (50 mL), dried over MgSO4, and concentrated in
vacuo. The resulting white solid was washed with a mix-
ture of hexane and EtOAc (9:1). To a solution of the
resulting solid (0.7 g, 1.6 mmol) in CH2Cl2 (70 mL)
was added Dess–Martin periodinane (1.0 g, 2.4 mmol).
4.8.6. ((1S)-1-Benzyl-2-hydroxy-3-oxo-3-(2,2,2-trifluoro-
ethylamino)propyl)carbamic acid 1,1-dimethylethyl ester
(16F). White solid. Mp 103.1–104.8 ꢁC. 1H NMR
(300 MHz, DMSO-d6) d 1.15 (s, 2H), 1.28 (s, 7H),
2.60–2.70 (m, 1.5H), 2.80 (m, 0.5H), 3.81–4.09 (m, 4H),
5.90 (d, 0.5H, J = 6.6), 6.01 (d, 0.5H, J = 6.0), 6.14 (d,
0.5H, J = 9.3), 6.64 (d, 0.5H, J = 8.7), 7.13–7.30 (m,
5H), 8.38–8.49 (m, 1H). MALDI-TOF-MS calcd for
C17H23F3N2O4 (M+Na)+, 399.1508, found, 399.1536.
4.8.7. ((1S)-1-Benzyl-3-(2-indanylamino)-2-hydroxy-3-oxo-
propyl)carbamic acid 1,1-dimethylethyl ester (16G).
1
White solid. Mp 145.0–145.9 ꢁC. H NMR (300 MHz,
DMSO-d6) d 1.28 (s, 4.5H), 1.31 (s, 4.5H), 2.55–2.68
(m, 1.5H), 2.84 (m, 2.5H), 3.07–3.18 (m, 2H), 3.80–

Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
4481
The mixture was stirred at room temperature for 18 h.
Aqueous 10% Na2S2O3 (35 mL) and saturated aqueous
NaHCO3 (35 mL) were added, and the mixture was
stirred for 30 min. The organic layer was separated,
washed with 1 M HCl, saturated NaHCO3, and satu-
rated NaCl, dried over MgSO4, and concentrated in va-
cuo. The residue was recrystallized from EtOAc/hexane
to give 18Aa (0.62 g, 88%) as colorless crystals. Mp
138.0–138.3 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.83 (d, 3H, J = 7.5), 0.85 (d, 3H, J = 7.2), 1.04 (t, 3H,
J = 7.1), 1.35 (m, 2H), 1.56 (m, 1H), 2.82 (m, 1H),
3.14 (m, 3H), 3.25 (s, 3H), 3.47 (t, 2H, J = 4.5), 4.04
(m, 3H), 5.19 (m, 1H), 7.16–7.33 (m, 6H), 8.24 (d, 1H,
J = 7.2), 8.70 (m, 1H). MALDI-TOF-MS calcd for
C22H33N3O6 (M+Na)+, 458.2267, found, 458.2361.
4.9.6. ((1S)-1-((((1S)-1-Benzyl-3-cyclopropylamino-2,3-di-
oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid
tetrahydro-4H-pyran-4-yl ester (18Bc). Colorless crys-
tals. Mp 137.0–138.2 ꢁC. H NMR (300 MHz, DMSO-
d6) d 0.58 (m, 2H), 0.65 (m, 2H), 0.84 (m, 6H), 1.35
(m, 2H), 1.48 (m, 3H), 1.80 (m, 2H), 2.79 (m, 2H),
3.11 (m, 1H), 3.41 (m, 2H), 3.79 (m, 2H), 4.03 (m,
1H), 4.65 (m, 1H), 5.18 (m, 1H), 7.15–7.30 (m, 6H),
8.23 (d, 1H, J = 6.9), 8.73 (d, 1H, J = 5.4). MALDI-
TOF-MS calcd for C25H35N3O6 (M+H)+, 474.2604,
found, 474.2643.
