Synthesis of chromone carboxamide derivatives with antioxidative and calpain inhibitory properties

Article abstract of DOI:10.1016/j.ejmech.2011.02.025

The overactivation of μ-calpain can cause serious cell damage in several diseases. Furthermore, cell death in a number of neurodegenerative disorders is linked to the overproduction of reactive oxygen species. Therefore, antioxidants and μ-calpain inhibit

Full text of DOI:10.1016/j.ejmech.2011.02.025

European Journal of Medicinal Chemistry 46 (2011) 1721e1728
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of chromone carboxamide derivatives with antioxidative and calpain
inhibitory properties
,
,
,b,
Sang Hoon Kim a b, Young Hoon Lee a b, Seo Yun Jung a, Hyoung Ja Kim c, Changbae Jin c, Yong Sup Lee a
*
a Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University, 1 Hoegi-dong Dongdaemoon-ku, Seoul 130-701, Republic of Korea
b Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea
c Life and Health Division, Korea Institute of Science and Technology, PO Box 131 Cheongryang, Seoul 130-650, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The overactivation of m-calpain can cause serious cell damage in several diseases. Furthermore, cell death
Received 18 November 2010
Received in revised form
10 February 2011
Accepted 12 February 2011
Available online 22 February 2011
in a number of neurodegenerative disorders is linked to the overproduction of reactive oxygen species.
Therefore, antioxidants and -calpain inhibitors could have the therapeutic potentials to treat cell death
related diseases. New chromone carboxamide derivatives 3 were synthesized to provide alternative
-calpain inhibitors to compound 2, a conformationally constrained structural variant of MDL 28,170.
Compounds 3h and 3l exhibited the most potent M),
-calpain inhibitory activities (IC50 ¼ 0.09e0.10
M). Compound 3i showed both potent -calpain
m
m
m
m
and were comparable to 2 in this respect (IC50 ¼ 0.07
m
m
Keywords:
Calpain inhibitor
Cell death
Neurodegenerative disease
Chromone carboxamide
Antioxidant
inhibitory activity (IC50 ¼ 0.28
m
M) and antioxidant activities in DPPH scavenging and lipid peroxidation
inhibition assays.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Most of the previously reported calpain inhibitors bind
competitively to its catalytic site and are usually derived from small
peptides, such as MDL 28,170 (1), which structurally mimic the
cleavage sites of calpain substrates [12,13]. However, MDL 28,170
suffers from the disadvantages of non-selectivity, instability during
storage, and a short half life in vivo due to its peptide character and
the high reactivity of its aldehyde group [14]. Concerning our work
The calpains (E.C. 3.4.22.17) are a family of cytosolic cysteine
proteases, which are expressed ubiquitously in cells and tissues [1].
Two major classes of calpains have been identified, calpain I (m-
calpains) and calpain II (m-calpains), which need micromolar and
millimolar concentrations of calcium ions, respectively, for activation
[2]. Calpains are involved in numerous cellular processes, such as,
signal transduction, cell migration, differentiation, and apoptosis [3].
However, in some neurological disorders such as stroke [4], Par-
kinson’s disease [5] and Alzheimer’s disease [6], calpains are over-
activated and cause serious cell damage or even cell death. These
enzymes have therefore attracted considerable interest as an
important class of targets in neurodegenerative diseases [7]. More-
over, recent reports suggest that calpain inhibition may be beneficial
for alleviating memory loss in Alzheimer’s disease [8] and for pre-
venting lissencephaly [9], heat stress-induced germ cell apoptosis
to identify a new scaffold for
m-calpain inhibitors, we recently
reported that chromone carboxamide or quinoline carboxamide,
which are conformationally restricted cyclic analogs of MDL 28170,
displayed the m-calpain inhibitory activity [15e17]. However, the
most potent chromone carboxamide (2, KYS 4516), which has
a primary amide group at its keto-amide position, was unstable
during storage and under acidic conditions, limiting our further
research pursuits. Thus, extended derivatization of 2 was needed to
produce alternative inhibitors with the same level of
m-calpain
inhibitory activity as 2. In this report, we describe the synthesis of
new chromone carboxamide derivatives 3 and their biological
[10], and TNF-a-induced inflammatory responses [11].
evaluation for their
m-calpain inhibitory activities (Fig. 1). We
modified the chromone ring (R1, R2) and amide region (R3) of 2, and
introduced three different keto-amides, a primary amide, a benzyl,
or 4-methoxyphenethyl amide to the warhead position (R3) of the
* Corresponding author. Kyung Hee East-West Pharmaceutical Research Institute,
Department of Pharmaceutical Science, College of Pharmacy, Kyung Hee University,
1 Hoegi-dong Dongdaemoon-ku, Seoul 130-701, Republic of Korea. Tel.: þ82 2 961
0370; fax: þ82 2 966 3885.
catalytic site of the enzyme and their
were later compared.
m-calpain inhibitory activities
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.025

1722
S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
Fig. 1. Design of chromone derivatives as new m-calpain inhibitors.
For substituents at R1 and R2, methoxy and hydroxyl groups
7a and b were obtained directly by the hydrolysis of the corre-
sponding ethyl esters 6a and b using KOH in aqueous ethanol. To
synthesize the hydroxyl-substituted chromone carboxamides
3gel, hydroxyl groups at the aromatic ring of chromone were
protected with MOM before hydroxyl-amide 12 coupling.
Compound 6c was reacted with methoxymethyl chloride (MOM-
Cl) and then hydrolyzed with KOH in aqueous ethanol to afford
7c. To synthesize the 7-hydroxy- and 6,7-dihydroxychromone
carboxamides 3iel, the required hydroxyl groups were generated
by the hydrolysis of the methyl ether group in 6a and b. Briefly,
the compounds 6a and b were treated with 48% HBr in the
presence of glacial acetic acid [25] and then converted to esters
(10a and b) using H2SO4 in MeOH. The hydroxyl esters 10a and
b were again transformed to MOM-protected chromone carbox-
ylic acids (7d and e) after MOM-protection followed by ester
hydrolysis.
were chosen, because both provide hydrogen-bonding donors or
acceptors to promote binding at the active sites of enzymes. Cell
death in a number of neurodegenerative disorders is also linked to
the overproduction of reactive oxygen species [18]. Therefore,
antioxidants that can scavenge oxygen free radicals have thera-
peutic potential for the treatment of neuronal cell death. The
chromone ring has been used as an important scaffold for the
synthesis of antitumor, insecticidal and pesticidal agents [19e21].
We also found that polyphenolic chromones can protect neuronal
cells against H2O2-induced oxidative injuries [22]. Thus, it was
expected that the substitution of hydroxyl groups on the chromone
ring of 3 would lead to antioxidant and m-calpain inhibitory activ-
ities. Accordingly, synthesized compounds were also assessed for
their antioxidant activities using two assay systems.
