Synthesis of cinnamoyl ketoamides as hybrid structures of antioxidants and calpain inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.03.077

The excessive calpain activation causes serious cellular damage or even cell death in neurological disorders such as stroke and Alzheimer's disease. Oxidative stress has also been implicated in the initiation or progression of neurodegenerative diseases. In the present studies, a series of cinnamoyl ketoamides 4a-4j were synthesized as hybrid structures of antioxidants and calpain inhibitors. Cinnamoyl ketoamides, possessing an alkyl chain at the α-position, showed potent μ-calpain inhibitory activities indicating that the cinnamoyl skeleton can be regarded as an acyclic variant of calpain inhibitory chromone carboxamide 2. Among synthesized, compound 4e was the most potent inhibitor of μ-calpain (IC₅₀ = 0.13 μM) and also exhibited strong antioxidant activities in DPPH and superoxide anion radical scavenging and lipid peroxidation inhibition assay systems.

Full text of DOI:10.1016/j.bmcl.2011.03.077

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2850–2854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of cinnamoyl ketoamides as hybrid structures of antioxidants
and calpain inhibitors
Yeong Jae Yoo a,b, Dong Hyuk Nam a,b, Seo Yun Jung a, Jae Wan Jang c, Hyoung Ja Kim c, Changbae Jin c,
Ae Nim Pae c, Yong Sup Lee a,b,
⇑
a Department of Pharmaceutical Science, College of Pharmacy Kyung Hee University, Seoul 130-701, Republic of Korea
b Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Republic of Korea
c Life & Health Division, Korea Institute of Science & 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 excessive calpain activation causes serious cellular damage or even cell death in neurological disor-
ders such as stroke and Alzheimer’s disease. Oxidative stress has also been implicated in the initiation or
progression of neurodegenerative diseases. In the present studies, a series of cinnamoyl ketoamides 4a–
4j were synthesized as hybrid structures of antioxidants and calpain inhibitors. Cinnamoyl ketoamides,
Received 25 January 2011
Revised 4 March 2011
Accepted 23 March 2011
Available online 30 March 2011
possessing an alkyl chain at the
the cinnamoyl skeleton can be regarded as an acyclic variant of calpain inhibitory chromone carboxamide
2. Among synthesized, compound 4e was the most potent inhibitor of -calpain (IC50 = 0.13 M) and also
a-position, showed potent l-calpain inhibitory activities indicating that
Keywords:
Calpain inhibitor
Cell death
Cinnamoyl ketoamides
Docking, Antioxidant
l
l
exhibited strong antioxidant activities in DPPH and superoxide anion radical scavenging and lipid perox-
idation inhibition assay systems.
Ó 2011 Elsevier Ltd. All rights reserved.
Calpain is a Ca2+-activated cysteine protease typically associ-
ated with cellular necrosis.1 Two isozymes of
- and m-calpains
show similar biochemical features, except for the calcium concen-
tration necessary for activation in vitro: - and m-calpains require
provide significant efficacy for preventing or retarding cell death
in neurodegenerative diseases.13 Accordingly, we considered a
combination of calpain inhibitory and antioxidant activities in
the design of neuroprotective agents so that they can protect
against cell death through dual mechanisms. Although some re-
ports on the linkage of two antioxidants14,15 or calpain inhibitory
moiety with an antioxidant through an amide bond has been re-
ported,16 synthesis of hybrid structures of calpain inhibitory and
antioxidative moieties was scarce. In connection with our efforts
l
l
micromolar and millimolar levels of calcium, respectively.2 Cal-
pains are involved in numerous physiological processes; signal
transduction, cell migration, differentiation, and apoptosis.3 How-
ever, in some pathological conditions in neurological disorders
such as in stroke,4 Parkinson’s disease5 and Alzheimer’s disease,6
calpains are overactivated to cause serious cell death. Thus the
inhibition of this enzyme has been considered as one of strategy
for treating neurodegenerative diseases.
Ph
O
O
Ph
O
O
Oxidative stress has also been implicated in the initiation or
progression of neurodegenerative diseases.7,8 Reactive oxygen spe-
cies (ROS) are highly reactive and can attack lipids, proteins, en-
zymes and DNA, and thus cause cell and tissue injuries.9
Especially, ROS is also known to induce calpain activation.10 There-
fore, antioxidants may provide a means for preventing or treating
oxidative stress-related neurodegenerative disorders.
