N. Fuchi et al. / Tetrahedron Letters 42 (2001) 1305–1308
1307
Hydrazide 4 R2-CHO 5 Cycloadduct 11
R2
R2
R1
=
R2
=
Yield (%)
R1
R1
BocHN
4a (H)
4a
4a
4a
4b (4-F)
4c (4-CF3)
4d (4-NO2)
4e (4-Me)
56
71
30
48
52
46
35
46
43
H
N
N
N
N
DBU
SO2p-Tolyl
iPr
Ph
4-OH-Ph
H
H
H
H
H
CH2Cl2
BocHN
R1
CO2Et
R2
CO2Et
O
O
11
10
4f (3-I)
R2
R1
ref 4
H2, Pd/C
N
N
N
N
O
MeOH
quant
BocHN
H2N
NH2
NH
O
CO2Et
O
O
O
12 (β-CO2Et)
14
H2N
NH
4
13 (α-CO2Et)
O
Scheme 2.
Table 1. Inhibition and selectivity of coagulation versus anticoagulation enzymes
Compd.
R1
R2
Thrombin Ki (nM) Trypsin Ki (nM) Factor VIIa Ki (nM)
Tryptase Ki (nM) Selectivity Kithr/Kitrp
14a
14b
14c
14d
4-F
H
H
H
i-Pr
41.2
17.7
6.5
26.2
11.3
7.8
9277
8277
2308
1096
164.4
62.6
15.8
15.4
1.57
1.57
0.83
4-CF3
4-Me
H
61.6
4.3
14.3
halogen group (4-F, 4-CF3) as the R1 substituent, the
tryptase inhibition activity decreased. These results
demonstrate the versatility of this template to inhibit a
wide range of serine proteases and importantly, that
specificity can be introduced using combinatorial intro-
duction of substituents. In summary, we have demon-
strated a practical synthetic route to a constrained
b-strand mimetic template via the regioselective 1,3-
dipolar cycloaddition of azomethine imines with vinyl
sulfone. The solid-phase version of this approach is
now underway in our laboratory and will be reported in
due course.
8. Caramella, P.; Albini, E.; Bandiera, T.; Corsico Coda, A.;
Grunanger, P.; Albini, F. M. Tetrahedron 1983, 39, 689–
699.
9. Shimizu, T.; Hayashi, Y.; Miki, M.; Teramura, K. J. Org.
Chem. 1985, 50, 904–907.
10. Stork, G.; Leong, A. Y. W.; Touzin, A. M. J. Org. Chem.
1976, 41, 3491–3493.
11. Najera, C.; Baldo, B.; Yus, M. J. Chem. Soc., Perkin
Trans. 1 1988, 1029–1033.
12. Compound 11a (R1=H, R2=H) 1H NMR (270 MHz,
CDCl3) l (ppm): 7.18–7.26 (m, 5H), 5.81 (t, 1H, J=2.6
Hz), 5.58 (brs., 1H), 4.21–4.38 (m, 3H), 3.92 (dd, 1H,
J=2.6, 14.9 Hz), 3.49 (d, 1H, J=13.5 Hz), 3.37 (d, 1H,
J=13.5 Hz), 2.95–3.05 (m, 2H), 2.54–2.68 (m, 1H), 2.40–
2.50 (m, 1H), 1.44 (s, 9H), 1.33 (t, 3H, J=7.3 Hz); 13C
NMR (67.8 MHz, CDCl3) l (ppm): 165.6, 160.6, 154.5,
135.9, 132.7, 130.5, 129.1, 127.0, 114.5, 79.4, 62.0, 60.9,
58.6, 53.5, 43.2, 34.2, 28.4, 14.1.
References
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13. All protease assays were performed at room temperature
in 96-well microplates using Bio-Rad microplate reader
(Model 3550), Molecular Devices SpectroMax 250, Lab-
systems Fluoroskan Ascent, or Wallac Victor2 fluores-
cence plate reader. Either 1 mM solutions of testing
compounds in water or 10 mM solutions of testing
compounds in DMSO served as the stock solutions for
the inhibition assays. For thrombin and trypsin assays,
the hydrolysis of chromogenic substrate, N-p-tosyl-Gly-
Pro-Arg-pNA (Sigma) or fluorogenic substrate N-p-tosyl-
Gly-Pro-Arg-AMC (Sigma) was monitored at 405 nm or
at Ex: 355 nm, Em: 460 nm, respectively. For tryptase
assay, the release of pNA from chromogenic substrate
S-2366, pyroGlu-Pro-Arg-pNA (Chromogenix) was mon-
itored at 405 nm. In Factor VIIa assays, the hydrolysis of
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fluorogenic substrate (D)Phe-Pro-Arg-AMC was moni-