1
4.9.7. ((1S)-1-((((1S)-1-Benzyl-3-(cyclopropylamino)-2,3-di-
oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid
5-methoxy-3-oxapentyl ester (18Bd). Colorless crystals.
1
Mp 127.9–128.7 ꢁC. H NMR (300 MHz, DMSO-d6) d
4.9.2. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(ethylamino)-
propyl)amino)carbonyl)-3-methylbutyl)carbamic acid (3S)-
tetrahydrofuran-3-yl ester (18Ab). Colorless crystal. Mp
158.9–160.7 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.83 (d, 3H, J = 6.6), 0.85 (d, 3H, J = 6.9), 1.04 (t, 3H,
J = 7.1), 1.35 (m, 2H), 1.55 (m, 1H), 1.83 (m, 1H),
2.08 (m, 1H), 2.82 (m, 1H), 3.14 (m, 3H), 3.61–3.78
(m, 4H), 4.01 (m, 1H), 5.07 (m, 1H), 5.19 (m, 1H),
7.17–7.33 (m, 6H), 8.22 (d, 1H, J = 7.2), 8.69 (t, 1H,
J = 5.7). MALDI-TOF-MS calcd for C23H33N3O6
(M+H)+, 448.2447, found, 448.2509.
0.54–0.66 (m, 4H), 0.81–0.86 (m, 6H), 1.30–1.42 (m,
2H), 1.57 (m, 1H), 2.73 (m, 1H), 2.82 (dd, 1H,
J = 14.3, 9.2), 3.11 (dd, 1H, J = 13.8, 4.2), 3.24 (s, 3H),
3.42–3.44 (m, 2H), 3.50–3.57 (m, 4H), 3.99–4.04 (m,
3H), 5.17 (m, 1H), 7.22–7.30 (m, 6H), 8.22 (d, 1H,
J = 6.9), 8.71 (d, 1H, J = 4.8). MALDI-TOF-MS calcd
for C25H37N3O7 (M+Na)+, 514.2530, found, 514.2944.
25
D
½aꢁ +13.9 (c 0.20, DMSO).
4.9.8. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(propylamino)-
propyl)amino)carbonyl)-3-methylbutyl)carbamic acid 2-
methoxyethyl ester (18Ca). Colorless crystals. Mp
108.8–109.9 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.83 (m, 9H), 1.35 (m, 2H), 1.46 (m, 2H), 1.55 (m,
1H), 2.83 (dd, 1H, J = 14.0, 9.2), 3.08 (m, 3H), 3.25 (s,
3H), 3.48 (t, 2H, J = 4.4), 4.04 (m, 3H), 5.19 (m, 1H),
7.22–7.28 (m, 6H), 8.24 (d, 1H, J = 6.9), 8.68 (t, 1H,
J = 5.6). MALDI-TOF-MS calcd for C23H35N3O6
(M+H)+, 450.2604, found, 450.2832.
4.9.3. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(ethylamino)-
propyl)amino)carbonyl)-3-methylbutyl)carbamic acid tetra-
hydro-4H-pyran-4-yl ester (18Ac). Colorless crystals. Mp
1
140.0–141.8 ꢁC. H NMR (300 MHz, DMSO-d6) d 0.84
(m, 6H), 1.04 (t, 3H, J = 7.2), 1.35 (m, 2H), 1.49 (m,
3H), 1.79 (m, 2H), 2.82 (m, 1H), 3.14 (m, 3H), 3.41
(m, 2H), 3.78 (m, 2H), 4.02 (m, 1H), 4.66 (m, 1H),
5.19 (m, 1H), 7.15–7.33 (m, 6H), 8.22 (d, 1H, J = 7.2),
8.69 (t, 1H, J = 5.7). MALDI-TOF-MS calcd for
C24H35N3O6 (M+Na)+, 484.2424, found, 484.2486.