Recently, combinations of compounds, each possessing a differ-
ent mechanism of action in preventing cell death, have shown syn-
ergistic effects in ischemic animal models.11,12 It was suggested
that a neuroprotectant with multiple mechanisms of action may
O
O
H
N
N
H
NH2
O
H
N
H
O
O
1, MDL 28170
2, KYS 4516
Ph
O
CH3
O
O
O
Ph
O
O
N
H
NH2
R1
R2
R4
N
H
N
H
H3C
O
O
R3
O
3
4
⇑
Corresponding author. Tel.: +82 2 961 0370; fax: +82 2 966 3885.
E-mail address: kyslee@khu.ac.kr (Y.S. Lee).
Figure 1. Design of new cinnamoyl ketoamides.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.077

Y. J. Yoo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2850–2854
2851
Ph
O
O
O
O
HO
HO
AcO
AcO
AcO
(a)
(b)
Ph
OH
OH
N
H
NHBn
OH
AcO
O
5
6a
8a
H2N
NHBn
OH
7a
Ph
O
O
Ph
O
O
O
HO
HO
(c)
(d)
N
H
NHBn
AcO
AcO
N
H
NHBn
O
9a
4a
IC50 = 6.07 0.24 µM for µ-calpain
Scheme 1. Reagents and conditions: (a) Ac2O, pyridine, 82%; (b) EDC, HOBt, DMF, 30%; (c) Dess–Martin periodinane, CH2Cl2, 34%; c-HCl, MeOH/H2O (1:2), 97%.
to discover neuroprotectants, we attempted to identify a new scaf-
fold possessing antioxidant activity as well as calpain inhibitory
activity. We recently reported calpain inhibitory chromone carbox-
amide 2,17 as a conformationally restricted cyclic analog of MDL
28170 (1) and 3,18 as an acyclic variants of 2 in order to elucidate
the structural requirements for inhibitor binding to the active site
cating that the cinnamoyl moiety possesses l-calpain inhibitory
activity, however, its potency was not high enough. Accordingly,
it was thought that the substitution of an alkyl chain to this skele-
ton may be required to further mimic the chromone ring as its acy-
clic variant and enhance activities (Table 1).
Alkyl-substituted cinnamic acid derivatives 6b–6g were pre-
pared as illustrated in Scheme 2. (Carbethoxymethylene)-triphen-
ylphosphorane (8c) was alkylated with alkyl iodides in refluxing
acetonitrile and the resulting alkylated ylides 10 were condensed
with benzaldehyde derivatives 11a–11c through the Wittig olefin-
ation reaction to provide alkyl-substituted cinnamic acid ester
derivatives 12b–12g.21 These consecutive reactions proceeded well
with n-alkyl iodides (two steps yield, 54–68%), however, when
using 1-iodo-2-methylpropane, the reaction afforded 12e in only
2% yield probably because of bulkiness of alkyl iodide.22 In the
model studies, acetyl was used for the protection of hydroxyl
groups in caffeic acid. However, for the synthesis of alkyl-substi-
tuted cinnamic acid derivatives 6b–6f, MOM protection was used
since this protecting group is stable in the next basic hydrolysis
step. The methyl ester group of compounds 12b–12g were hydro-
lyzed with KOH in aqueous ethanol to afford cinnamic acid deriv-
atives 6b–6g.
of l-calpain. In this work, we designed a cinnamic acid moiety for
the purpose of introducing calpain inhibitory activities since it can
be regarded as an acyclic variant of the chromone ring in 2 (Fig. 1).
Furthermore, hydroxycinnamic acids such as caffeic acid are al-
ready well known for their antioxidant and neuroprotective
activities.19,20
Before synthesizing a series of cinnamoyl ketoamides 4, we first
examined the importance of an alkyl group at the
a-position of the
cinnamoyl group on -calpain inhibitory activities by the synthesis
l
of 4a, which possesses no alkyl group (Scheme 1).
