Exploration of cornea permeable calpain inhibitors as anticataract agents

Article abstract of DOI:10.1016/S0968-0896(02)00612-0

To explore cornea permeable calpain inhibitors, four compounds displaying different characteristics were designed and synthesized based on the known potent calpain inhibitor, peptidyl aldehyde SJA6017. Two approaches were adopted; an improvement in the physicochemical properties, and conversion of the active aldehyde. The water-soluble peptidyl aldehyde 1 containing a pyridine ring at the P3 site showed a modest inhibition against calpains and an improvement of corneal permeability in comparison with SJA6017. Replacement of the aldehyde of SJA6017 by an α-ketoamide provided compound 2 that was approximately equipotent with SJA6017, but it was extremely water-insoluble. However, compound 3, in which the aldehyde was converted into a cyclic hemiacetal, proved to be a less potent calpain inhibitor than SJA6017, but demonstrated excellent transcorneal permeability. Further modification generating the cyclic hemiacetal 4 containing a thiourea linker between the P3 and P2 sites exhibited potent inhibitory activities, high cornea permeability and excellent efficacy in the rat lens culture cataract model.

Full text of DOI:10.1016/S0968-0896(02)00612-0

Bioorganic & Medicinal Chemistry 11 (2003) 1371–1379
Exploration of Cornea Permeable Calpain Inhibitors as
Anticataract Agents
Masayuki Nakamura,* Masazumi Yamaguchi, Osamu Sakai and Jun Inoue
Research Laboratory, Senju Pharmaceutical Co., Ltd., 1-5-4 Murotani Nishiku Kobe 651-2241, Japan
Received 25 July 2002;accepted 20 November 2002
Abstract—To explore cornea permeable calpain inhibitors, four compounds displaying different characteristics were designed and
synthesized based on the known potent calpain inhibitor, peptidyl aldehyde SJA6017. Two approaches were adopted;an improve-
ment in the physicochemical properties, and conversion of the active aldehyde. The water-soluble peptidyl aldehyde 1 containing a
pyridine ring at the P3 site showed a modest inhibition against calpains and an improvement of corneal permeability in comparison
with SJA6017. Replacement of the aldehyde of SJA6017 by an a-ketoamide provided compound 2 that was approximately equi-
potent with SJA6017, but it was extremely water-insoluble. However, compound 3, in which the aldehyde was converted into a
cyclic hemiacetal, proved to be a less potent calpain inhibitor than SJA6017, but demonstrated excellent transcorneal permeability.
Further modification generating the cyclic hemiacetal 4 containing a thiourea linker between the P3 and P2 sites exhibited potent
inhibitory activities, high cornea permeability and excellent efficacy in the rat lens culture cataract model.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
vent induced-cataract in lens culture models,4 they have
yet to be launched commercially.
Calpains are nonlysosomal calcium-dependent cysteine
proteases and the family of these enzymes has grown
rapidly in recent years.1 These well-known m-calpain
and m-calpain enzymes are ubiquitously found in
mammalian cells, and they are implicated in a variety of
biological processes and numerous diseases.2
To investigate novel calpain inhibitors as anticataract
agents that have the superior cornea permeability, we
attempted a synthetic program based on the lead com-
pound SJA6017 using two approaches. The one
approach was to obtain a water-soluble calpain inhibitor
by conversion of the practically insoluble SJA6017 to
incorporate a pyridine ring at the P3 site with the view of
studying the physicochemical properties (Fig. 1). It is
well known that solubility is the most important factor
for studying the absorption of the drug, since only com-
pounds dissolved in solution are able to permeate across
the barrier.5 The other approach was employed to con-
vert the active aldehyde group into a further functional
moiety. The aldehyde group is an essential active site and
forms a hemithioacetal with the active SH on the cysteine
residue of the enzyme. However, the chemical reactivity
of the aldehyde is considered to be potentially high, and
it may superfluously react with various substances under
physiological conditions. Therefore, as an exploration of
calpain inhibitors without the aldehyde, a-ketoamide
and cyclic hemiacetal moieties were introduced into the
SJA6017 backbone. An original a-ketoamide calpain
inhibitor, AK-275 was demonstrated to be an effective-
ness in an animal model of stroke.6 Then cyclic hemi-
acetals have been reported by the Mitsubishi group as
Cataract is a major disease of the lens and the leading
cause of blindness in the world. Proteolysis of lens
proteins, crystallins, by activated calpain has been
hypothesized to be one cause of cataract formation,
which involves interrupting the normal interaction
between lens proteins leading to aggregation, insolubi-
lization and light scattering.3 Although there is a pos-
sibility of topical and oral administrations as the
anticataract drugs, topical administration is more
advantageous than oral administration from the view-
point of systemic side effect. The transcorneal perme-
ability of the drug is an important factor to treat
intraocular diseases like cataract with ophthalmic
solution. In fact, synthetic calpain inhibitors such as
SJA6017 and E-64-d have been demonstrated to pre-
*Corresponding author. Tel.:+81-78-997-1010;fax:+81-78-997-1016;
e-mail: masayuki-nakamura@senju.co.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(02)00612-0

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M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
through a change in the physicochemical properties or
conversion of the aldehyde as the essential active site of
the inhibitor. In this report, we describe the synthesis of
SJA6017-based calpain inhibitors, their inhibitory
activity and the relationship between the structure and
the physicochemical properties in addition to cornea
permeability. Furthermore, we report the discovery of a
thiourea compound in our efforts to improve the inhib-
itory activity as well as the cornea permeability and
demonstrate its anticataract efficacy in the rat lens cul-
ture cataract model.
Results and Discussion
Chemistry
Synthesis of the water-soluble aldehyde 1 is detailed in
Scheme 1. Peptidic condensation of commercially avail-
able N-carbobenzoxy-l-valine N-hydroxysuccinimide
ester (5) and l-leucinol afforded 6, which was then
oxidized with DMSO in the presence of sulfurtrioxide/
pyridine complex (SO3/pyridine) to give peptidyl
aldehyde 7. The aldehyde was protected by the formation
of an acetal with ethylene glycol, and subsequent
hydrogenolysis of the carbobenzoxy-protecting group
afforded intermediate 9. Acylation of 9 with 3-pyridyl-
acetic acid followed by hydrolysis of the acetal provided
aldehyde 1.