4.9.9. ((1S)-1-((((1S)-1-Benzyl-3-(cyclobutylamino)pro-
pyl)amino)carbonyl)-2,3-dioxo-3-methylbutyl)carbamic acid
2-methoxyethyl ester (18Da). Colorless crystals. Mp
114.2–115.3 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.84 (m, 6H), 1.34 (m, 2H), 1.49–1.72 (m, 3H), 2.10
(m, 4H), 2.81 (dd, 1H, J = 13.8, 9.3), 3.10 (m, 1H),
3.25 (s, 3H), 3.47 (m, 2H), 4.03 (m, 3H), 4.22 (m, 1H),
5.15 (m, 1H), 7.24 (m, 6H), 8.24 (d, 1H, J = 7.2), 8.91
(d, 1H, J = 7.8). MALDI-TOF-MS calcd for
C24H35N3O6 (M+Na)+, 484.2424, found, 484.2400.
4.9.4. ((1S)-1-((((1S)-1-Benzyl-3-(cyclopropylamino)pro-
pyl)amino)carbonyl)-2,3-dioxo-3-methylbutyl)carbamic acid
2-methoxyethyl ester (18Ba). Colorless crystals. Mp
112.4–113.5 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.58 (m, 2H), 0.65 (m, 2H), 0.83 (d, 3H, J = 6.6), 0.85
(d, 3H, J = 6.6), 1.35 (m, 2H), 1.56 (m, 1H), 2.68–2.88
(m, 2H), 3.11 (m, 1H), 3.25 (s, 3H), 3.47 (t, 2H,
J = 4.5), 4.04 (m, 3H), 5.17 (m, 1H), 7.17–7.34 (m,
6H), 8.25 (d, 1H, J = 7.2), 8.73 (d, 1H, J = 4.8). MAL-
4.9.10. ((1S)-1-((((1S)-1-Benzyl-3-butylamino-2,3-dioxo-
propyl)amino)carbonyl)-3-methylbutyl)carbamic acid 2-
methoxyethyl ester (18Ea). Colorless crystals. Mp
DI-TOF-MS calcd for C23H33N3O6 (M+Na)+,
25
D
470.2267, found, 470.2441. ½aꢁ +6.3 (c 0.20, DMSO).
1
94.0–95.2 ꢁC. H NMR (300 MHz, DMSO-d6) d 0.85
4.9.5. ((1S)-1-((((1S)-1-Benzyl-3-(cyclopropylamino)-2,3-
dioxopropyl)amino)carbonyl)-3-methylbutyl)carbamic
acid (3S)-tetrahydrofuran-3-yl ester (18Bb). Colorless
crystals. Mp 169.2–170.5 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 0.58 (m, 2H), 0.65 (m, 2H), 0.83 (d, 3H,
J = 8.1), 0.85 (d, 3H, J = 6.9), 1.34 (m, 2H), 1.55 (m,
1H), 1.83 (m, 1H), 2.08 (m, 1H), 2.79 (m, 2H), 3.12
(m, 1H), 3.61–3.80 (m, 4H), 4.02 (m, 1H), 5.08 (m,
1H), 5.17 (m, 1H), 7.22–7.35 (m, 6H), 8.24 (d, 1H,
J = 6.6), 8.74 (d, 1H, J = 5.1). MALDI-TOF-MS
calcd for C24H33N3O6 (M+Na)+, 482.2267, found,
482.2586.
(m, 9H), 1.25 (m, 2H), 1.35 (m, 2H), 1.42 (m, 2H),
1.56 (m, 1H), 2.83 (dd, 1H, J = 13.8, 9.0), 3.10 (m,
3H), 3.25 (s, 3H), 3.47 (t, 2H, J = 4.5), 4.04 (m, 3H),
5.18 (m, 1H), 7.21–7.29 (m, 6H), 8.23 (d, 1H, J = 6.6),
8.67 (t, 1H, J = 6.0). MALDI-TOF-MS calcd for
C24H37N3O6 (M+H)+, 464.2760, found, 464.2870.