The hydroxyl groups in caffeic acid (5) were protected as acetyls
and the resulting compound 6a was coupled with hydroxyamide
7a using EDC and HOBt to afford 8a. Compound 8a was oxidized
with Dess–Martin periodinane and then deprotected under the
acidic condition to provide a caffeoyl ketoamide 4a. The
inhibitory activity of 4a was moderate (IC50 = 6.07 0.24
l
-calpain
lM) indi-
Table 1
The yields of coupling, oxidation, and deprotection steps and the
l
-calpain inhibitory activities of 4a–4j and their parent compounds 2 and 3
Ph
O
O
R1
R2
N
H
NHR4
O
R3
4a~4j
Compds
R1, R2
R3
R4
Yields (%)
Oxidation
Calpain inhibitiona (IC50
, lM)
EDC coupling
Deprotection
4a
4b
4c
4d
4e
4f
4g
4h
4i
6.07 0.24
0.61 0.06
0.46 0.06
0.36 0.01
0.13 0.00
0.67 0.01
0.50 0.04
0.32 0.02
0.63 0.03
1.38 0.05
0.07 0.00
0.52 0.01
R1 = R2 = OH
Methyl
Methyl
Ethyl
Benzyl
4-Methoxyphenethyl
Benzyl
4-Methoxyphenethyl
Benzyl
4-Methoxyphenethyl
Benzyl
72
58
94
66
81
22
56
71
64
47
92
89
97
99
23
99
96
96
82
83
83
69
64
62
69
69
—
Ethyl
n-Propyl
n-Propyl
iso-Propyl
n-Propyl
n-Propyl
R1 = OMe, R2 = OH
R1 = R2 = OMe
4-Methoxyphenethyl
4-Methoxyphenethyl
4j
2
3
a
IC50 values (defined as concentrations that inhibited activity by 50%) were calculated using GraphPad Prism using data obtained from at least three independent
experiments. The IC50 values are expressed as the means SEM.

2852
Y. J. Yoo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2850–2854
O
O
(b)
R1
R2
(a)
O
OEt
Ph3P
Ph3P
OEt
OEt
O
R3
R3
10
R1
R2
H
12b, R1 = R2 = OH, R3 = methyl, 63%
12c, R1 = R2 = OH, R3 = ethyl, 64%
12d, R1 = R2 = OH, R3 = n-propyl, 54%
11a, R1 = R2 = OH
, R1 = R2 = OH, R3 = iso-propyl, 2%
11b, R1 = OMe, R2 = OH
11c, R1 = R2 = OMe
12e
12f
, R1 = OMe, R2 = OH, R3 = n-propyl, 68%
12g, R1 = R2 = OMe, R3 = n-propyl, 67%
6b, R1 = R2 = OMOM, R3 = methyl, 88%
6c, R1 = R2 = OMOM, R3 = ethyl, 94%
O
(c)
R1
R2
OH
6d, R1 = R2 = OMOM, R3 = n-propyl, 93%
1
, R = R2 = OMOM, R3 = iso-propyl, 93%
R3
6e
6f
, R1 = OMe, R2 = OMOM, R3 = n-propyl, 97%
6g, R1 = R2 = OMe, R3 = n-propyl, 96%
Scheme 2. Reagents and conditions: (a) (i) R3-CH2-I, CH3CN; (ii) NaOH; (b) toluene, reflux; (c) for 12b–12f; (i) MOM-Cl, NaH, THF; (ii) KOH, EtOH–H2O; for 12g, KOH, EtOH–
H2O.
The synthesis of cinnamoyl ketoamides 4b–4j is shown in
Ph
Scheme 3. The cinnamic acid derivatives 6g were coupled with
hydroxyamide 7a or 7b using general coupling reagents EDC and
HOBt at room temperature followed by oxidation of the resulting
hydroxylamides with Dess–Martin periodinane to afford cinnam-
O
H2N
NHR4
OH
O
7a,b
Dess-Martin
periodinane
R1
R2
EDC, HOBt
DMF
OH
oyl
a-ketoamide 4j. Cinnamoyl ketoamides 4b–4i, which contain
hydroxyl-substituents on its aromatic ring, were obtained by the
same procedures as for 4j with additional deprotection of the
MOM group at the final stage by treating with HCl/MeOH (1%) at
reflux temperature. The chemical yields at each step are summa-
rized in Table 1.
R3
CH2Cl2
6b~6g
The l-calpain inhibitory activities of the cinnamoyl ketoamides
Ph
O
4a–4j were evaluated using human calpain I isolated from erythro-
cytes. Suc-Leu-Tyr-AMC was used as the fluorogenic substrate.23
Parent chromone carboxamide 2 and its acyclic variant 3 were also
tested for comparison and assay results are summarized in Table 1.