The synthesis of chromone carboxamide derivatives 3ael is
illustrated in Scheme 3. The chromone carboxylic acids 7a and
b were coupled with three types of hydroxy-amides 12aec using an
EDC/HOBt system followed by oxidation of the resulting hydrox-
ylamides with DesseMartin periodinane to afford chromone car-
boxamides 3aef [17]. Chromone carboxamides 3gel, which contain
a hydroxyl-substituent on their aromatic rings, were obtained by
removing the MOM group during the final stage with 1% HCl in
MeOH under reflux. However, the hydroxy-substituted chromone
carboxamides derived from hydroxyl-amide 12a, which has
a primary group, could not be obtained, confirming their instability
under acidic MOM-deprotection conditions. The chemical yields at
each step are summarized in Table 1.
2. Chemistry
The synthesis of chromone carboxamide derivatives 3 was
accomplished using a general pathway, which involves a coupling
reaction between a chromone carboxylic acid 7 and a hydroxyl-
amide 12 using an EDC/HOBt system, and subsequent oxidation of
the resulting hydroxylamides under DesseMartin periodinane
conditions, as illustrated in Scheme 3. For the synthesis of 3, which
contains a hydroxyl-substituent on its aromatic ring, a final depro-
tection step was required.
Phenols 4a and 4c were transformed to 2-hydroxypropio-
phenones 5a and 5c by Fries rearrangement by propionyl chloride
treatment in the presence of BF3$Et2O at reflux temperature [23].
For phenol 4b, toluene was used as a co-solvent because of its low
solubility in BF3$Et2O [24]. A chromone ring was constructed by
acylating 5aec with ethyl oxalyl chloride followed by in situ cycli-
zation of the resulting ester in the presence of pyridine at reflux
temperature to afford chromone carboxylic acid ethyl esters 6aec
(Scheme 1).
3. Pharmacology
The m-calpain inhibitory activities of the chromone carboxamide
derivatives 3ael were evaluated using human calpain I isolated from
erythrocytes, and Suc-Leu-Tyr-AMC as the fluorogenic substrate
[16,26]. Parent chromone carboxamide 2 was tested for comparison
purposes and assay results are summarized in Table 1. The chromone
derivatives were also evaluated for antioxidant activities by exam-
ining their DPPH scavenging and lipid peroxidation inhibitory effects
The chromone carboxylic acids 7, which have a methoxy or
MOM-protected hydroxyl group on the phenyl ring were
prepared from three chromone carboxylic acid ethyl esters 6
(Scheme 2). The methoxy-substituted chromone carboxylic acids
Scheme 1. Synthesis of chromone carboxylic acid esters 6aec.

S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
1723
Scheme 2. Synthesis of chromone carboxylic acids 7aee.
using rat liver homogenate (Table 2); ascorbic acid and trolox were
included as positive controls.
conditions. Accordingly, we attempted to synthesize alternative
inhibitors that retained the inhibitory activity of 2 for -calpain by
m
replacing the primary amide of 2 with another amide group.
First, we introduced substituents on the aromatic ring of chro-
4. Results and discussion
mone carboxamides to examine their influence on
m-calpain
inhibitory activity. When methoxy groups were substituted at the
C-6 and/or C-7 positions of the chromone ring, the primary amide-
The calpains are promising targets for the treatment of cell death
related diseases. During the course of our program to identify potent
derived compounds 3a and d were found to inhibit
m-calpain
m-calpain inhibitors, we recently found that chromone carboxamide
almost as well as 2. However, replacing the primary amide group in
the warhead region with benzyl or 4-methoxyphenethyl decreased
inhibitory activity by 2e4 fold. Hydroxy-chromone carboxamides
derived from the primary amide group were not available due to
2 is a potent -calpain inhibitor. However, during our efforts to
m
enhance its activities and improve its physicochemical characteris-
tics, we found that further modification of its aromatic ring is diffi-
cult due to the instability of a primary amide moiety under acidic
Scheme 3. Synthesis of chromone carboxamide derivatives 3ael.

1724
S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
Table 1
The yields of the coupling, oxidation, and deprotection steps and the m-calpain inhibitory activities of 3ael and that of the parent compound 2.
O
O
R1
R2
O
O
R3
N
H
N
H
O
3a~3l
Compds
R1, R2
R3
Yields (%)
Caipaln Inhibition (IC50, m
M)a
EDC coupling
Oxidation
Deprotection
3a
3b
3c
R1 ¼ R2 ¼ OCH3
H
63
75
29
30
39
62
0.09 ꢀ 0.00
0.19 ꢀ 0.01
0.27 ꢀ 0.01
Benzyl
4-Methoxyphenethyl
3d
3e
3f
R1 ¼ OCH3, R2 ¼ H
H
54
89
31
31
45
71
0.12 ꢀ 0.02
0.43 ꢀ 0.07
0.23 ꢀ 0.01
Benzyl
4-Methoxyphenethyl
3g
3h
R1 ¼ OCH3, R2 ¼ OH
R1 ¼ R2 ¼ OH
Benzyl
4-Methoxyphenethyl
46
82
47
51
60
44
0.11 ꢀ 0.00
0.10 ꢀ 0.01
3i
3j
Benzyl
4-Methoxyphenethyl
89
86
64
44
61
47
0.28 ꢀ 0.00
1.29 ꢀ 0.04
3k
3l
R1 ¼ OH, R2 ¼ H
R1 ¼ R2 ¼ H
Benzyl
4-Methoxyphenethyl
80
92
60
69
56
51
0.13 ꢀ 0.00
0.09 ꢀ 0.01
2
H
0.07 ꢀ 0.00
a
IC50 was defined as the concentration resulting in 50% inhibition. Data are presented as the means ꢀSDs of three independent experiments.
their instabilities. However, benzyl and 4-methoxyphenethyl
derived hydroxyl-chromone carboxamides were readily obtained
after removing the hydroxyl-protecting group under acidic condi-
tions. Fortunately, the potencies of the monohydroxyl-substituted
compounds 3geh and 3kel were not decreased even after benzyl
or 4-methoxyphenethyl groups were incorporated. On the other
hand, the potencies of the dihydroxyl-substituted compounds 3i
and j were 4e18 fold less than that of 2. Of the synthesized
compounds, 3h and l showed the greatest inhibitory effects
assay system. However, compounds 3i and j, which both possess
a catechol group at the C-6 and C-7 positions of the chromone ring,
exhibited DPPH scavenging activity levels similar to that of ascorbic
acid and more potent lipid peroxidation inhibitory activities than
trolox. Although compound 3ishowed
m-calpaininhibitoryactivity4-
fold less than compound 2 (IC50 0.28 versus 0.07
mM), its antioxidant
activity was greater than those of ascorbic acid and trolox.