4.9.11. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(2,2,2-trifluoro-
ethylamino)propyl)amino)carbonyl)-3-methylbutyl)carbamic
acid 2-methoxyethyl ester (18Fa). Colorless crystals. Mp
1
152.5–153.9 ꢁC. H NMR (300 MHz, DMSO-d6) d 0.84
(m, 6H), 1.34 (m, 2H), 1.55 (m, 1H), 2.86 (dd, 1H,

4482
Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
J = 14.0, 8.6), 3.10 (dd, 1H, J = 14.1, 4.8), 3.25 (s, 3H),
3.48 (t, 2H, J = 4.7), 3.90 (m, 2H), 4.04 (m, 3H), 5.14
(m, 1H), 7.21–7.31 (m, 6H), 8.34 (d, 1H, J = 6.9), 9.29
(m, 1H). MALDI-TOF-MS calcd for C22H30F3N3O6
(M+H)+, 490.2165, found, 490.2434.
DI-TOF-MS calcd for C24H35N3O6 (M+H)+,
462.2604, found, 462.2742.
4.9.17. ((1S)-1-((((1S)-3-Cyclopropylamino-2,3-dioxo-1-
(phenylethyl)propyl)amino)carbonyl)-3-methylbutyl)car-
bamic acid (3S)-tetrahydrofuran-3-yl ester (19Bb). Color-
1
4.9.12. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(2-indanyl-
amino)propyl)amino)carbonyl)-3-methylbutyl)carbamic acid
2-methoxyethyl ester (18Ga). Colorless crystals. Mp
141.9–143.5 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.83 (d, 3H, J = 6.9), 0.86 (d, 3H, J = 6.9), 1.36 (m,
2H), 1.57 (m, 1H), 2.80–2.96 (m, 3H), 3.10–3.18 (m,
3H), 3.24 (s, 3H), 3.47 (t, 2H, J = 4.7), 4.04 (m, 3H),
4.50 (m, 1H), 5.19 (m, 1H), 7.13–7.30 (m, 10H), 8.29
(d, 1H, J = 6.9), 8.97 (d, 1H, J = 7.2). MALDI-TOF-
MS calcd for C29H37N3O6 (M+H)+, 524.2760, found,
524.2810.
less crystals. Mp 115.8–116.2 ꢁC. H NMR (300 MHz,
DMSO-d6) d 0.56–0.59 (m, 4H), 0.88 (t, 6H, J = 6.3),
1.42 (t, 2H, J = 7.4), 1.60–1.91 (m, 3H), 2.09 (m, 2H),
2.56–2.76 (m, 3H), 3.63–3.81 (m, 4H), 4.05–4.13 (m,
1H), 4.87 (m, 1H), 5.09–5.13 (m, 1H), 7.20–7.35 (m,
6H), 8.34 (d, 1H, J = 6.9), 8.69 (d, 1H, J = 5.1). MAL-
DI-TOF-MS calcd for C25H35N3O6 (M+H)+,
474.2604, found, 474.2598.
4.9.18. ((1S)-1-((((1S)-1-Benzyl-3-butylamino-2,3-dioxo-
propyl)amino)carbonyl)-3-methylbutyl)carbamic acid ben-
zyl ester (18Ee). Colorless crystals. Mp 150.1–153.8
1
4.9.13. ((1S)-1-((((1S)-1-Benzyl-2,3-dioxo-3-(2-methoxy-
ethylamino)propyl)amino)carbonyl)-3-methylbutyl)carbamic
acid 2-methoxyethyl ester (18Ha). Colorless crystals. Mp
ꢁC. H NMR (300 MHz, DMSO-d6) d 0.85 (m, 9H),
1.24 (m, 2H), 1.33–1.49 (m, 4H), 1.58 (m, 1H), 2.83
(m, 1H), 3.11 (m, 3H), 4.07 (m, 1H), 5.01 (s, 2H), 5.19
(m, 1H), 7.23 (m, 5H), 7.35 (m, 6H), 8.28 (d, 1H,
J = 7.2), 8.67 (t, 1H, J = 5.9). MALDI-TOF-MS calcd
for C28H37N3O5 (M+Na)+, 518.2631, found, 518.2847.
