2360
K. D. Rice et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2357±2360
432.7 mmol) in DCM (100 mL). The mixture was warmed to
Acknowledgements
room temperature over 30 min followed by addition of aq HCl
(0.1 N, 200 mL) then drying the organic layer over anhydrous
magnesium sulfate. Filtration and concentration gave tert-
butyl 1-piperazinecarboxylate-4-carbonyl chloride (45.6 g,
85%) as a pale-yellow solid. 1H NMR (300 MHz, CDCl3) d
3.70 (m, 2H), 3.60 (m, 2H), 3.50 (m, 4H), 1.50 (s, 9H).
The authors wish to thank Mark Dreyer and Liling
Fang for analytical support as well as Heinz Gschwend
and Mike Venuti. We would also like to acknowledge
Bayer AG for their ®nancial support of tryptase
research at Axys Pharmaceuticals, Inc.
19. cis-1,5-Cyclooctanediol (20.2 g, 0.14 mol) and potassium
carbonate (41.4 g, 0.3 mol) were taken into acetonitrile
(250 mL) then cooled to 0 ꢀC followed by dropwise addition
of phosgene (1.9 M in toluene, 220 mL, 0.42 mol) over 1 h
followed by warming to rt and stirring over 12 h. Ether (1 L)
was added and the suspension ®ltered. Concentration followed
by recrystallization from hexane gave cis-1,5-cycloocetylene
bis-chloroformate as a colorless crystalline solid. 1H NMR
(300 MHz, CDCl3) d 5.00±4.85 (m, 2H), 2.20±1.60 (m, 12H).
20. Stock solutions of enzyme (0.06 mg/mL) were prepared
with 10 mM 2-(N-morpholino)ethane sulfonic acid (MES)
containing 2 mM calcium chloride, 20% glycerol and 0.05 mg/
mL heparin. Approximately 1 mg of inhibitor was dissolved in
DMSO (0.2 mL) and diluted 10-fold into buer containing
50 mM Tris hydrochloride (pH 8.2), 100 mM sodium chloride,
and 0.05% Tween-20. Seven additional threefold dilutions
were made from the initial dilution into the same buer sup-
plemented with 10% DMSO. Aliquots (0.05 mL) from each of
the eight dilutions were transferred to individual wells in a 96-
well U-bottom microtiter plate. Enzyme stock solution (25 mL)
was added to each well and incubated for 1 h at rt. The enzy-
matic reaction was initiated with addition of tosyl-Gly-Pro-
Lys-p-nitroanilide (25 mL, 0.5 mM ®nal concentration) and the
reaction was followed spectrophotometrically at 405 nm.
Initial velocity measurements calculated from the progress
curves by a kinetic analysis program (BatchKi; Peter Kuzmic,
University of Wisconsin, Madison, WI) were used to deter-
mine apparent inhibition constants. The standard error for
single determinations is a factor of 2.
References and Notes
1. Welle, M. J. Leukoc. Biol. 1997, 61, 233.
2. Yong, L. C. J. J. Exp. Toxic Pathol. 1997, 49, 409.
3. Zhang, M. Q.; Timmerman, H. Mediators of In¯ammation
1997, 6, 311.
4. Rossi, G. L.; Olivieri, D. Chest 1997, 112, 523.
5. Kozik, A.; Moore, R. B.; Potempa, J.; Imamura, T.;
Rapala-Kozik, M.; Travis, J. J. Biol. Chem. 1998, 273, 33224.
6. Dery, O.; Bunnett, N. W. Biochem. Soc. Trans. 1999, 27,
246.
7. Mirza, H.; Schmidt, V. A.; Derian, C. K.; Jesty, J.; Bahou,
W. F. Blood 1997, 90, 3914.
8. Steinho, M.; Vergnolle, N.; Young, S. H.; Tognetto, M.;
Amadesi, S.; Ennes, H. S.; Trevisani, M.; Hollenberg, M. D.;
Wallace, J. L.; Caughey, G. H.; Mitchell, S. E.; Williams, L.
M.; Geppetti, P.; Mayer, E. A.; Bunnett, N. W. Nature Medi-
cine 2000, 6, 151.
9. He, S.; Gaca, M. D. A.; Walls, A. F. J. Pharmacol. Exp.
Ther. 1998, 286, 289.
10. Krishna, M. T.; Chauhan, A. J.; Little, L.; Sampson, K.;
Mant, T. G. K.; Hawksworth, R.; Djukanovic, R.; Lee, T. H.;
Holgate, S. T. Am. J. Respir. Crit. Care Med. 1998, 157, A456.
11. Sturzebecher, J.; Prasa, D.; Sommerho, C. P. Biol. Chem.
Hoppe-Seyler 1992, 373, 1025.
12. Caughy, G. H.; Raymond, W. W.; Bacci, E.; Lombardy,
R. J.; Tidwell, R. R. J. Pharmacol. Exp. Ther. 1993, 264, 676.
13. Ono, S.; Kuwahara, S.; Takeuchi, M.; Sakashita, H.;
Naito, Y.; Kondo, T. Bioorg. Med. Chem. Lett. 1999, 9, 3285.
14. Burgess, L. E.; Newhouse, B. J.; Ibrahim, P.; Rizzi, J.;
Kashem, M. A.; Hartman, A.; Brandhuber, B. J.; Wright, C.
D.; Thomson, D. S.; Vigers, G. P. A.; Koch, K. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 8348.
15. Richter, R.; Tucker, B. J. Org. Chem. 1983, 48, 2625.
16. Sa®er, S. R.; Kushner, S.; Brancone, L. M.; Subbarrow,
Y. J. Org. Chem. 1948, 13, 924.
17. Krapcho, P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559.
18. To triphosgene (25 g, 84.2 mmol) in DCM (200 mL) at
0 ꢀC was slowly added a solution of tert-butyl 1-piper-
azinecarboxylate (40 g, 214.8 mmol) and pyridine (35 mL,
21. Ki value for the tight binding inhibitor 27 was determined
by the following procedure. Tryptase (0.25, 0.50, 1.0, and
2.0 nM) and inhibitor (40, 110, 340, and 680 pM) were varied
in preincubation mixtures for 1 h at rt followed by addition of
substrate as before. The IC50 of the compound was then
determined at each concentration of tryptase. The y-intercept
of a replot of IC50 (ordinate) versus tryptase concentration
(abscissa) produces a value for the apparent dissociation con-
stant from which the true dissociation constant of the tight
binding competitive inhibitor was calculated.
22. Sommerho, C. P.; Bode, W.; Matschiner, G.; Bergner,
A.; Fritz, H. Biochim. Biophys. Acta 2000, 1477, 75.
23. Sommerho, C. P.; Bode, W.; Pereira, P. J. B.; Stubbs, M.
T.; Sturzebecher, J.; Piechottka, G.; Matschiner, G.; Bergner,
A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10984.