5. Conclusions
(IC50
¼
0.09e0.10
mM) and almost matched compound 2
(IC50 ¼ 0.07
mM), indicating that these compounds can be consid-
Here, new chromone carboxamide derivatives 3 were synthe-
sized as conformationally constrained structural variants of MDL
28,170 to provide alternative m-calpain inhibitors, by replacing the
primary amide group in the warhead position of 2 with other amide
groups. Of the derivatives synthesized, compounds 3h and l, which
possess a 4-methoxyphenethyl group at the keto-amide position,
ered alternatives for 2 for further in vitro studies.
Reactive oxygen species are also involved in several cell death
related neuronal diseases [18]. At the initiation of this study, we
considered that compounds with m-calpain inhibitory and antioxi-
dant activities would synergistically protect cells from damage, and
thus, synthesized compounds were also tested for their abilities to
scavenge DPPH and inhibit lipid peroxidation. Methoxy or mono-
hydroxyl-substituted compounds, like the parent compound 2,
most potently inhibited
comparable to that of the parent compound 2. Interestingly, unlike
3h and l, compound 3i showed both potent -calpain inhibitory
activity (IC50 ¼ 0.28 M) and DPPH scavenging and lipid perox-
idation inhibitory effects, whereas MDL 28,170 and compound 2
exhibited no antioxidant activity at concentrations under 100 M.
m-calpain (IC50 ¼ 0.09e0.10
mM) to levels
m
showednoantioxidantactivityatconcentrationsof<100 mMineither
m
m
Table 2
The antioxidant activities of 3iej and 2.
These biological assay results support that the compounds 3h, i,
and l should be considered as alternatives of 2, which can be used
for further in vitro studies.
Compds
DPPH scavenging
(IC50 M)
Lipid peroxidation inhibition
(IC50
M)a
,
m
, m
3i
3j
2
26.51 ꢀ 0.30
41.35 ꢀ 1.14
>100
48.68 ꢀ 4.57
52.46 ꢀ 3.56
>100
6. Experimental
Ascorbic acid
Trolox
30.81 ꢀ 1.01
>100
71.44 ꢀ 5.54
NTb
6.1. Instrumentation and chemicals
a
IC50 was defined as the concentration resulting in 50% inhibition.
Not tested. Data are presented as the means ꢀSDs of three independent
b
The high resolution mass spectra were recorded using a Jeol
AccuTOF (JMS-T100TD) equipped with a DART (direct analysis in real
experiments.

S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
1725
time) ion source (from IonSense, Tokyo, Japan) in the positive modes.
1H and 13C NMR spectra were recorded on a Gemini Varian-300 (at
300 and 75 MHz, respectively). Analytical thin layer chromatography
(TLC) was carried out using precoated silica gel (E. Merck Kiesegel
60F254 layer thickness 0.25 mm), and flash column chromatography
was performed using Merck Kiesegel 60 Art 9385 (230e400 mesh).
All solvents were purified using standard procedures.
6.2.3.2. 7-Methoxy-3-methyl-4-oxo-4H-chromen-2-carboxylic acid
(7b). Yield 24%, 1H NMR (400 MHz, pyridine-d5)
8.50 (1H, brs,
d
eOH), 8.32 (1H, d, J ¼ 8.8 Hz), 7.50 (1H, dd, J ¼ 8.4, 2.4 Hz), 7.02 (1H,
d, J ¼ 2.4 Hz), 3.72 (3H, s), 2.71 (3H, s).
6.2.4. Procedure for the preparation of 7-methoxy-6-(methoxy-
methoxy)-3-methyl-4-oxo-4H-chromen-2-carboxylic acid (7c)
Methoxymethyl chloride (633
a solution of 6c (780 mg, 2.8 mmol) and diisopropylethylamine
(2.44
l,14 mmol)inCH2Cl2 (20 ml)at0 ꢁC and stirredatrtfor2 h. The
ml, 8.41 mmol) was added slowly to
6.2. Syntheses
m
6.2.1. General procedure for the preparation of 2-hydroxypropiophe-
nones (6aec)
mixture was then diluted with EtOAc and washed successively with
water and brine. The organic layer was dried over MgSO4 and
concentrated to afford 8c (877 mg, 2.72 mmol, 97%), which was
hydrolyzed with KOH using the procedure described for 7a to afford
A mixture of phenols 4 (1.0 eq.), propionyl chloride (1.5 eq.), and
BF3$Et2O (1.5 eq.) was heated at 80e85 ꢁC for 2e6 h. In the reaction
of phenol 4b, toluene was used as a co-solvent. After reaction
completion, mixtures were cooled to rt, poured into water, and
extracted with EtOAc. The organic layer was dried over MgSO4 and
then concentrated to afford 5.
7c in 61% yield. 8c: 1H NMR (400 MHz, pyridine-d5)
d 8.07 (1H, s), 7.11
(1H, s), 5.29 (2H, s), 4.43e4.37 (2H, m), 3.79 (3H, s), 3.39 (3H, s), 2.49
(3H, s),1.28 (3H, t, J ¼ 7.2 Hz); 7c: 1H NMR (400 MHz, DMSO-d6)
d 7.53
(1H, s), 7.18 (1H, s) 5.26 (2H, s), 3.93 (3H, s), 3.41 (3H, s), 2.21 (3H, s).
6.2.1.1. 1-(2-Hydroxy-4,5-dimethoxyphenyl)propan-1-one (5a). Yield
6.2.5. Procedure for the preparation of 6,7-bis(methoxymethoxy)-
3-methyl-4-oxo-4H-chromen-2-carboxylic acid (7d)
88%,1H NMR (400 MHz, CDCl3)
d 7.10 (1H, s), 6.46 (1H, s), 3.91 (3H, s),
3.86 (3H, s), 2.98e2.92 (2H, m), 1.24 (3H, t, J ¼ 7.3 Hz).