1
127.0–127.9 ꢁC. H NMR (300 MHz, DMSO-d6) d 0.83
(d, 3H, J = 6.9), 0.86 (d, 3H, J = 6.9), 1.35 (m, 2H), 1.56
(m, 1H), 2.83 (dd, 1H, J = 13.8, 9.0), 3.11 (dd, 1H,
J = 14.0, 4.4), 3.24 (s, 3H), 3.25 (s, 3H), 3.16–3.34 (m,
2H), 3.39 (m, 2H), 3.48 (t, 2H, J = 4.5), 4.04 (m, 3H),
5.20 (m, 1H), 7.18–7.30 (m, 6H), 8.21 (d, 1H, J = 6.9),
8.66 (t, 1H, J = 5.4). MALDI-TOF-MS calcd for
C23H35N3O7 (M+Na)+, 488.2373, found, 488.2680.
4.10. Determination of water-solubility
A suspension of inhibitor (1–5 mg) in pH 7 buffer solu-
tion (2.0 mL) was shaken at 25 ꢁC for 5 h. The suspen-
sion was filtered through CHROMATODISKꢂ
(0.45 lm, GL Sciences), and the filtrate (1.0 mL) was
quantified by the HPLC–UV method.
4.9.14. ((1S)-1-((((1S)-2,3-Dioxo-3-ethylamino-1-(phenyl-
ethyl)propyl)amino)carbonyl)-3-methylbutyl)carbamic acid
2-methoxyethyl ester (19Aa). Colorless crystals. Mp
119.1–120.4 ꢁC. 1H NMR (300 MHz, DMSO-d6) d
0.89 (t, 6H, J = 6.3), 1.03 (t, 3H, J = 7.2), 1.43 (t, 2H,
J = 7.2), 1.61–1.85 (m, 2H), 2.07 (m, 1H), 2.56–2.74
(m, 2H), 3.07–3.17 (m, 2H), 3.25 (s, 3H), 3.49 (t, 2H,
J = 4.7), 4.05–4.14 (m, 3H), 4.89 (m, 1H), 7.16–7.36
(m, 5H), 7.34 (d, 1H, J = 8.4), 8.33 (d, 1H, J = 6.9),
8.65 (t, 1H, J = 5.9). MALDI-TOF-MS calcd for
C23H35N3O6 (M+H)+, 450.2604, found, 450.2701.
4.11. Inhibition assays for calpains
Inhibition assays were performed as described in the
previous literature10,11 using commercial l-calpain (hu-
man erythrocyte, Calbiochem) and m-calpain (porcine
kidney, Calbiochem). Assay solution including 0.5 mg/
mL casein, 20 mM dithiothreitol, 50 mM Tris–HCl
(pH 7.4), and 1.0 nmol of enzyme was used. The assay
solution (200 lL) and DMSO (2.5 lL) including inhibi-
tor of different concentrations were placed in each well.
Reaction was started by addition of 20 mM CaCl2
(50 lL) in a test well and 1 mM EDTA (50 lL) in a
blank well. After incubation for 60 min at 30 ꢁC, the
mixture (100 lL) was transferred to another plate in
which H2O (100 lL) and Bio-Rad protein assay dye re-
agent (50 lL) were placed in each well. After incubation
at room temperature for 15 min, the OD of the mixture
was taken at 595 nm with a plate reader (Multiscan
Multisoft, Labsystems). The percent inhibition was cal-
culated from the difference of OD between the presence
and absence of the compound. The IC50 was obtained
from the graphical analysis of the concentration and
the inhibition.
4.9.15. ((1S)-1-((((1S)-2,3-Dioxo-3-ethylamino-1-(phenyl-
ethyl)propyl)amino)carbonyl)-3-methylbutyl)carbamic acid
(3S)-tetrahydrofuran-3-yl ester (19Ab). Colorless crys-
1
tals. Mp 111.9–114.5 ꢁC. H NMR (300 MHz, DMSO-
d6) d 0.89 (t, 6H, J = 6.3), 1.03 (t, 3H, J = 7.2), 1.43 (t,
2H, J = 7.4), 1.60–1.91 (m, 3H), 2.09 (m, 2H), 2.56–
2.76 (m, 2H), 3.07–3.17 (m, 2H), 3.63–3.82 (m, 4H),
4.02–4.13 (m, 1H), 4.88 (m, 1H), 5.09–5.13 (m, 1H),
7.16–7.31 (m, 5H), 7.34 (d, 1H, J = 8.4), 8.34 (d, 1H,
J = 6.9), 8.66 (t, 1H, J = 5.7). MALDI-TOF-MS calcd
for C24H35N3O6 (M+H)+, 462.2604, found, 462.2870.