Figure 1. Structures of SJA6017 and calpain inhibitors 1–4.
calpain inhibitors.7 Since this hemiacetal is capable of
forming a cyclized structure between the aldehyde and
the hydroxy group of the P1 residue, this formation
potentially may offer moderate protection in the reactiv-
ity of the aldehyde. We predicted that undesirable che-
mical reaction of the aldehyde might be prevented and an
improvement in the cornea permeability could be
achieved by such modifications.
Synthesis of peptidyl a-ketoamide 2 is illustrated in
Scheme 2. Sulfonylation of l-valine (11) with 4-fluoro-
phenylsulfonylchloride, and condensation of the result-
ing intermediate 12 and N-hydroxysuccinimide (HOSu)
in the presence of 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide hydrochloride (EDCHCl) provided
active ester 13. Treatment of 13 with l-leucinol followed
We conducted studies to establish the most effective
method for an improvement in cornea permeability,
Scheme 1. Conditions: (a) l-leucinol, EtOAc;(b) SO 3/pyridine, DIPEA, DMSO;(c) ethylene glycol p-TsOH/pyridine, toluene;(d) Pd/C, EtOH;(e)
.
3-pyridine acetic acid HCl, HOBt, Et3N, EDC HCl, DMF/CH2Cl2;(f) 1 M HCl;(g) 4 M HCl/EtOAc.
.
.
Scheme 2. Conditions: (a) 4-fluorophenylsulfonylchloride, NaOH, THF/H2O;(b) HOSu, EDC HCl, THF/CH2Cl2;(c) l-leucinol, EtOAc;(d) SO 3/
pyridine, DIPEA, DMSO;(e) n-butylisocyanide, TFA, pyridine, CH2Cl2;(f) Dess–Martin periodinane, CH Cl2.
2

M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
1373
by a DMSO oxidation in the presence of SO3/pyridine
provided peptidyl aldehyde 15. Passerini reaction of
aldehyde 15, butylisocyanide and trifluoroacetic acid
and subsequent Dess–Martin oxidation produced the
a-ketoamide 2.8
tetrahydrofuran moiety in the structure of 3. Therefore,
we took an interest in cyclic hemiacetal and applied it in
the synthesis with the objective of producing more
potent calpain inhibitors.
In the next stage, a cyclic hemiacetal containing a
thiourea linker between the P3 and P2 sites, compound
4 was synthesized and tested. This resulting compound
was about 10-fold more potent against both m- and
m-calpain than compound 3. An investigation into the
physicochemical properties of hemiacetals 3 and 4
revealed almost identical solubilities and logP (partition
coefficient) values for both compounds. Therefore, we
have successfully produced various types of calpain
inhibitors by altering the physicochemical properties or
by replacement of the aldehyde for use in cornea per-
meability experiments.
Cyclic hemiacetal 3 was synthesized from active ester 13
as shown in Scheme 3. Thus, treatment of compound 13
with a-amino-g-butyrolactone gave lactone 17, and
reduction of this compound in the presence of diiso-
butylaluminum hydride (DIBAL-H) afforded target
compound 3.
The synthesis of cyclic hemiacetal 4 containing a
thiourea linker is shown in Scheme 4. Boc-l-leucine (18)
was coupled with a-amino-g-butyrolactone to give lac-
tone 20 via the active N-hydroxysuccinimide ester 19.
Boc-deprotection of 20 and treatment with phenyl-
isothiocyanate followed by reduction with DIBAL-H
provided thiourea compound 4.
Transcorneal permeation
Plots of the aqueous humor concentration of drugs after
topical administration to albino rabbits are shown in
Figure 2. The area under the curves (AUC0!3 h) of
aldehyde SJA6017, water-soluble aldehyde 1, hemiacetal
3 and thiourea-contained hemiacetal 4 were 0.051, 0.31,
0.39 and 1.7 mg/mLh, respectively. The a-ketoamide 2
was not tested since its extremely low solubility would
be expected to result in low permeability.
Enzyme inhibition and physicochemical properties
Although SJA6017 is a potent calpain inhibitor, it is
poorly soluble in water (Table 1). Replacement of the
P3 site of SJA6017 by a pyridine ring provided the
water-soluble peptidyl aldehyde 1, but this compound
demonstrated modest inhibition of calpains. Further-
more, compound 1 displayed a slight undesirable inhib-
itory activity against proteasome.
Table 1. Inhibitory activity and physicochemical properties
Compd
IC50 (mM)
Water-solubilityd
(mg/mL)
Compound 2, which possessed an a-ketoamide in the
place of an aldehyde, showed almost identical activity
against m-calpain as SJA6017 and marginally superior
activity against m-calpain (2-fold). However, compound
2 was found to be remarkably insoluble in water as
compared to SJA6017 (0.0053 vs 0.10 mg/mL). In con-
trast, conversion to the cyclic hemiacetal 3 resulted in a
loss of potency against m- and m-calpain. Interestingly,
compound 3 has a 15-fold higher water-solubility than
SJA6017, even though it is an uncharged compound
with a very similar structure to SJA6017. This phenom-
enon appears to be a consequence of the presence of a
LogP
m-Calpaina m-Calpainb
20S
Proteasomec
1
2
3
0.083
0.021
0.88
0.086
0.022
0.33
0.021
2.6
0.19
0.049
15
>100
>100
>100
>100
>100
0.0053
1.5
1.5
0.10
0.025
>3
0.60
0.70
1.7
4
SJA6017
aHuman erythrocyte m-calpain.
bPorcine kidney m-calpain.
cHuman recombinant 20S proteasome.
dWater-solubility in pH 7 buffer, at 25 ꢀC.
.
Scheme 3. Conditions: (a) S-a-amino-g-butyrolactone HCl, Et3N, DMF;(b) DIBAL-H, CH Cl2.
2
.
.
Scheme 4. Conditions: (a) HOSu, EDC HCl, THF/CH2Cl2;(b) S-a-amino-g-butyrolactone HBr, Et3N, DMF;(c) 4 M HCl/EtOAc;(d) phenyl-
isothiocyanate, Et3N, EtOAc;(e) DIBAL-H, CH Cl2.