O
R1
R2
c-HCl
MeOH
N
H
NHR4
O
R3
The introduction of alkyl chains to the
group increased -calpain inhibitory activities in every compound
when compared to that of 4a. The degree of -calpain inhibitory
a-position of the cinnamoyl
4b~4j
l
l
Scheme 3.
activities was increased in the order of propyl ffi iso-buty-
l > ethyl ffi butyl as alkyl substituents. Compounds 4b–4h possess-
ing hydroxyl groups at both C-6 and C-7 positions of the aromatic
ring showed more potent activities than methoxy-substituted
compounds (4i, 4j). Among synthesized, compound 4e showed
jor improvement of the inhibitory activity of compound 4e was
caused by additional van der Waals interactions of the propyl
and 4-methoxyphenylethyl groups of the inhibitor 4e with the res-
idues, Ala241 and Trp266, located in the S2 and S10 subsites,
respectively, (Goldscore.External.Vdw of 4e = 43.5779 and Gold-
score.External.Vdw of 4a = 25.2249).26 The interaction between
Ala241 and an alkyl group of the co-crystallized ligand in the S2
the most potent inhibitory activity (IC50 = 0.13
pain and its potency was ca. 4-fold higher than that of acyclic var-
iant 3 (IC50 = 0.52 M) and ca. 2-fold lower than that of parent
compound 2 (IC50 = 0.07 M).
The docking studies of 4a and 4e into the active site of
lM) against l-cal-
l
l
l-calpain
subsite has also been observed in several
l-calpain crystal struc-
were performed to understand the structural basis of the differ-
ence in the inhibitory activities of the cinnamoyl ketoamides. A
covalent constraint implemented in GOLD v4.0 was used for dock-
tures including the 2G8J. The stacking between the 4-meth-
p–p
oxyphenylethyl and Trp266 in the S10 subsite might increase
potency similarly to the interaction between the adenine group
of the co-crystallized ligand and Trp266 in the crystal structure
of 2R9C, which was important for the increased activity of the
inhibitors.27 Thus, the substitutions in the cinnamoyl ketoamides
that can occupy the S2 hydrophobic pocket and extend into the
ing simulation and the crystal structure of
l-calpain (PDB code:
2G8J) was obtained from the protein data bank.24,25 The docking
study showed that both compounds 4a and 4e are stabilized within
the active site located near Cys83 residue, called the S1 subsite
through hydrogen bond interactions with the residues and the
water molecules (Fig. 2).24 The compound 4e showed higher
docking score (GOLD fitness score = 8.1000) than that of compound
4a (GOLD fitness score = 3.1271). The hydrogen bond interaction
scored by Gold of 4e was slightly higher than that of 4a, implying
that the angle and distance required for hydrogen bond is more
favorable in the compound 4e (Goldscore.External.Hbond of
4e = 11.2312 and Goldscore.External.Hbond of 4a = 9.7694). How-
ever, comparison between two docking poses suggested that a ma-
S10 subsite in the cinnamoyl ketoamides of
might enhance -calpain inhibitory activities.
l-calpain inhibitor
l
The cinnamoyl ketoamides 4a–4j were evaluated for antioxi-
dant activities using DPPH and superoxide anion radical scaveng-
ing, and lipid peroxidation inhibition assays (Table 2).28 For
comparison purpose, the antioxidant activities of ascorbic acid
and trolox were included as positive controls. Every compound
exhibited a similar level of antioxidant activities each other in
three assay systems, while the parent chromone carboxamide 2

Y. J. Yoo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2850–2854
2853
Figure 2. The cinnamoyl ketoamides 4a (A) and 4e (B) docked into the active site of l-calpain crystal structure (PDB ID: 2G8J). The inhibitor is colored as green, and residues
involved in the hydrogen bonding and van der Waals contact are shown and represented as a white stick form. The hydrogen bond interactions are represented as black
dashed lines. Note that the propyl and 4-methoxyphenethyl substituents in 4e, whereas 4a does not have, show a good contact with Ala241 and Trp266, respectively. The
image was generated using PyMol (http://www.pymol.org).
Table 2
The antioxidant activities of 4a–4j and their parent compound 2 and 3
Compds
DPPH scavenginga
Superoxide anion scavengingb
Lipid peroxidation inhibitionc
d
IC50
(lM)
4a
4b
4c
4d
4e
4f
4g
4h
40.43 1.61
45.48 0.72
46.05 0.72
42.18 1.59
48.64 0.17
36.72 2.14
38.87 2.42
44.65 1.92
>100
31.19 1.01
15.48 0.82
29.22 3.92
27.12 2.08
37.99 1.10
3093 1.50
41.44 1.41
41.68 1.91
>100
7.53 0.24
6.09 0.24
6.40 0.33
6.15 0.12
5.71 0.20
7.34 1.06
5.88 0.28
6.06 1.07
36.11 3.71
>100
4i
4j
>100
>100
2
>100
>100
>100
3
>100
>100
>100
Ascorbic acid
Trolox
30.81 0.60
—
>100
—
>100
71.44 5.54
a
b
c
DPPH radical scavenging activity.
Scavenging activity of superoxide anion radicals generated in the xanthine/xanthine oxidase system.