4.9.16. ((1S)-1-((((1S)-2,3-Dioxo-3-cyclopropylamino-1-
(phenylethyl)propyl)amino)carbonyl)-3-methylbutyl)car-
bamic acid 2-methoxyethyl ester (19Ba). Colorless
crystals. Mp 109.7–111.1 ꢁC. 1H NMR (300 MHz,
4.12. Metabolic stability
DMSO-d6)
d 0.53–0.68 (m, 4H), 0.87–0.91 (m,
6H), 1.43 (t, 3H, J = 7.2), 1.59–1.85 (m, 2H), 2.01–2.13
(m, 1H), 2.56–2.74 (m, 3H), 3.25 (s, 3H), 3.48–3.51 (m,
2H), 4.05–4.14 (m, 3H), 4.87 (m, 1H), 7.17–7.36 (m,
6H), 8.34 (d, 1H, J = 6.6), 8.69 (d, 1H, J = 5.1). MAL-
Human hepatic S9 fraction (S9) incubations were per-
formed in the presence of an NADPH-generating system
composed of 3 mM MgCl2, 1 mM NADP+, 5 mM glu-
cose-6-phosphate, and 1 Unit/mL glucose-6-phosphate

Y. Shirasaki et al. / Bioorg. Med. Chem. 13 (2005) 4473–4484
4483
dehydrogenase in a 50 mM potassium phosphate buffer
(pH 7.4). All concentrations are relative to the final
incubation volume. The compounds were added in ace-
tonitrile to a final concentration of 5 lM. Incubations
were conducted at 37 ꢁC. After 30 min, aliquots of incu-
bations (1 mL) were terminated by addition of 4 mL of
acetonitrile. The precipitated proteins were removed by
centrifugation, and supernatants were evaporated and
the residue was dissolved in the mobile phase. The solu-
tion was analyzed by the HPLC–UV method.
a UV detector (monitoring at 250 nm). Aliquots (5–
20 lL) of the sample solution were injected onto the
HPLC column. The retention time (tR) of each com-
pound was determined and the k0IAM values were calcu-
lated using the following equation:
k0IAM ¼ ðtR ꢀ t0Þ=t0;
where tR is the retention times (min) of the test com-
pound and t0 corresponds to the void volume time.
The Logk0IAM (LogK0) value of propranolol was deter-
mined to be 1.95.
4.13. Pharmacokinetic studies in monkey
Acknowledgments
The in vivo pharmacokinetic results were determined
after oral (10 mg/kg, n = 2) single agent dosing to female
cynomolgus monkeys. The compounds were adminis-
trated using a 0.5% carboxymethyl cellulose suspension
formulation by nasogastric tube. Blood samples from
each dosing were taken at pre-determined times (0.5,
1, 2, and 4 h) for analysis. All plasma samples were fro-
zen and stored in ꢀ30 ꢁC until analysis. The plasma con-
centration of each test compound was determined by the
HPLC–UV method.
The authors would like to thank Yuji Sakamoto, Atsu-
ko Fujii, Misae Ohtaka, and Kazuko Nishiyama for
their support of this work. Also, we would like to thank
Drs. Yutaka Kawamatsu and Mitsuyoshi Azuma for
useful discussions.
References and notes
1. (a) Ono, Y.; Sorimachi, H.; Suzuki, K. In Calpain:
Pharmacology and toxicology of calcium-dependent prote-
ase; Wang, K. K. W., Yuen, P.-W., Eds.; Taylor &
Francis: Philadelphia, 1999, pp 1–23; (b) Huang, Y.;
Wang, K. K. W. Trends Mol. Med. 2001, 7, 355.