2

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M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
the lens by day 5 of culture (Fig. 3, A23187). Most
importantly, hemiacetal (100 mM) delayed the
4
appearance of the dense nuclear opacity against
A23187-induced cataract (Fig. 3, A23187+4). Thus, we
have demonstrated the efficiency of hemiacetal 4 as an
anticataract agent in the rat lens culture model.
Conclusions
We have provided a novel strategy for exploration of
cornea permeable calpain inhibitors. Among the four
synthesized inhibitors with different characteristics,
a-ketoamide 2 was the most potent calpain inhibitor,
however this compound was extremely water-insoluble.
Although water-soluble aldehyde 1 having a pyridine
ring at the P3 site, and compound 3 in which the alde-
hyde of SJA6017 was replaced with a cyclic hemiacetal,
both displayed reduced inhibitory activities against cal-
pains, they showed improved transcorneal permeability.
From these results, it was concluded that existence of
the aldehyde and the solubility affected the cornea per-
meability in the case of SJA6017. Hemiacetal 4 con-
taining a thiourea linker between P3 and P2 sites was
superior to hemiacetal 3 in view of both the inhibitory
activity (m-calpain: 10-fold, m-calpain: 14-fold) and
corneal permeability (AUC: 3-fold). Furthermore,
hemiacetal 4 exhibited the efficacy against A23187-
induced nuclear cataract in the rat lens culture model.
Future work is aimed at generating inhibitors that
demonstrate superior activity combined with excellent
cornea permeability based on our knowledge presented
in this paper. The expansion of this series of calpain
inhibitors will be reported in due course.
Figure 2. Mean aqueous humor levels after topically administration of
0.5% compound 1 in aqueous solution, 0.5% compound 3 in aqueous
suspension and 0.5% compound 4 in aqueous suspension. Each dosing
volume was 50 mL (single instillation, n=3 eyes).
Water-soluble aldehyde 1 and hemiacetal 3 showed an
improved permeability as compared to SJA6017, and
the AUC values increased to approximately 6-fold and
8-fold, respectively. Both the increased solubility and
the conversion of the aldehyde may have contributed to
this increase in transcorneal permeability. It appears
that while SJA6017 has limiting physicochemical prop-
erties, the aldehyde lowers the permeability by adsorb-
ing to certain other substances present in cornea.
However, the improvement of permeability of hemi-
acetal 3, was considered to be a consequence of not only
removal of the aldehyde, but also the effect of increased
solubility. Interestingly, the thiourea-contained hemi-
acetal 4 revealed a marked improvement in the perme-
ability, with the increase in AUC values of about
33-fold of SJA6017 and 4-fold of hemiacetal 3.
Although hemiacetals 3 and 4 have a similar structure
and comparable physicochemical properties such as
water-solubility and logP (Table 1), the two compounds
have significantly different melting points (3: mp
162–164 ꢀC, 4: mp 62–64 ꢀC). A transdermal absorption
study has reported a correlation between the melting
point and transdermal absorption.9 Similarly to trans-
dermal absorption, transcorneal permeation through
ophthalmic aqueous suspension might be also affected
by the melting point of drugs, and the higher permea-
tion of hemiacetal 4 might be attributed to the lower
melting point.
Experimental
General. Melting points were determined on a Yanaco
1
micro melting point apparatus without correction. H
NMR spectra were recorded on a Varian Gemini-2000
spectrometer. Chemical shifts are reported in parts per
million, and coupling constants (J) are reported in
Hertz. The NMR spectra of compounds 3 and 4 were
extremely complicated due to the presence of anomeric
carbon. The major and minor peaks of the same proton
were separately listed, in case each peak was identified.
Elemental analyses were performed on an Elementar
Vario EL analyzer. FAB high-resolution mass spectra
were obtained by Jeol JMS-700T mass spectrometer.
Matrix-assisted laser desorption ionization time-of-
flight mass spectra (MALDI-TOF MS) were obtained
on a Perseptive Voyager DE PRO mass spectrometer
using a-cyano-4-hydroxycinamic acid as the matrix.
Optical rotations were measured in a Horiba SEPA-
2000 model.
Cultured lens study
The effectiveness of hemiacetal 4 as anticataract agent
was evaluated on the calcium ionophore (A23187)
induced rat lens culture cataract model, and the results
are summarized in Figure 3. Lenses that were cultured
for 5 days in the medium remained clear and were
similar in appearance to fresh lenses (Fig. 3, Normal).
On the other hand, the addition of 10 mM A23187
caused a dense opacity to appear in the central region of
(2S)-2-(Benzyloxycarbonylamino)-N-((1S)-1-(hydroxy-
methyl)-3-methylbutyl)-3-methylbutanamide (6). To a
solution of N-carbobenzoxy-l-valine N-hydroxy-
succinimide ester (30 g, 86 mmol) in EtOAc (400 mL)

M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
1375
Figure 3. Representative photomicroscopy of A23187-induced cataract and inhibition by hemiacetal 4 (100 mM) on day 5 of culture. Darker areas
are cataracts in these backlit lenses. Insert bar graph shows increased density of nuclear opacity in cultured lenses treated with A23187 and reduction
of opacity by hemiacetal 4. Data are mean ÆSD (n=5). *P<0.01 relative to A23187.
was added l-leucinol (12 g, 100 mmol). The mixture was
stirred at room temperature for 18 h. Resulting pre-
cipitate was dissolved by addition of EtOAc, and the
solution was washed with 1 M HCl, saturated NaHCO3
and saturated NaCl, dried over MgSO4, and con-
centrated in vacuo. The residue was crystallized from
hexane to give 6 (19 g, 63%) as colorless crystals. Mp
mmol) in DMSO (70 mL) were added under the ice-cool
condition. The mixture was stirred for 30 min under the
same condition. The reaction 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 resulting white
solid was crystallized from EtOAc/hexane to give 7 (12
65–66 ꢀC. H NMR (300 MHz, DMSO-d6) d 0.80–0.87
g, 63%) as colorless crystals. Mp 123–125 ꢀC. H NMR
1
1
(m, 12H), 1.26–1.37 (m, 2H), 1.59 (m, 1H), 1.92 (m,
1H), 3.18 (m, 1H), 3.30 (m, 1H), 3.77–3.83 (m, 2H), 4.61
(m, 1H), 4.99–5.08 (m, 2H), 7.21 (d, 1H, J=8.7), 7.31–
7.36 (m, 5H), 7.51 (d, 1H, J=8.7). Anal. calcd for
C19H30N2O4: C, 65.12;H, 8.63;N, 7.99;found: C,
65.06;H, 8.44;N, 7.79.