HBr (48%) in glacial acetic acid (11.64 ml) was added to a solu-
tion of 6a (1.7 g, 5.82 mmol) in acetic acid (23 ml) and heated at
reflux for 12 h. The reaction mixture was then diluted with EtOAc
and washed successively with water and brine. The organic layer
was concentrated to afford 9a (644 mg, 2.73 mmol, 47%). H2SO4
(0.5 ml) was then added to a solution of 9a (3.6 g, 15.24 mmol) in
MeOH (60 ml) and heated at reflux for 12 h. The reaction mixture
was then diluted with EtOAc and washed successively with water
and brine. The organic layer was dried over MgSO4 and concen-
trated to afford 10a (2.8 g, 11.21 mmol, 74%). Compound 10a was
transformed to 11a (94%) and then to 7d (94%) using procedures
similar to those described for 8c and 7c, respectively. 9a: 1H NMR
6.2.1.2. 1-(2-Hydroxy-4-methoxyphenyl)propan-1-one (5b). Yield
88%, 1H NMR (400 MHz, CDCl3)
dd, J ¼ 9.2, 2.4 Hz), 6.47 (1H, d, J ¼ 2.36 Hz), 3.91 (3H, s), 3.11e3.06
(2H, m), 1.38 (3H, t, J ¼ 7.4 Hz).
d
4.65 (1H, d, J ¼ 9.3 Hz), 6.57 (1H,
6.2.1.3. 1-(2,5-Dihydroxy-4-methoxyphenyl)propan-1-one (5c). Yield
86%, 1H NMR (200 MHz, CDCl3)
d 7.15 (1H, s), 6.36 (1H, s), 3.84 (3H,
s), 2.83 (2H, q, J ¼ 7.2 Hz), 1.14 (3H, t, J ¼ 7.2 Hz).
6.2.2. General procedure for the preparation of ethyl 4-oxo-4H-
chromen-2-carboxylate derivatives (6aec)
(400 MHz, DMSO-d6)
(1H, s), 2.18 (3H, s); 10a: 1H NMR (400 MHz, DMSO-d6)
s, eOH), 9.91 (1H, s, eOH), 7.29 (1H, s), 6.85 (1H, s), 3.92 (3H, s), 2.20
(3H, s); 11a: 1H NMR (400 MHz, CDCl3)
7.59 (1H, s), 7.28 (1H, s),
5.41 (2H, s), 5.30 (2H, s), 3.43 (3H, s), 3.42 (3H, s), 2.23 (3H, s); 7d:
1H NMR (400 MHz, DMSO-d6)
7.59 (1H, s), 7.29 (1H, s), 5.42 (2H,
d
10.59 (1H, s), 9.85 (2H, brs), 7.28 (1H, s), 6.85
Ethyl oxalyl chloride (2.0 eq.) was added a solution of 5 (1.0 eq.) in
pyridine and heated at reflux for 12 h. After reaction completion, the
mixturewascooledtort, pouredintowater, andextractedwithEtOAc.
The organic layer was dried over MgSO4, concentrated, and purified
by flash column chromatography (EtOAc/hexane ¼ 1:5) to afford 6.
d
10.62 (1H,
d
d
s), 5.31 (2H, s), 3.44 (3H, s), 3.43 (3H, s), 2.23 (3H, s).
6.2.2.1. Ethyl 7-methoxy-3-methyl-4-oxo-4H-chromen-2-carboxylate
(6a). Yield 27%, 1H NMR (400 MHz, CDCl3)
s), 4.47e4.42 (2H, m), 3.97 (2H, s), 3.96 (2H, s), 2.35 (3H, s), 1.43 (3H,
t, J ¼ 7.1 Hz).
d
7.47 (1H, s), 6.90 (1H,
6.2.6. Procedure for the preparation of 7-methoxymethoxy-3-
methyl-4-oxo-4H-chromen-2-carboxylic acid (7e)
The compound 7e was prepared from 6b according to the
procedure described for 7d. 9b: Yield 51%, 1H NMR (400 MHz,
6.2.2.2. Ethyl 6,7-dimethoxy-3-methyl-4-oxo-4H-chromen-2-carbox-
DMSO)
d
7.88 (1H, d, J ¼ 8.7 Hz), 6.92 (1H, dd, J ¼ 8.7, 1.6 Hz), 6.81
ylate (6b). Yield 46%, 1H NMR (400 MHz, CDCl3)
d
8.06 (1H, d,
(1H, d, J ¼ 1.6 Hz), 2.19 (3H, s); 10b: Yield 86%, 1H NMR (400 MHz,
J ¼ 8.9 Hz), 6.94 (1H, dd, J ¼ 8.9, 2.4 Hz), 6.86 (1H, d, J ¼ 2.4 Hz),
DMSO-d6)
d
10.90 (1H, s, eOH), 7.86 (1H, d, J ¼ 8.8 Hz), 7.03 (1H, dd,
4.47e4.42 (2H, m), 3.88 (3H, s), 2.33 (3H, s), 1.43 (3H, t, J ¼ 7.1 Hz).
J ¼ 8.8, 2.2 Hz), 6.77 (1H, d, J ¼ 2.2 Hz), 3.91 (3H, s), 2.17 (3H, s); 11b:
Yield 95%, 1H NMR (400 MHz, CDCl3)
d
8.03 (1H, d, J ¼ 8.8 Hz), 7.04
6.2.2.3. Ethyl 6-hydroxy-7-methoxy-3-methyl-4-oxo-4H-chromen-2-
(1H, d, J ¼ 2.3 Hz), 6.98 (1H, dd, J ¼ 8.8, 2.3 Hz), 5.19 (2H, s), 3.92
carboxylate (6c). Yield 32%, 1H NMR (400 MHz, DMSO-d6)
d 7.27
(3H, s), 3.43 (3H, s), 2.28 (3H, s); 7e: Yield 97%, 1H NMR (400 MHz,
(1H, s), 7.09 (1H, s), 4.41e4.36 (2H, m), 3.92 (3H, s), 2.19 (3H, s), 1.35
(3H, t, J ¼ 7.2 Hz).
pyridine-d5)
d
7.97 (1H, d, J ¼ 8.8 Hz), 7.18 (1H, d, J ¼ 2.0 Hz), 7.13
(1H, dd, J ¼ 8.8, 2.0 Hz), 5.38 (2H, s), 3.43 (3H, s), 2.22 (3H, s).
6.2.3. General procedure for the preparation of 4-oxo-4H-chromen-
2-carboxylic acid derivatives (7aeb)
KOH (2 eq.) was added to solutions of 6a or b (1 eq.) in 50%
aqueous ethanol, and stirred at rt for 1 h. Mixtures were treated
with 3 N HCl to a pH of 2, and the resulting precipitates were
filtered and dried to afford 7a or b.
