1334
K.Y. Lee et al. / European Journal of Medicinal Chemistry 44 (2009) 1331e1334
[17] W. Lubisch, H.P. Hofmann, H.J. Treiber, A. Moller, Bioorg. Med. Chem.
Lett. 10 (2000) 2187e2191.
¨
contains a primary amide, most potently inhibited m-calpain
with an IC50 value of 2.81 ꢀ 1.26 mM. It was less potent than
that of the parent compound, MDL 28,170 (1). These results
of the activity testing of 2, which has limited conformational
flexibility in the P2eP3 region of the inhibitors encourage us
to further investigate the molecular recognition requirements
of the active site of m-calpain.
[18] A.D. Abell, K.M. Brown, J.M. Coxon, M.A. Jones, S. Miyamoto,
A.T. Neffe, J.M. Nikkel, B.G. Stuart, Peptides 26 (2005) 251e258.
[19] I.O. Donkor, Curr. Med. Chem. 7 (2000) 1171e1188.
[20] R.T. Bartus, K.L. Baker, A.D. Heiser, S.D. Sawyer, R.L. Dean, P.J. Elliot,
J.A. Straub, J. Cereb. Blood Flow Metab. 14 (1994) 537e544.
[21] A. Loupy, N. Philippon, P. Pigeon, H. Galons, Heterocycles 32 (1991)
1947e1953.
[22] Spectral data of selected compounds. Compound 6: 1H NMR (200 MHz,
CDCl3) d 7.57 (1H, d, J ¼ 8.4 Hz, H-4), 7.35 (1H, d, J ¼ 8.4 Hz, H-5),
4.45 (2H, q, J ¼ 7.2 Hz, eOCH2CH3), 2.54 (3H, s, eCH3), 1.45 (3H,
t, J ¼ 7.2 Hz, eOCH2CH3); 13C NMR (75 MHz, CDCl3) d 165.2,
148.4 148.0, 142.5, 133.6, 126.6, 62.0, 19.1, 14.3. Compound 7: 1H
NMR (200 MHz, CDCl3) d 7.29 (1H, d, J ¼ 9.2 Hz, H-4), 6.70 (1H, d,
J ¼ 9.4 Hz, H-5), 4.39 (2H, q, J ¼ 7.4 Hz, eOCH2CH3), 2.40 (3H, s, e
CH3), 1.40 (2H, t, J ¼ 7.4 Hz, eOCH2CH3); 13C NMR (75 MHz,
CDCl3) d 161.7, 161.5, 145.6, 129.9, 126.3, 121.4, 62.7, 18.3, 14.2. Com-
pound 8: 1H NMR (200 MHz, D2O) d 7.48 (1H, d, J ¼ 9.2 Hz, H-4), 6.48
(1H, d, J ¼ 9.2 Hz, H-5), 2.17 (3H, s, eCH3); 13C NMR (75 MHz, D2O)
d 167.9, 162.7, 147.7, 137.6, 119.5, 118.3, 16.0. Compound 2a: 1H NMR
(400 MHz, DMSO-d6) d 8.67 (1H, br s, eNH ), 8.12 (1H, s, eNH ), 8.87
(1H, s, eNH ), 7.47 (1H, d, J ¼ 8.4 Hz, eCOCHCHe), 7.29e7.18 (7H,
m, aromatic), 6.36 (1H, d, J ¼ 8.4 Hz, eCOCHCHe), 5.46 (1H, m, e
NHCHCH2Ph), 3.22 (1H, dd, J ¼ 4.4, 14.0 Hz, eNHCHCH2Ph), 2.93
(1H, dd, J ¼ 8.8, 14.0 Hz, eNHCHCH2Ph), 2.19 (3H, s, eC]Ce
CH3); 13C NMR (100 MHz, DMSO-d6) d 197.2, 165.7, 163.3, 161.4,
144.6, 137.9, 129.8, 129.2, 127.4, 55.8, 36.1, 18.1.