(300 MHz, DMSO-d6) d 0.84–0.91 (m, 12H), 1.37–1.65
(m, 3H), 2.00 (m, 1H), 3.90 (m, 1H), 4.12 (m, 1H), 5.04
(s, 2H), 7.31–7.37 (m, 6H), 8.34 (d, 1H, J=6.9), 9.39 (s,
1H). FAB-HRMS calcd for C19H29N2O4 [M+H]+:
349.2127, found: 349.2119.
(2S) - N - ((1S) - 1 - (2,5 - Dioxolanyl) - 3 - methylbutyl) - 3 -
methyl-2-(benzyloxycarbonylamino)butanamide (8). To a
solution of 7 (11 g, 31 mmol) in toluene (300 mL) was
added ethylene glycol (9.8 g, 160 mmol) and pyridinium
toluenesulfonate (1.6 g, 6.3 mmol). The mixture was
stirred at 80 ꢀC for 4 h. The mixture was washed with
(2S)-2-(Benzyloxycarbonylamino)-N-((1S)-1-formyl-3-
methylbutyl)-3-methylbutanamide (7). Compound 6 (19
g, 54 mmol) was dissolved in DMSO (150 mL) and
CH2Cl2 (70 mL). N,N-Diisopropylethylamine (28 g, 220
mmol) and a suspension of SO3/pyridine (35 g, 220

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M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
1 M HCl, saturated NaHCO3 and saturated NaCl, dried
over MgSO4, and concentrated in vacuo. Resulting
white solid was crystallized from EtOAc/hexane to give
1H), 3.51 (d, 1H, J=14.1), 3.60 (d, 1H, J=14.1), 4.12
(m, 1H), 4.21 (m, 1H), 7.32 (m, 1H), 7.67 (d, 1H,
J=7.5), 8.28 (d, 1H, J=9.0), 8.41–8.47 (m, 3H), 9.39 (s,
25
8 (12 g, 97%) as colorless crystals. Mp 106–107 ꢀC. H
1H). ½aꢁD ꢂ24.1ꢀ (c=0.203). This compound was dis-
1
NMR (300 MHz, DMSO-d6) 0.79–0.87 (m, 12H), 1.21
(m, 1H), 1.38 (m, 1H), 1.57 (m, 1H), 1.93 (m, 1H), 3.73–
3.9 (m, 5H), 4.01 (m, 1H), 4.71 (d, 1H, J=2.4), 4.98-
5.09 (m, 2H), 7.18 (d, 1H, J=9.0), 7.31–7.35 (m, 5H),
7.54 (d, 1H, J=9.3). Anal. calcd for C21H32N2O5: C,
64.26;H, 8.22;N, 7.14;found: C, 64.17;H, 7.95;N,
7.00.
solved in EtOAc, and 4 M HCl/EtOAc (0.40 mL) was
added under the ice-cool condition. Resulting pre-
cipitate was collected and washed with EtOAc to give 1
(0.40 g, 86%) as colorless crystals. Mp 79–80 ꢀC. Anal.
calcd for C18H27N3O3HClH2O: C, 55.73;H, 7.80;N,
10.83;found: C, 55.58;H, 7.66;N, 10.63. MALDI-TOF
MS calcd for C18H27N3O3Na [M+Na]+: 356.195,
found: 356.196.
(2S)-2-Amino-N-((1S)-1-(2,5-dioxolanyl)-3-methylbutyl)-
3-methylbutanamide (9). Compound 8 (12 g, 31 mmol)
was dissolved in EtOH (250 mL) and hydrogenated at
room temperature under atmosphere pressure over pal-
ladium carbon powder (Pd: 10%) (2.0 g). After stirring
for 72 h, palladium carbon was filtered off, and the
mixture was concentrated in vacuo to give 9 (7.5 g,
N-((4-Fluorophenyl)sulfonyl)-L-valine (12). To a solution
of l-valine (11) (82 g, 700 mmol) in 1 M NaOH (700
mL), THF (500 mL) and water (500 mL), a solution of
4-fluorobenzenesulfonylchloride (135 g, 700 mmol) in
THF (700 mL) and 1 M NaOH (700 mL) were added
slowly at the same time under the ice-cool condition.
The mixture was stirred at room temperature for 18 h,
and diluted with EtOAc. 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 12 (193 g, 76%) as
1
quant) as colorless oil. H NMR (300 MHz, DMSO-d6)
d 0.76 (d, 3H, J=6.9), 0.82 (d, 3H, J=6.6), 0.87 (d, 6H,
J=6.6), 1.23 (m, 1H), 1.37 (m, 1H), 1.55 (m, 1H), 1.68
(s, 2H), 1.90 (m, 1H), 2.96 (d, 1H, J=5.1), 3.74–3.91 (m,
4H), 4.01 (m, 1H), 4.72 (d, 1H, J=3.3), 7.62 (d, 1H,
J=9.6).
colorless crystals. Mp 116–118 ꢀC. H NMR (300 MHz,
1
DMSO-d6) d 0.79 (d, 3H, J=6.6), 0.83 (d, 3H, J=6.6),
1.94 (m, 1H), 3.52 (dd, 1H, J=9.3, 6.0), 7.35–7.43 (m,
2H), 7.80–7.87 (m, 2H), 8.08 (d, 1H, J=9.3), 12.61 (brs,
1H). Anal. calcd for C11H14FNO4S: C, 47.99;H, 5.12;
N, 5.09;found: C, 47.86;H, 5.06;N, 5.06.
(2S)-N-((1S)-1-(2,5-Dioxolanyl)-3-methylbutyl)-3-methyl
- 2 - ((3 - pyridylmethyl)carbonylamino)butanamide (10).