6.2.7. Procedure for the preparation of (S)-N-(4-amino-3,4-dioxo-
1-phenylbutan-2-yl)-6,7-dimethoxy-3-methyl-4-oxo-4H-chromen-
2-carboxamide (3a)
To a solution of 7a (129 mg, 0.49 mmol) and 1-hydroxybenzo-
triazole (HOBt, 93.8 mg, 0.69 mmol) in DMF (5 ml) were added
successively (2R, 3S)-3-amino-2-hydroxy-4-phenylbutanamide
(12a, 95 mg, 0.49 mmol) and 1-[3-(dimethylamino)propyl]-3-eth-
ylcarbodiimide$HCl (EDC, 133 mg, 0.69 mmol) at 0 ꢁC. The mixture
was then stirred at rt for 2 h, treated with water, and the resulting
precipitate was filtered, washed with ether, and dried to afford
6.2.3.1. 6,7-Dimethoxy-3-methyl-4-oxo-4H-chromen-2-carboxylic
acid (7a). Yield 60%, 1H NMR (400 MHz, pyridine-d5)
d
11.17 (1H, brs,
eOH), 7.73 (1H, s), 7.10 (1H, s), 3.81 (1H, s), 3.74 (1H, s), 2.77 (3H, s).

1726
S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
a coupling adduct, N-((2S, 3R)-4-amino-3-hydroxy-4-oxo-1-phe-
nylbutan-2-yl)-6,7-dimethoxy-3-methyl-4-oxo-4H-chromen-2-
carboxamide (134 mg, 63%). This compound (100 mg, 0.23 mmol)
was then dissolved in DMF (5 ml), treated with DesseMartin
periodinane (193 mg, 0.46 mmol) at 0 ꢁC, and stirred at rt for 2 h.
10% Na2S2O3 aqueous solution (1 ml) was then added and stirred
for 5 min. The resulting solid was filtered and purified by flash
column chromatography (CH2Cl2:MeOH ¼ 30:1) to afford 3a
3.91 (3H, s), 3.29 (1H, dd, J ¼ 14.4, 4.4 Hz), 2.96 (1H, dd, J ¼ 14.4,
10.0 Hz), 1.94 (3H, s); 13C NMR (100 MHz, DMSO-d6)
196.1, 177.1,
164.5, 161.5, 161.1, 157.0, 152.3, 138.9, 137.7, 129.6, 128.9, 128.8,
127.8, 127.4, 127.2, 127.0, 119.3, 116.3, 115.7, 100.8, 56.6, 56.5, 42.6,
35.3, 9.9; DART-MS m/z 499.1870 (calcd for C32H32N2O8 [M þ H]þ,
499.1864).
d
6.2.12. (S)-7-Methoxy-N-(4-(4-methoxyphenethylamino)-3,4-
dioxo-1-phenylbutan-2-yl)-3-methyl-4-oxo-4H-chromen-2-
carboxamide (3f)
(23 mg, 23%). 1H NMR (400 MHz, DMSO-d6)
d 9.34 (1H, d,
J ¼ 7.6 Hz), 8.18 (1H, s, eNH), 7.92 (1H, s, eNH), 7.37e7.19 (6H, m),
7.18 (1H, s), 5.49e5.44 (1H, m), 3.93 (3H, s), 3.86 (3H, s), 3.28 (1H,
dd, J ¼ 14.0, 3.6 Hz), 2.90 (1H, dd, J ¼ 14.0, 10.1 Hz), 1.95 (3H, s); 13C
Compound 3f was prepared from 7b (57 mg, 0.24 mmol) and
12c (80 mg, 0.24 mmol) as described for 3a. 1H NMR (400 MHz,
NMR (100 MHz, DMSO-d6)
d
196.1, 176.3, 162.5, 161.1, 154.6, 150.8,
DMSO-d6)
d
9.36 (1H, d, J ¼ 7.3 Hz), 8.90 (1H, t, J ¼ 5.4 Hz),
147.6, 137.3, 129.6, 128.9, 128.4, 126.6, 118.2, 115.2, 103.5, 100.3,
100.2, 56.4, 55.8, 34.7, 9.6; DART-MS m/z 439.1492 (calcd for
C23H22N2O7 [M þ H]þ, 439.1500).
7.35e6.80 (12H, m), 5.48e5.44 (1H, m), 3.91 (3H, s), 3.67 (3H, s),
3.18 (1H, dd, J ¼ 13.8, 3.0 Hz), 2.85 (1H, dd, J ¼ 13.6, 10.3 Hz), 1.93
(3H, s); 13C NMR (100 MHz, DMSO-d6)
d 195.6, 176.6, 164.0,160.9,
160.3, 157.7, 156.5, 151.9, 137.2, 130.8, 129.6, 128.9, 128.4, 126.7,
126.5, 118.6, 115.8, 115.2, 113.7, 100.3, 56.1, 55.9, 54.8, 40.4, 34.6,
33.6, 9.4; DART-MS m/z 543.2134 (calcd for C32H32N2O8 [M þ H]þ,
543.2126).
6.2.8. (S)-N-(4-Benzylamino-3,4-dioxo-1-phenylbutan-2-yl)-6,7-
dimethoxy-3-methyl-4-oxo-4H-chromen-2-carboxamide (3b)
Compound 3b was prepared from 7a (120 mg, 0.45 mmol) and
(2R, 3S)-3-amino-N-benzyl-2-hydroxy-4-phenylbutanamide (12b,
129 mg, 0.45 mmol) using the procedure described for 3a. 1H NMR
6.2.13. (S)-N-(4-Benzylamino-3,4-dioxo-1-phenylbutan-2-yl)-6-
hydroxy-7-methoxy-3-methyl-4-oxo-4H-chromen-2-carboxamide
(3g)
(400 MHz, DMSO-d6)
d
9.41 (1H, d, J ¼ 7.2 Hz), 9.38 (1H, t,
J ¼ 5.6 Hz), 7.35e7.14 (11H, m), 7.10 (1H, s), 5.49e5.44 (1H, m), 4.37
(2H, t, J ¼ 5.6 Hz), 3.93 (3H, s), 3.86 (3H, s), 3.30 (1H, dd, J ¼ 14.0,
4.4 Hz), 2.97 (1H, dd, J ¼ 14.0, 9.6 Hz), 1.97 (3H, s); 13C NMR
(S)-N-(4-Benzylamino-3,4-dioxo-1-phenylbutan-2-yl)-7-
methoxy-6-(methoxymethoxy)-3-methyl-4-oxo-4H-chromen-2-
carboxamide was obtained by coupling 7c (150 mg, 0.51 mmol) and
12b (145 mg, 0.51 mmol) followed by DesseMartin periodinane
oxidation as described for 3a. A portion of the above coupling
compound (40 mg, 0.07 mmol) obtained was then dissolved in 1%
methanolic HCl (10 mL) and heated at 70 ꢁC for 2 h. This mixture
was then diluted with EtOAc and washed successively with water
and brine, and the organic layer was dried over MgSO4, concen-
trated, and purified by column chromatography (CH2Cl2:MeOH ¼
30:1) to afford 3g (22 mg, 60%). 1H NMR (400 MHz, DMSO-d6)
(100 MHz, DMSO-d6)
d 195.7, 176.3, 161.1, 160.6, 154.6, 151.4, 150.8,
147.6, 138.4, 137.2, 129.0, 128.4, 128.2, 127.3, 126.9, 126.6, 118.4,
115.2, 103.5, 100.1, 56.3, 55.9, 55.8, 42.1, 34.8, 9.5; DART-MS m/z
529.1961 (calcd for C30H28N2O7 [M þ H]þ, 529.1969).