Iron-dependent lipid peroxidation inhibition activity using rat liver homogenate.
d
IC50 values (defined as concentrations that inhibited activity by 50%) were calculated using GraphPad Prism using data obtained from at least three independent
experiments. The IC50 values are expressed as the means SD.
and its acyclic variant 3 showed no antioxidant activities under
Acknowledgment
100 lM concentrations. The cinnamoyl ketoamides 4a–4h showed
DPPH radical scavenging activities comparable to ascorbic acid and
ca. 10-fold higher lipid peroxidation inhibitory activities than
trolox. Superoxide radical scavenging activities (IC50 = 15.48–
This work was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by MEST (2010-0016080).
41.68 lM) were also observed from every synthesized
compound.29
References and notes
In conclusion, cinnamoyl ketoamides were synthesized as hy-
brid structures of antioxidants and calpain inhibitors. This study
demonstrates that the alkylated cinnamoyl skeleton can be-
regarded as an acyclic variant of the calpain inhibitory chromone
ring. The alkyl and 4-methoxyphenylethyl substituents in cinnam-
oyl ketoamides are likely to increase calpain inhibitory activities
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p–p stacking interaction, respectively,
with the residues in the active site of the enzyme from modeling
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29. Spectral data of selected compounds. Compound 4d: 1H NMR (400 MHz, DMSO-
d6) d 9.22 (1H, t, J = 6.3 Hz, –NH), 9.17 (1H, br s, –OH), 9.09 (1H, br s, –OH), 8.39
(1H, d, J = 7.1 Hz, –NH), 7.32–7.20 (10H, m, aromatic), 6.84 (1H, s, vinyl-H), 6.77
(1H, d, J = 1.6 Hz, Ar-H20), 6.76 (1H, d, J = 8.3 Hz, Ar-H50), 6.62 (1H, dd, J = 1.6,
8.3 Hz, Ar-H60), 5.20–5.14 (1H, m, –CH–CH2–Ph), 4.39–4.29 (2H, m, –NH–CH2–
Ph), 3.17 (1H, dd, J = 4.0, 13.7 Hz, –CH–CH2–Ph), 2.94–2.88 (1H, m, –CH–CH2–
Ph), 2.36 (2H, t, J = 7.5 Hz, –CH2–CH2–CH3), 1.35–1.26 (2H, m, –CH2–CH2–CH3),
0.82 (3H, t, J = 7.2 Hz, –CH2–CH2–CH3); 13C NMR (100 MHz, DMSO-d6) d 196.7,
169.8, 161.1, 145.6, 145.0, 138.5, 138.0, 134.1, 132.9, 129.0 (2C), 128.44, 128.24
(2C), 128.19 (2C), 127.3 (2C), 126.9, 126.87, 126.4, 121.0, 116.1, 115.6, 56.2,
42.0, 34.6, 29.1, 21.4, 13.9. Compound 4e: 1H NMR (400 MHz, DMSO-d6) d 9.20
(1H, br s, –OH), 9.12 (1H, br s, –OH), 8.78 (1H, t, J = 6.0 Hz, –NH), 8.35 (1H, d,
J = 7.2 Hz, –NH), 7.33–7.11 (9H, m, aromatic), 6.86 (1H, s, vinyl-H), 6.84 (1H,
J = 2.0 Hz, Ar-H20), 6.76 (1H, d, J = 8.4 Hz, Ar-H50), 6.67 (1H, dd, J = 2.0, 8.4 Hz,
Ar-H60), 5.23–5.18 (1H, m, –CH–CH2–Ph), 3.68 (3H, s, –OCH3), 3.08 (1H, dd,
J = 3.6, 14.0 Hz, –CH–CH2–Ph), 2.85–2.79 (1H, m, –CH–CH2–Ph), 2.73–2.69 (2H,
m, –NH–CH2–CH2–), 2.43–2.40 (2H, m, –CH2–CH2–Ar), 2.38–2.34 (2H, m, –
CH2–CH2–CH3), 1.33–1.28 (1H, m, –CH2–CH2–CH3), 0.85 (3H, t, J = 7.2 Hz, –
CH2–CH2–CH3); 13C NMR (100 MHz, DMSO-d6) d 196.8, 169.8, 160.9, 157.7,
145.5, 145.1, 138.1, 134.3, 132.7, 130.9, 129.5 (2C), 129.0 (2C), 128.2 (2C),
126.9, 126.4, 120.9, 116.2, 115.6, 113.7 (2C), 56.1, 54.9, 40.3, 34.5, 33.7, 29.2,
21.4, 13.9..
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