Compound 9 (0.80 g, 3.1 mmol), 3-pyridylacetic acid
hydrochloride (0.59 g, 3.4 mmol), 1-hydroxy-
benzotriazole (HOBt) (0.46 g, 3.4 mmol) and Et3N (0.34
g, 3.4 mmol) were dissolved in DMF (20 mL). A solu-
tion of EDCHCl (0.65 g, 3.4 mmol) in CH2Cl2 (20 mL)
was added under the ice-cool condition. The mixture
was stirred at room temperature for 18 h and con-
centrated in vacuo. The residue was dissolved in EtOAc,
and the solution was washed with saturated NaHCO3
and saturated NaCl, dried over MgSO4, and con-
centrated in vacuo. The residue was crystallized from
hexane to give 10 (0.92 g, 79%) as colorless crystals. Mp
N-((4-Fluorophenyl)sulfonyl)-L-valine N-Hydroxysuccini-
mide ester (13). Compound 12 (146 g, 530 mmol) and
HOSu (73 g, 636 mmol) were dissolved in THF (1200
mL), and a suspension of EDCHCl (122 g, 636 mmol) in
CH2Cl2 (1200 mL) was added thereto under the ice-cool
condition. The mixture was stirred at room temperature
for 18 h. After concentration in vacuo, the residue was
dissolved in 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 13 (197 g, 94%) as
90–91 ꢀC. H NMR (300 MHz, DMSO-d6) d 0.77–0.84
1
(m, 12H), 1.21 (m, 1H), 1.35 (m, 1H), 1.51 (m, 1H), 1.92
(m, 1H), 3.51 (d, 1H, J=14.1), 3.58 (d, 1H, J=14.1),
3.73–3.87 (m, 4H), 3.99 (m, 1H), 4.18 (dd, 1H, J=8.9,
7.2), 4.69 (d, 1H, J=3.6), 7.31 (m, 1H), 7.65–7.71 (m,
2H), 8.19 (d, 1H, J=8.9), 8.41–8.46 (m, 2H). FAB-
HRMS calcd for C20H32N3O4 [M+H]+: 378.2393,
found: 378.2403.
colorless crystals. Mp 147–149 ꢀC. H NMR (300 MHz,
1
DMSO-d6) d 0.88 (d, 3H, J=6.9), 0.91 (d, 3H, J=6.9),
2.10 (m, 1H), 2.79 (s, 4H), 4.14 (m, 1H), 7.36–7.41 (m,
2H), 7.85–7.89 (m, 2H), 8.67 (d, 1H, J=8.7). Anal.
calcd for C15H17FN2O6S: C, 48.38;H, 4.60;N, 7.52;
found: C, 48.22;H, 4.63;N, 7.41.
(2S)-N-((1S)-1-Formyl-3-methylbutyl)-3-methyl-2-((3-
pyridylmethyl)carbonylamino)butanamide hydrochloride
(1). Compound 10 (0.89 g, 2.4 mmol) was dissolved in
1 M HCl (20 mL). The mixture was stirred at 50 ꢀC for 4
h and purified by HPLC system (column: YMC-Pack
ODS-A 250Â20 mm, mobile phase CH3CN/H2O/
TFA=20:80:0.1). The main fractions were collected,
neutralized by addition of NaHCO3 and extracted with
EtOAc. The organic layer was washed with saturated
NaCl, dried over MgSO4, and concentrated in vacuo.
The residue was crystallized from hexane to give pyri-
dine free of 1 (0.50 g, 64%) as colorless crystals. Mp
109–111 ꢀC. 1H NMR (300 MHz, DMSO-d6) d 0.82–
0.88 (m, 12H), 1.40 (m, 1H), 1.49–1.61 (m, 2H), 1.99 (m,
(2S)-2-((4-Fluorophenyl)sulfonylamino)-N-((1S)-1-(hy-
droxymethyl)-3-methylbutyl)-3-methylbutanamide (14).
To a solution of 13 (185 g, 496 mmol) in EtOAc (4000
mL) was added l-leucinol (70 g, 590 mmol). The mix-
ture was stirred at room temperature for 18 h and
washed with 1 M HCl, saturated NaHCO3 and satu-
rated NaCl, dried over MgSO4, and concentrated in
vacuo. Resulting white solid was crystallized from hex-
ane/EtOAc (9:1) to give 14 (183 g, 98%) as colorless
crystals. Mp 120–122 C. H NMR (300 MHz, DMSO-
d6) d 0.66 (d, 3H, J=6.0), 0.75–0.85 (m, 9H), 0.98 (m,
1H), 1.10–1.19 (m, 2H), 1.81 (m, 1H), 3.06 (m, 1H), 3.18
(m, 1H), 3.48–3.57 (m, 2H), 4.55 (t, 1H, J=5.4), 7.32–
7.41 (m, 2H), 7.50 (d, 1H, J=8.4), 7.74 (d, 1H, J=9.0),
ꢀ
1

M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
1377
7.80–7.89 (m, 2H). Anal. calcd for C17H27FN2O4S: C,
54.53;H, 7.26;N, 7.48;found: C, 54.21;H, 7.22;N,
7.45.
J=5.7), 0.79–0.88 (m, 12H), 1.15–1.45 (m, 7H), 1.82 (m,
1H), 3.04-3.11 (m, 2H), 3.59 (m, 1H), 4.75 (m, 1H),
7.33–7.39 (m, 2H), 7.78–7.83 (m, 2H), 7.88 (d, 1H,
J=9.3), 8.15 (d, 1H, J=6.3), 8.65 (t, 1H, J=5.9). Anal.
calcd for C22H34FN3O5S: C, 56.03;H, 7.27;N, 8.91;
found: C, 55.99;H, 7.00;N, 8.91. MALDI-TOF MS
calcd for C22H34FN3O5SNa [M+Na]+: 494.210, found:
(2S)-2-((4-Fluorophenyl)sulfonylamino)-N-((1S)-1-for-
myl-3-methylbutyl)-3-methylbutanamide (15). Com-
pound 14 (182 g, 486 mmol) was dissolved in DMSO
(800 mL) and CH2Cl2 (400 mL). N,N-Diisopropylethyl-
amine (251 g, 1940 mmol) and a suspension of SO3/
pyridine (309 g, 1940 mmol) in DMSO (500 mL) were
added under the ice-cool condition. The mixture was
stirred for 30 min under the same condition. The reac-
tion 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. Resulting white solid was washed with hexane,
and crystallized from EtOAc to give 15 (67 g, 37%) as
colorless crystals. Mp 154 ꢀC. 1H NMR (300 MHz,
DMSO-d6) d 0.74 (d, 3H, J=6.3), 0.81–0.88 (m, 9H),
1.15–1.45 (m, 3H), 1.87 (m, 1H), 3.59 (dd, 1H, J=9.2,
6.6), 3.84 (m, 1H), 7.34–7.42 (m, 2H), 7.79–7.85 (m,
2H), 7.96 (d, 1H, J=9.2), 8.27 (d, 1H, J=7.2), 9.14 (s,
1H). Anal. calcd for C17H25FN2O4S: C, 54.82;H, 6.77;
N, 7.52;found: C, 54.67;H, 6.63;N, 7.41.