6.2.9. (S)-6,7-Dimethoxy-N-(4-(4-methoxyphenethyl amino)-3,4-
dioxo-1-phenylbutan-2-yl)-3-methyl-4-oxo-4H-chromen-2-
carboxamide (3c)
Compound 3c was prepared from 7a (64 mg, 0.24 mmol) and
(2R, 3S)-3-amino-2-hydroxy-N-(4-methoxyphenethyl)-4-phenyl-
butanamide (12c, 80 mg, 0.24 mmol) using the procedure
d
9.86(1H, s, eOH), 9.39e9.35 (2H, m), 7.32e7.21 (11H, m), 7.11 (1H,
s), 5.47e5.41 (1H, m), 4.36 (2H, t, J ¼ 5.6 Hz), 3.92 (3H, s), 3.28 (1H,
described for 3a. 1H NMR (400 MHz, DMSO-d6)
d 9.34 (1H, d,
dd, J ¼ 14.0, 4.4 Hz), 2.95 (1H, dd, J ¼ 14.0, 10.0 Hz), 1.93 (3H, s); 13C
J ¼ 7.3 Hz), 8.90 (1H, t, J ¼ 5.3 Hz), 7.34e6.83 (11H, m), 5.49e5.43
(1H, m), 3.92 (3H, s), 3.86 (3H, s), 3.67 (3H, s), 3.18 (1H, dd, J ¼ 13.7,
3.1 Hz), 2.86 (1H, dd, J ¼ 13.7, 10.4 Hz), 1.95 (3H, s); 13C NMR
NMR (100 MHz, DMSO-d6) d 195.7, 176.3, 161.2, 160.6, 154.1, 151.4,
149.9, 145.5138.4, 137.2, 129.0, 128.4, 128.2, 127.3, 126.9, 126.6, 117.9,
115.6, 107.0, 100.1, 56.2, 55.9 42.1 34.8, 9.5; DART-MS m/z 515.1836
(calcd for C32H32N2O8 [M þ H]þ, 515.1813).
(100 MHz, DMSO-d6)
d 195.7, 176.3, 161.1, 160.3, 157.7, 154.6, 151.6,
150.8, 147.5, 137.3, 130.8, 129.6, 128.9, 128.4, 126.7, 118.1, 115.2, 113.7,
103.4, 100.1, 56.3, 55.9, 55.7, 54.9, 40.4, 34.6, 33.6, 9.6; DART-MS m/z
573.2248 (calcd for C32H32N2O8 [M þ H]þ, 573.2232).
6.2.14. (S)-N-(4-(4-Methoxyphenethylamino)-3,4-dioxo-1-
phenylbutan-2-yl)-6-hydroxy-7-methoxy-3-methyl-4-oxo-4H-
chromen-2-carboxamide (3h)
6.2.10. (S)-N-(4-Amino-3,4-dioxo-1-phenylbutan-2-yl)-7-
methoxy-3-methyl-4-oxo-4H-chromen-2-carboxamide (3d)
Compound 3d was prepared from 7b (110 mg, 0.47 mmol) and
12a (91.3 mg, 0.47 mmol) as described for 3a. 1H NMR (400 MHz,
Compound 3h was prepared from 7c (108 mg, 0.37 mmol)
and 12c (120 mg, 0.37 mmol) as described for 3g. 1H NMR
(400 MHz, DMSO-d6)
d
9.33 (1H, d, J ¼ 7.4 Hz, eNH), 8.92 (1H, t,
J ¼ 5.8 Hz, eNH), 7.35e6.80 (11H, m), 5.46 (1H, m), 3.91 (3H, s),
3.67 (3H, s), 3.17 (1H, dd, J ¼ 13.9, 3.8 Hz), 2.85 (1H, dd, J ¼ 13.9,
9.6 Hz), 2.72e2.74 (2H, m), 1.93 (3H, s); 13C NMR (100 MHz,
DMSO-d6)
d
9.36 (1H, d, J ¼ 7.6 Hz), 8.18 (1H, s, eNH2), 7.33e7.15
(5H, m), 7.12 (1H, d, J ¼ 2.4 Hz), 7.08 (1H, dd, J ¼ 8.8, 2.4 Hz),
5.49e5.43 (1H, m), 3.92 (3H, s), 3.28 (1H, dd, J ¼ 14.0, 4.0 Hz), 2.90
(1H, dd, J ¼ 14.0, 10.0 Hz), 1.93 (3H, s); 13C NMR (100 MHz, DMSO-
DMSO-d6)
d 195.7, 176.3, 161.2, 160.3, 157.7, 154.1, 151.6, 149.9,
145.4, 137.3, 130.8, 129.6 (2C), 129.0 (2C), 128.5 (2C), 126.6, 117.8,
115.6, 113.76 (2C), 106.9, 100.1, 56.2, 55.9, 54.9, 40.4 34.6, 33.6,
9.5; DART-MS m/z 559.2080 (calcd for C32H32N2O8 [M þ H]þ,
559.2075).
d6) d 196.1, 176.6, 164.0, 162.4, 160.9, 156.5, 151.9, 137.3, 129.2, 129.0,
128.4, 128.1, 126.5, 118.6, 115.8, 100.3, 56.1, 55.7, 34.7, 9.4; DART-MS
m/z 409.1377 (calcd for C32H32N2O8 [M þ H]þ, 409.1394).
6.2.11. (S)-N-(4-Benzylamino-3,4-dioxo-1-phenylbutan-2-yl)-7-
methoxy-3-methyl-4-oxo-4H-chromen-2-carboxamide (3e)
Compound 3e was prepared from 7b (150 mg, 0.64 mmol) and
12b (182 mg, 0.64 mmol) as described for 3a. 1H NMR (400 MHz,
6.2.15. (S)-N-(4-Benzylamino-3,4-dioxo-1-phenylbutan-2-yl)-6,7-
dihydroxy-3-methyl-4-oxo-4H-chromen-2-carboxamide (3i)
Compound 3i was prepared from 7d (150 mg, 0.46 mmol) and
12b (131.5 mg, 0.46 mmol) as described for 3g above. 1H NMR
DMSO-d6)
d
9.42 (1H, d, J ¼ 7.2 Hz), 9.37 (1H, t, J ¼ 6.4 Hz), 7.95
(400 MHz, DMSO-d6) d 9.38e9.33 (2H, m, two eNH), 7.33e7.21
(1H, d, J ¼ 8.8 Hz), 7.33e7.18 (10H, m), 7.10 (1H, dd, J ¼ 7.2, 2.4 Hz),
(11H, m), 6.91 (1H, s), 5.38 (1H, m), 4.36 (2H, t, J ¼ 6.4 Hz), 3.27 (1H,
7.07 (1H, d, J ¼ 2.4 Hz), 5.48e5.43 (1H, m), 4.37 (2H, t, J ¼ 5.6 Hz),
dd, J ¼ 13.8, 3.6 Hz), 2.96 (1H, dd, J ¼ 13.8, 9.6 Hz), 1.94 (3H, s); 13C

S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
1727
NMR (100 MHz, DMSO-d6)
d
195.8, 176.3, 161.3, 160.7, 152.9, 150.8,
methylcoumarin, was measured using a spectrofluorimeter at
lex ¼ 380 nm and lem ¼ 460 nm against calpain free blank. IC50
values were calculated based on percent inhibitions of enzyme
activity.