2. (a) Wang, K. K. W.; Yuen, P.-W. Trends Pharmacol. Sci.
1994, 15, 412; (b) Stracher, A. Ann. NY Acad. Sci. 1999,
884, 52.
3. (a) Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med.
Chem. 2000, 43, 305; (b) Donkor, I. O. Curr. Med. Chem.
2000, 7, 1171.
4. (a) Bartus, R. T.; Baker, K. L.; Heiser, A. D.; Sawyer, S.
D.; Dean, R. L.; Elliott, P. J.; Straub, J. A. J. Cereb. Blood
Flow Metab. 1994, 14, 537; (b) Herbeson, S. L.; Abelleira,
S. M.; Akiyama, A.; Barrett, R., III; Carroll, R. M.;
Strauh, J. A.; Tkacz, J. N.; Wu, C.; Mussi, G. F. J. Med.
Chem. 1994, 37, 2918.
5. (a) Bartus, R. T.; Hayward, N. J.; Elliott, P. J.; Sawyer, S.
D.; Baker, K. L.; Dean, R. L.; Akiyama, A.; Straub, J. A.;
Harbenson, S. L. Stroke 1994, 25, 2265; (b) Li, Z.; Ortega-
Vilain, A.; Patil, G. S.; Chu, D.; Foreman, J. E. J. Med.
Chem. 1996, 39, 4089.
6. 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. J. Med.
Chem. 2003, 46, 2404.
7. (a) Fukiage, C.; Azuma, M.; Nakamura, Y.; Tamada, Y.;
Nakamura, M.; Shearer, T. R. Biochim. Biophys. Acta
1997, 1361, 304; (b) Fukiage, C.; Azuma, M.; Nakamura,
Y.; Tamada, Y.; Shearer, T. R. Curr. Eye Res. 1998, 17,
623; (c) Tamada, Y.; Fukiage, C.; Mizutani, K.; Yamag-
uchi, M.; Nakamura, Y.; Azuma, M.; Shearer, T. R. Curr.
Eye Res. 2001, 22, 280; (d) Inoue, J.; Nakamura, M.; Cui,
Y.; Sakai, Y.; Sakai, O.; Hill, J. R.; Wang, K. K. W.;
Yuen, P.-W. J. Med. Chem. 2003, 46, 868.
4.14. Caco-2 permeability studies18
Caco-2 cells were cultivated under aseptic conditions at
37 ꢁC in an atmosphere of 90% relative humidity (5%
CO2). The culture medium consisted of 10% fetal bovine
serum, antibiotic–antimicotic solution (GIBCO-BRL),
non-essential amino acids solution (GIBCO-BRL), and
2 M L-glutamate. The culture medium was replaced
every 2–3 days. The Caco-2 cells were seeded onto 24-
well transwell plates (1 · 105 cells/cm2) and grown for
22 days until monolayer was apparent.
The chamber was preincubated for 1 h at 37 ꢁC in HBSS
(apical, pH 6.5, 300 lL: basolateral, pH 7.4, 1000 lL).
The test compounds and permeability reference markers
(propranolol and 14C-mannitol) were dissolved in HBSS
with 0.1% DMSO (final concentration: 10 lM).
Independently, the test compounds and the reference
markers were added to the apical side of the cell mono-
layer. Aliquots (500 lL) were removed from this com-
partment at 60 and 120 min. The concentration of the
test samples were determined by LC/MS/MS or a liquid
scintillation counter (14C-mannitol) and Papp values were
calculated as described in the literature. The Papp values
of propranolol were determined to be 6.8 · 10ꢀ6 cm/s
(at 60 min) and 8.5 · 10ꢀ6 cm/s (at 120 min) while those
of 14C-mannitol were found to be 0.8 · 10ꢀ6 cm/s (at
60 min) and 0.6 · 10ꢀ6 cm/s (at 120 min).
4.15. IAM Analysis19
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