25
494.243. ½ꢀꢁD +42.4ꢀ (c=0.203, DMSO).
(2S) - 2 - ((4 - Fluorophenyl)sulfonylamino) - 3 - methyl - N -
((3S)-tetrahydro-2-oxo-3-furanyl)butanamide (17). To a
solution of compound 13 (4.8 g, 13 mmol) in DMF (80
mL) was added S-a-amino-g-butyrolactone hydrochlo-
ride (1.4 g, 14 mmol) and Et3N (2.6 g, 16 mmol). The
mixture was stirred at room temperature for 18 h. After
dilution with EtOAc, 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 17 (2.0 g, 43%) as
colorless crystals. Mp 116–118 ꢀC. H NMR (300 MHz,
1
DMSO-d6) d 0.84 (d, 6H, J=6.6), 1.76–1.95 (m, 2H),
2.16 (m, 1H), 3.45 (t, 1H, J=8.1), 4.17 (m, 1H), 4.30 (m,
1H), 4.48 (m, 1H), 7.34–7.42 (m, 2H), 7.77–7.84 (m,
2H), 7.99 (d, 1H, J=8.7), 8.38 (d, 1H, J=8.1). Anal.
calcd for C15H19FN2O5S: C, 50.27;H, 5.34;N, 7.82;
found: C, 50.14;H, 5.34;N, 7.65.
(3S)-N-Butyl-3-((2S)-2-((4-fluorophenyl)sulfonylamino)-3
-methylbutanoylamino)-2-hydroxy-5-methylhexanamide
(16). TFA (2.4 g, 21 mmol) was added dropwise to a
cooled solution of 15 (4.0 g, 11 mmol), n-butylisocya-
nide (1.0 g, 12 mmol) and pyridine (3.4 g, 43 mmol) in
CH2Cl2 (100 mL). The mixture was stirred at room
temperature for 18 h. After concentration in vacuo, the
residue was dissolved in EtOAc, and the solution was
washed with 1 M HCl, saturated NaHCO3 and satu-
rated NaCl, dried over MgSO4, and concentrated in
vacuo. The residue was purified by HPLC system (col-
umn: YMC Pack ODS-A 250Â20 mm, mobile phase:
CH3CN/H2O/TFA=40:60:0.1), and crystallized from
hexane to give 16 (1.0 g, 20%) as colorless crystals. Mp
(2S) - 2 - ((4 - Fluorophenyl)sulfonylamino) - 3 - methyl - N -
((3S)-tetrahydro-2-hydroxy-3-furanyl)butanamide
(3).
To a solution of compound 17 (1.9 g, 5.3 mmol) in
CH2Cl2 (250 mL) was added DIBAL-H (1.0 M solution
in toluene) (17 mL, 17 mmol) at ꢂ78 ꢀC. The mixture
was kept below ꢂ70 ꢀC and stirred for 3 h, and then
aqueous saturated NH4Cl (4.0 mL) was added and stir-
red for 30 min. MgSO4 and EtOAc (100 mL) were
added at room temperature, and inorganic was filtered
off. After concentration in vacuo, the residue was pur-
ified by HPLC system (column: YMC-Pack ODS-A
250Â20
mm,
mobile
phase:
CH3CN/H2O/
192–194 ꢀC. H NMR (300 MHz, DMSO-d6) d 0.69 (d,
TFA=20:80:0.1). The main fractions were collected,
and extracted with EtOAc. The solution was washed
with saturated NaHCO3 and saturated NaCl, dried over
MgSO4, and concentrated in vacuo, and crystallized
from hexane to give 3 (0.19 g, 9.9%) as colorless crys-
1
3H, J=6.9), 0.70 (d, 3H, J=5.4), 0.75 (d, 3H, J=6.0),
0.79 (d, 3H, J=6.6), 0.85 (t, 3H, J=7.4), 0.94 (m, 1H),
1.05–1.09 (m, 2H), 1.18–1.27 (m, 2H), 1.30–1.39 (m,
2H), 1.82 (m, 1H), 2.92–3.07 (m, 2H), 3.63 (dd, 1H,
J=9.2, 5.7), 3.72 (dd, 1H, J=6.0, 2.6), 3.93 (m, 1H),
5.65 (d, 1H, J=6.0), 7.31–7.36 (m, 3H), 7.53 (t, 1H,
J=5.9), 7.59 (d, 1H, J=9.2), 7.80–7.85 (m, 2H). Anal.
calcd for C22H36FN3O5S: C, 55.79;H, 7.66;N, 8.87;
found: C, 55.60;H, 7.40;N, 8.73.
tals. Mp 162–164 ꢀC. H NMR (300 MHz, DMSO-d6) d
1
(major/minor) 0.77/0.76–0.84 (d/m, 3H, J=6.3), 0.80/
0.76–0.84 (d/m, 3H, J=6.6), 1.53 (m, 1H), 1.75–1.87 (m,
2H,), 3.53–3.87 (m, 4H), 4.99/4.83 (t/d, 1H, J=4.4/4.8),
6.34–6.35/6.08 (m/d, 1H, J=4.5), 7.35–7.41 (m, 2H),
7.68/7.90 (d/d, 1H, J=7.8/6.3), 7.77–7.84 (m, 3H).
major/minor=3.3:1.0. Anal. calcd for C15H21FN2O5S:
C, 49.99;H, 5.87;N, 7.77;found: C, 49.89;H, 5.97;N,
7.55. MALDI-TOF MS calcd for C15H21FN2O5SNa
(3S)-N-Butyl-3-((2S)-2-((4-fluorophenyl)sulfonylamino)-
3-methylbutanoylamino)-5-methyl-2-oxohexanamide (2).