149.9, 144.9,138.4, 137.3, 129.1 (2C), 128.4 (2C), 128.36 (2C), 127.36
(2C), 126 9, 126.6, 118.0, 114.96 (2C), 107.4, 102.7, 56.1, 42.1, 34.7,
9.4; DART-MS m/z 501.1677 (calcd for C32H32N2O8 [M þ H]þ,
501.1656).
6.3.2. DPPH radical scavenging activity assay
6.2.16. (S)-6,7-Dihydroxy-N-(4-(4-methoxyphenethylamino)-3,4-
dioxo-1-phenylbutan-2-yl)-3-methyl-4-oxo-4H-chromen-2-
carboxamide (3j)
The antioxidant activities of the synthesized compounds were
assessed by examining their abilities to scavenge the 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free radical. Reaction mixtures
Compound 3j was prepared from 7d (119 mg, 0.37 mmol) and
containing test samples (dissolved in EtOH) and 100 mM of
12c (120 mg, 0.37 mmol) as described for 3g. 1H NMR (400 MHz,
ethanolic DPPH solution in 96-well plates were incubated at
37 ꢁC for 30 min. Absorbances were measured at 515 nm. Percent
inhibitions were calculated versus ethanol-treated controls. IC50
values denote the concentration required to scavenge 50% of
DPPH radicals.
DMSO-d6)
d
9.28 (1H, d, J ¼ 7.3 Hz), 8.92 (1H, t, J ¼ 5.6 Hz),
7.31e6.83 (11H, m), 5.42e5.37 (1H, m), 3.67 (3H, s), 3.25e3.21 (1H,
m), 3.16 (1H, dd, J ¼ 13.8, 3.7 Hz), 3.10e3.04 (1H, m), 2.86 (1H, dd,
J ¼ 13.8, 10.4 Hz), 2.75e2.71 (2H, m), 1.94 (3H, s); 13C NMR
(100 MHz, DMSO-d6)
d 195.7, 176.2, 160.3, 157.6, 152.8, 149.8, 144.9,
137.3, 130.7, 130.4, 129.5, 128.9, 128.2, 127.5, 126.6, 117.8, 114.8,113.6,
107.3, 102.7, 54.8, 54.7, 40.6 33.5, 33.4, 9.4; DART-MS m/z 545.1908
(calcd for C32H32N2O8 [M þ H]þ, 545.1919).
6.3.3. Inhibition of lipid peroxidation
The effects of the synthesized compounds on lipid perox-
idation induced by an iron-ascorbic acid mix were determined in
rat liver homogenate. In brief, rat liver homogenate (300
ml,
6.2.17. (S)-N-(4-Benzylamino-3,4-dioxo-1-phenylbutan-2-yl)-7-
hydroxy-3-methyl-4-oxo-4H-chromen-2-carboxamide (3k)
Compound 3k was prepared from 7e (200 mg, 0.76 mmol) and
12b (215 mg, 0.76 mmol) as described for 3g. 1H NMR (400 MHz,
11 mg protein/ml) was incubated with 10 M Fe2SO4, 0.4 mM
m
ascorbic acid and various concentrations of the test compounds
in 50 mM TriseHCl (pH 7.5) in a total volume of 1 ml at 37 ꢁC for
30 min. After incubation, lipid peroxidation levels were deter-
mined by measuring the formation of thiobarbituric acid-reac-
tive substance (TBARS). Reactions were terminated by adding
2 ml of a solution of 0.375% thiobarbituric acid in 15% tri-
chloroacetic acid containing 0.25 N HCl and 0.01% butylated
hydroxytoluene (TBAeTCA reagent). Mixtures were then heated
at 95 ꢁC for 30 min, cooled, and centrifuged at 5000 ꢂ g for
10 min. The absorbances of supernatants were measured at
535 nm.
DMSO-d6)
d
9.43e9.38 (2H, m, eNH), 7.88 (1H, d, J ¼ 8.8 Hz),
7.33e7.21 (10H, m), 6.97 (1H, d, J ¼ 2.4 Hz), 6.95 (1H, s), 5.43e5.38
(1H, m), 4.37 (2H, t, J ¼ 6.0 Hz), 3.28 (1H, dd, J ¼ 14.0, 4.4 Hz), 2.97
(1H, dd, J ¼ 14.0, 10.0 Hz), 1.94 (3H, s); 13C NMR (100 MHz, DMSO-
d6)
d 195.7, 176.6, 163.1, 161.1, 160.8, 156.5, 151.3, 138.4, 137.3, 129.1,
128.3, 128.2, 127.3, 126.9, 126.8, 126.6, 118.7, 115.6, 114.8, 102.1, 56.1,
42.1 34.7, 9.4; DART-MS m/z 485.1735 (calcd for C32H32N2O8
[M þ H]þ, 485.1707).
Protein contents in liver homogenates were determined using
the Bradford method using bovine serum albumin as a standard.
6.2.18. (S)-7-Hydroxy-N-(4-(4-methoxyphenethylamino)-3,4-
dioxo-1-phenylbutan-2-yl)-3-methyl-4-oxo-4H-chromen-2-
carboxamide (3l)
Acknowledgments
Compound 3l was prepared from 7e (64 mg, 0.24 mmol) and
12c (80 mg, 0.30 mmol) as described for 3g. 1H NMR (400 MHz,
This work was supported by the Basic Science Research Program
of the Korean National Research Foundation (NRF) funded by MEST
(#2010-0016080).