Acknowledgements
This research was supported by the Mid-Term Technological
Development Project funded by the Korean Ministry of Com-
merce, Industry and Energy (grant no. 10027898-2007-22).
References
[1] H. Sorimachi, S. Ishiura, K. Suzuki, Biochem. J. 328 (1997) 721e732.
[2] T.J. Stalker, C.B. Skvarka, R. Scalia, FASEB J. 17 (2003) 1511e1513.
[3] 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.
[4] B. Boland, V. Campbell, Neurobiol. Aging 24 (2003) 179e186.
[5] S. Mehdi, Trends Biochem. Sci. 16 (1991) 150e153.
[6] Y. Shirasaki, M. Yamaguchi, H. Miyashita, J. Ocular Pharmacol. Ther. 22
(2007) 417e424.
[7] J.A. Fehrentz, B. Castro, Synthesis (1983) 676e678.
[23] Calpain inhibitory activity was assayed using a slight modification of a re-
ported procedure [24]. Briefly, calpain-I (Calbiochem) from human
erythrocytes and the fluorogenic calpain substrate (Suc-Leu-Tyr-7-
amino-4-methylcoumarin, Calbiochem) were used as an enzyme and
substrate, respectively. In a typical experiment, 1 ml of a compound solu-
tion at various concentrations, 25 ml of 1 mM calpain substrate, 10 ml of
1 M NaCl and 11 ml of 1 mM CaCl2 were added to each well of 96-well
plates. All chemicals except for the compounds concerned were dis-
solved in 50 mM TriseHCl (pH 7.5) buffer containing 1 mM dithiothrei-
tol (DTT) and used immediately. The compounds were dissolved in
DMSO, but the DMSO concentrations in the final experimental solutions
did not exceed 1%. Incubations were initiated by adding 53 ml of 34 nM
calpain and the mixture was incubated for 30 min at room temperature.
After incubation, the fluorescence of the cleavage product, 7-amino-4-
[8] R. Tripathy, Z.-Q. Gu, D. Dunn, S.E. Senadhi, M.A. Ato, S. Chatterjee,
Bioorg. Med. Chem. Lett. 8 (1998) 2647e2652.
[9] W. Lubisch, A. Moller, Bioorg. Med. Chem. Lett. 12 (2002) 1335e1338.
¨
[10] M. Nakamura, J. Inoue, Bioorg. Med. Chem. Lett. 12 (2002) 1603e1606.
[11] 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.
[12] 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.
[13] M.H. Norman, J. Zhu, C. Fotsch, Y. Bo, N. Chen, P. Chakrabarti,
E.M. Doherty, N.R. Gavva, N. Nishimura, T. Nixey, V.I. Ognyanov,
R.M. Rzasa, M. Stec, S. Surapaneni, R. Tamir, V.N. Viswanadhan,
J.J.S. Treanor, J. Med. Chem. 50 (2007) 3515e3527.
[14] B. Le Bourdonnec, A.J. Goodman, M. Michaut, H.-F. Ye, T.M. Graczyk,
S. Belanger, T. Herbertz, G.P.A. Yap, R.N. DeHaven, R.E. Dolle, J. Med.
Chem. 49 (2006) 7278e7289.
methylcoumarin, was measured using
a
spectrofluorimeter at
lex ¼ 380 nm and lem ¼ 460 nm against a blank sample not containing
calpain. The IC50 values were calculated using percent inhibitions of
enzyme activity.
[15] X. Yang, S. Luo, F. Fang, P. Liu, Y. Lu, M. He, H. Zhai, Tetrahedron 62
(2006) 2240e2246.
[24] T. Sasaki, T. Kikuchi, N. Yumoto, N. Yoshimura, T. Murachi, J. Biol.
Chem. 259 (1984) 12489e12494.
[16] I.O. Donkor, X. Zheng, D.D. Miller, Bioorg. Med. Chem. Lett. 10 (2000)
2497e2500.