To a solution of 16 (1.0 g, 2.1 mmol) in CH2Cl2 (100
mL) was added Dess–Martin periodinane (1.3 g, 3.2
mmol). The mixture was stirred at room temperature
for 18 h. Aqueous 10% Na2S2O3 (50 mL) and aqueous
10% NaHCO3 (50 mL) were added, and the mixture
was stirred for 10 min. The organic layer was separated,
washed with 1 M HCl, saturated NaHCO3 and satu-
rated NaCl, dried over MgSO4, and concentrated in
vacuo. The residue was crystallized from EtOAc/hexane
to give 2 (0.80 g, 80%) as colorless crystals. Mp 108–
25
[M+Na]+: 383.105, found: 383.103. ½aꢁD ꢂ35.2ꢀ
(c=0.203).
N-(tert-Butoxy)carbonyl-L-leucine N-hydroxysuccinimide
ester (19). Boc-l-leucine (27 g, 110 mmol) and HOSu
(16 g, 140 mmol) were dissolved in THF (180 mL), and
a suspension of EDCHCl (27 g, 140 mmol) in CH2Cl2
(180 mL) was added thereto under the ice-cool condi-
tion. The mixture was stirred at room temperature for
18 h, and concentrated in vacuo. The residue was dis-
110 ꢀC. H NMR (300 MHz, DMSO-d6) d 0.73 (d, 3H,
1

1378
M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
solved in EtOAc, and the solution was washed with 1 M
HCl, saturated NaHCO3 and saturated NaCl, dried
over MgSO4, and concentrated in vacuo. Resulting
white solid was crystallized from hexane to give 19 (33
system (column: YMC-Pack ODS-A 250Â20 mm,
mobile phase: CH3CN/H2O/TFA=30:70:0.1). The
main fractions were collected, and extracted with
EtOAc. The solution was washed with saturated
NaHCO3 and saturated NaCl, dried over MgSO4, and
concentrated in vacuo. The residue was crystallized
from hexane to give 4 (0.46 g, 27%) as colorless crystals.
g, 93%) as colorless crystals. Mp 74–76 ꢀC. H NMR
1
(300 MHz, DMSO-d6) d 0.88 (d, 3H, J=6.3), 0.91 (d,
3H, J=6.3), 1.39 (s, 9H), 1.54–1.75 (m, 3H), 2.80 (s,
4H), 4.32 (m, 1H), 7.62 (d, 1H, J=8.4). FAB-HRMS
calcd for C15H25N2O6 [M+H]+: 329.1713, found:
329.1695.
Mp 62–64 ꢀC. H NMR (300 MHz, DMSO-d6) d 0.91
1
(d, 6H, J=6.0), 1.49–1.91 (m, 4H), 2.14 (m, 1H), 3.64–
4.00 (m, 3H), 4.91 (m, 1H), 5.00/5.11 (d/t, 1H, J=4.5/
4.7), 6.16/6.34 (d/d, 1H, J=4.8/4.8), 7.10 (m, 1H), 7.29–
7.34 (m, 2H), 7.49–7.53 (m, 2H), 7.72/7.78 (d/d, 1H,
J=8.4/8.7), 8.26/7.87 (d/d, 1H, J=6.3/7.5), 9.69 (s, 1H),
major:minor=1.0:0.85. Anal. calcd for C17H25N3O3S:
C, 58.09;H, 7.17;N, 11.96;found: C, 58.03;H, 7.26;N,
11.60. MALDI-TOF MS calcd for C17H25N3O3SNa
(2S)-2-((tert-Butoxy)carbonylamino)-4-methyl-N-((3S)-
tetrahydro-2-oxo-3-furanyl)pentanamide (20). To a solu-
tion of 19 (20 g, 61 mmol) in DMF (120 mL) was added
S-a-amino-g-butyrolactone hydrobromide (17 g, 91
mmol) and Et3N (18 g, 180 mmol) under the ice-cool
condition. 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 con-
centrated in vacuo. The residue was crystallized from
hexane to give 20 (19 g, 99%) as colorless crystals. Mp
92–93 ꢀC. 1H NMR (300 MHz, DMSO-d6) d 0.85 (d,
3H, J=6.3), 0.88 (d, 3H, J=6.6), 1.28–1.50 (m, 2H),
1.38 (s, 9H), 1.61 (m, 1H), 2.14 (m, 1H), 2.39 (m, 1H),
3.96 (m, 1H), 4.20 (m, 1H), 4.34 (m, 1H), 4.60 (m, 1H),
6.88 (d, 1H, J=8.4), 8.30 (d, 1H, J=8.4). FAB-HRMS
calcd for C15H27N2O5 [M+H]+: 315.1920, found:
315.1925.
25
[M+Na]+: 358.174, found: 358.169. ½ꢀꢁD +23.6ꢀ
(c=0.211).
Inhibition assayfor calpains. This inhibition assay was
performed as described in the previous literature10 using
commercially m-calpain (human erythrocyte, Cosmo Bio
Co., Ltd.) and m-calpain (porcine kidney, Cosmo Bio
Co., Ltd.). Assay solution including 0.5 mg/mL casein,
20 mM DTT, 50 mM Tris–HCl (pH 7.4) and 1.0 nmol
of enzyme was used. In each well was placed 200 mL of
assay solution and 2.5 mL of DMSO including inhibitor
of different concentration. Reaction was started by
addition of 50 mL of 20 mM CaCl2 in test well and 50
mL of 1 mM EDTA in blank well. After incubation for
60 min at 30 ꢀC, the mixture (100 mL) was transferred to
another plate in which 100 mL of H2O and 50 mL of Bio-
Rad protein assay dye reagent were placed in each well.
After incubation at room temperature for 15 min, the
mixture was read OD at 595 nm with plate reader
(Multiskan Multisoft, Labsystems). The percentage
inhibition was calculated 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.