DMSO-d6)
d
10.9 (1H, brs, eOH), 9.31 (1H, d, J ¼ 7.4 Hz, eNH), 8.90
(1H, t, J ¼ 5.7 Hz, eNH), 7.89 (1H, d, J ¼ 8.8 Hz), 7.33e6.83 (11H, m),
5.41 (1H, m), 3.68 (3H, s), 3.17 (1H, dd, J ¼ 13.6, 3.6 Hz), 2.86 (1H, dd,
J ¼ 13.6, 9.6 Hz), 2.72e2.75 (2H, m),1.94 (3H, s); 13C NMR (100 MHz,
DMSO-d6) d 195.7, 176.6, 162.9, 161.1, 160.3, 157.7, 156.5, 151.4, 137.3,
References
130.8, 129.6 (2C), 129.0 (2C), 128.4 (2C), 126.9, 126.6, 118.6, 115.5,
114.8, 113.7 (2C), 102.1, 56.1, 54.9, 40.4 34.5, 33.6, 9.4; DART-MS m/z
529.1934 (calcd for C32H32N2O8 [M þ H]þ, 529.1969).
[1] K. Suzuki, H. Sorimachi, T. Yoshizawa, K. Kinbara, S. Ishiura, Biol. Chem.
Hoppe-Seyler 376 (1995) 523e529.
[2] D.E. Croall, G.N. DeMartino, Physiol. Rev. 71 (1991) 813e847.
[3] D.E. Goll, V.F. Thompson, H. Li, W. Wei, J. Cong, Physiol. Rev. 83 (2003)
731e801.
6.3. Biological activity testing
[4] C.G. Markgraf, N.L. Velayo, M.P. Johnson, D.R. McCarty, S. Medhi, J.R. Koehl,
P.A. Chmielwski, M. Linnik, Stroke 29 (1998) 152e158.
[5] S. Samantaray, J.T. Butler, S.K. Ray, N.L. Banik, N.Y. Ann, Acad. Sci. 1139 (2008)
331e336.
[6] B. Barry, C. Veronica, Neurobiol. Aging 23 (2003) 179e186.
[7] N.O. Carragher, Curr. Pharm. Des. 12 (2006) 615e638.
[8] F. Trinchese, M. Fa, S. Liu, H. Zhang, A. Hidalgo, S.D. Schmidt, H. Yamaguchi,
N. Yoshii, P.M. Mathews, R.A. Nixon, O. Arancio, J. Clin. Invest. 118 (2008)
2796e2807.
6.3.1. Calpain inhibitory activity
Calpain inhibitory activity was assayed using a slight modifica-
tion of a previously reported procedure [26]. Briefly, calpain I
(Calbiochem) from human erythrocytes and the fluorogenic calpain
substrate (Suc-Leu-Tyr-7-amino-4-methylcoumarin, Calbiochem)
were used as enzyme and substrate, respectively. In a typical
[9] M. Yamada, Y. Yoshida, D. Mori, T. Takitoh, M. Kengaku, H. Umeshima,
K. Takao, T. Miyakawa, M. Sato, H. Sorimachi, A. Wynshaw-Boris, S. Hirotsune,
Nat. Med. 15 (2009) 1202e1207.
[10] C. Lizama, C.F. Lagos, R. Lagos-Cabre, L. Cantuarias, F. Rivera, P. Huenchunir,
T. Perez-Acle, F. Carrion, R.D. Moreno, J. Cell. Physiol. 221 (2009) 296e305.
[11] A.J. Wiemer, M.A. Lokuta, J.C. Surfus, S.A. Wernimont, A. Huttenlocher, Mol.
Immunol. 47 (2010) 894e902.
[12] S. Mehdi, Trends Biochem. Sci. 16 (1991) 150e153.
[13] Y. Shirasaki, M. Yamaguchi, H. Miyashita, J. Ocul. Pharmacol. Ther. 22 (2006)
417e424.
[14] D. Leung, G. Abbenante, D.P. Fairlie, J. Med. Chem. 43 (2000) 305e341.
[15] K.S. Lee, S.H. Seo, Y.H. Lee, H.D. Kim, M.H. Son, B.Y. Chung, J.Y. Lee, C. Jin,
Y.S. Lee, Bioorg. Med. Chem. Lett. 15 (2005) 2857e2860.
experiment, 1
trations), 25
11 l of 1 mM CaCl2 were added to the wells of 96-well plates. All
chemicals except for the test compounds were made up in 50 mM
TriseHCl (pH 7.5) buffer containing 1 mM dithiothreitol immedi-
ately before use. Test compounds were dissolved in DMSO, but
DMSO concentrations in the final experimental solutions did not
exceed 1%. Incubations were initiated by adding 53 ml of 34 nM
calpain and mixtures were then incubated for 30 min at rt. After
incubation, the fluorescence of the cleavage product, 7-amino-4-
m
l of a test compound solution (at different concen-
m
l of 1 mM calpain substrate, 10 l of 1 M NaCl, and
m
m

1728
S.H. Kim et al. / European Journal of Medicinal Chemistry 46 (2011) 1721e1728
[16] D.H. Nam, K.S. Lee, S.H. Kim, S.M. Kim, S.Y. Jung, S.H. Chung, H.J. Kim, N.D. Kim,
C. Jin, Y.S. Lee, Bioorg. Med. Chem. Lett. 18 (2008) 205e209.
[17] K.Y. Lee, K.S. Lee, C. Jin, Y.S. Lee, Eur. J. Med. Chem. 44 (2009) 1331e1334.
[18] K.P. Loh, S.H. Huang, R. De Silva, B.K.H. Tan, Y.Z. Zhu, Curr. Alzheimer Res. 3
(2006) 327e337.
[22] S.H. Kim, Ch.N. Kumar, H.J. Kim, D. Han Kim, J. Cho, C. Jin, Y.S. Lee, Bioorg. Med.
Chem. Lett. 19 (2009) 6009e6013.
[23] J.A. Donnelly, D.E. Maloney, Tetrahedron 35 (1979) 2883e2888.
[24] S. Kim, D.W. Sohn, Y.C. Kim, S.-A. Kim, S.K. Lee, H.S. Kim, Arch. Pharm. Res. 30
(2007) 18e21.
[19] W. Huang, Y. Ding, Y. Miao, M.-Z. Liu, Y. Li, G.-F. Yang, Eur. J. Med. Chem. 44
(2009) 3687e3696.
[25] W.-H. Huang, P.-Y. Chien, C.-H. Yang, A.-R. Lee, Chem. Pharm. Bull. 51 (2003)
339e340.
[20] P.-L. Zhao, J. Li, G.-F. Yang, Bioorg. Med. Chem. 15 (2007) 1888e1895.
[21] Z.-Z. Zhou, G.-F. Yang, Bioorg. Med. Chem. 14 (2006) 8666e8674.
[26] T. Sasaki, T. Kikuchi, N. Yumoto, N. Yoshimura, T. Murachi, J. Biol. Chem. 51
(1984) 12489e12494.