(2S)-4-Methyl-2-(3-phenylthioureido)-N-((3S)-tetrahydro-
2-oxo-3-furanyl)pentanamide (21). To a solution of 20
(3.4 g, 11 mmol) in EtOAc (100 mL) was added 4 M
HCl/EtOAc (8.0 mL) under the ice-cool condition. The
mixture was stirred at room temperature for 18 h, and
concentrated in vacuo. EtOAc was added, and then
removed in vacuo (twice). The residue was suspended
with EtOAc, and phenylisothiocyanate (1.5 g, 11
mmol) and Et3N (3.3 g, 32 mmol) were added thereto.
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 concentrated
in vacuo. The residue was crystallized from hexane,
and washed with hexane to give 21 (1.8 g, 48%) as
Inhibition assayfor proteasome. 20S proteasome assay
kit (QunatiZyme1 Assay System AK-740, BIOMOL
Research Lab., Inc.) was purchased, and the procedure
was performed according to the guided method. In each
well was placed 5.0 mL of 10% DMSO including inhi-
bitor of different concentration, 90 mL of assay buffer
(25 mM HEPES, pH 7.5, 0.5 mM EDTA, 0.05% NP-
40, 0.03% SDS) and 5.0 mL of enzyme solution (0.02
mg/mL 20S proteasome from human erythrocyte in
assay buffer). The reaction was started by addition of 10
mL substrate solution (0.29 mg/mL Suc-LLVY-AMC in
assay buffer) at 30 ꢀC, and the fluorescence measure-
ments were performed with plate reader (excitation: 360
nm, emission: 450 nm, CYTOFLUOR series 4000, Per-
septive Biosystems) for 60 min. The percent inhibition
was calculated from the difference between the inhibitor
slope and control slope (no inhibitor). The IC50 was
obtained from the graphical analysis of the concen-
tration and the inhibition.
colorless crystals. Mp 78–79 ꢀC. H NMR (300 MHz,
1
DMSO-d6) d 0.92 (d, 3H, J=6.3), 0.93 (d, 3H, J=6.6),
1.55–1.70 (m, 3H), 2.20 (m, 1H), 2.40 (m, 1H), 4.22 (m,
1H), 4.36 (m, 1H), 4.62 (m, 1H), 4.96 (m, 1H), 7.10 (m,
1H), 7.29–7.34 (m, 2H), 7.51–7.54 (m, 2H), 7.78 (d, 1H,
J=8.1), 8.64 (d, 1H, J=8.1), 9.70 (s, 1H). FAB-HRMS
calcd for C17H24N3O3S [M+H]+: 350.1538, found:
350.1542.
(2S)-4-Methyl-2-(3-phenylthioureido)-N-((3S)-tetrahydro-
2-hydroxy-3-furanyl)pentanamide (4). To a solution of
21 (1.7 g, 4.9 mmol) in CH2Cl2 (200 mL) was added
DIBAL-H (1.0 M solution in toluene) (17 mL, 17 mmol)
at ꢂ78 ꢀC. The mixture was kept below ꢂ70 ꢀC and
stirred for 3 h, and then saturated NH4Cl solution (4.0
mL) was added and stirred for 30 min. MgSO4 and
EtOAc (150 mL) were added at room temperature, and
inorganic was filtered off using Celite. After concen-
tration in vacuo, the residue was purified by HPLC
Determination of water-solubility. A suspension of
inhibitor (1–5 mg) in pH 7 buffer solution (2.0 mL)

M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 1371–1379
1379
was shaken at 25 ꢀC for 5 h. The suspension was fil-
Acknowledgements
tered through CHROMATODISK1 (0.45 mm, GL
Sciences), and the filtrate (1.0 mL) was quantified by
HPLC.
The authors would like to acknowledge Dr. Ying-she
Cui and Mr. Yusuke Sakai for helpful discussions.
Determination of partition coefficient (water/octanol).
To a solution of inhibitor (generally 0.1 mg/mL) in pH
7 buffer solution (2.0 mL) was added octanol (2.0 mL).
The mixture was shook at 25 ꢀC for 1 h, and centrifuged
(3000 rpm). The concentration of inhibitor in each
phase was determined by HPLC.
References and Notes
1. (a) Ono, Y.;Sorimachi, H.;Suzuki, K. In Calpain: Phar-
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Wang, K. K. W.;Yuen, P.-W., Eds.;Taylor & Francis;Phila-
delphia, 1999;pp 1–23. (b) Huang, Y.;Wang, K. K. W. Trends
Mol. Med. 2001, 7, 355.
Transcorneal permeation. Eye drops of compound 1 was
prepared as 0.5% aqueous solution. Eye drops of com-
pounds 3 and 4 were prepared as 0.5% aqueous sus-
pensions. Each eye drops (50 mL, 1 drop) was exactly
applied to eyes of albino rabbits with micropipet (single
instillation, n=3 eyes). At 0.5, 1, 2 and 4 h after instil-
lation, the animals were sacrificed with pentobarbital
overdosage and the aqueous humor (250 mL) was sam-
pled from each eye. The aqueous humor specimens were
subjected to measurement of drug concentration by
HPLC analysis.
2. (a) Wang, K. K. W.;Yuen, P.-W. Trends Pharmacol. Sci.
1994, 15, 412. (b) Stracher, A. Ann. N. Y. Acad. Sci. 1999, 884,
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3. (a) Shih, M.;David, L. L.;Lampi, K. J.;Ma, H.;Fukiage,
C.;Azuma, M.;Shearer, T. R. Curr. Eye. Res. 2001, 22, 458.
(b) Shearer, T. R.;David, L. L.;Anderson, R. S. Curr. Eye.
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4. (a) Fukiage, C.;Azuma, M.;Nakamura, Y.;Tamada, Y.;
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Lens culture. Lenses from 5-week-old Sprague–Dawley
rats were cultured at 37 ꢀC under 5% CO2 in 4 mL
Eagle’s minimum essential medium (MEM Gibco) with
10% fetal bovine serum (Gibco) (Normal group). Cal-
cium ionophore (A23187) (10 mM) was present on day 1
only (A23187 group), and 100 mM hemiacetal 4 was
present continuously (A23187 + 4). After 5 days of
culture, lenses were photographed under a dissecting
microscope, and density of lens opacity was quantitated
using computerized image analysis (Image 1.31 soft-
ware, Twilight clone BBC, Silver Springs, MD).