1752 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Białas et al.
Crystallization from an ethyl acetate/hexane yielded a pure chlo-
rmethyl ketone as a crystalline white solid.
and emission at 445 nm were used to maximize the free AMC (7-
amino-4-methylcoumarin) fluorescence signal. First-order inactiva-
tion rate constants (kobs) were obtained from the plots showing the
dependence of -ln(V/V0) versus time. The second-order rate constant
(kass) was calculated from the equation kass ) kobs/[I]. The measure-
ments were repeated at least twice for every compound tested. Mean
values of kass, which differed by less than 20% for potent inhibitors
(kobs/[I] > 104 M-1 s-1) and less than 30% for weaker ligands,
were calculated for every compound.
General Procedures for Preparation of the Protected Chlo-
romethyl Ketones: R2-Arg(Z)2-CH2Cl (12), R2-Lys(Z)-CH2Cl
(13), and R3-Phe-Lys(Z)-CH2Cl (14). Method A: Acylation with
an Appropriate Acid Chloride. H-Arg(Z)2-CH2Cl (10), H-Lys-
(Z)-CH2Cl (11), or H-Phe-Lys(Z)-CH2Cl hydrochloridates were
obtained from Boc-Arg(Z)2-CH2Cl (8), Boc-Lys(Z)-CH2Cl (9), or
Boc-Phe-Lys(Z)-CH2Cl (13e) (2 mmol), respectively, dissolved in
a minimum quantity of THF and treated with an excess of 7.5 M
HCl in methanol (13.4 mL, 10 mmol) within 40 min for arginine
and 15 min for lysine derivative at 0 °C. After evaporation, the
obtained salt was suspended in anhydrous THF (30 mL). N-
Methylmorpholine (0.55 mL, 5 mmol) and an acid chloridate (2.4
mmol, 5 min later) were added consecutively to the stirred mixture
and cooled in a water/ice bath. Acid chloridates were either
commercial or synthesized from the appropriate acid upon reaction
with thionyl chloride in the standard way. After 24 h of stirring,
the mixture was concentrated under reduced pressure. The residue
was taken up with ethyl acetate (50 mL) and water (10 mL) and
washed consecutively with 5% NaHCO3 (40 mL), 5% citric acid
(2 × 40 mL), and brine (40 mL). The organic layer was then dried
over anhydrous Na2SO4. After removal of the solvent, the residue
was purified on silica gel column. A solution of ethyl acetate/hexane
was used as the eluent. The obtained material was additionally
recrystallized from an ethyl acetate/hexane mixture, giving pure
chloromethyl ketones as white crystals.
Acknowledgment. This work is supported by the Committee
of Scientific Research (KBN, Poland, grant 6 P04B 025 21).
J.P. acknowledges grants DE 09761 and 3 P04A 003 24 from
the National Institutes of Health and the Committee of Scientific
Research (KBN, Poland), respectively. J.P. was the recipient
of an award SUBSYDIUM PROFESORSKIE from the Founda-
tion for Polish Science (FNP, Warszawa, Poland). We thank
Rachael Riley and David Shultis (University of Virginia,
Charlottesville) for critical reading of the manuscript.
Supporting Information Available: NMR, IR, and elemental
analysis data and HPLC chromatograms of the protected and final
products. This material is available free of charge via the Internet
References
Method B: Mixed Anhydrides. To a stirred solution of an acid
(2 mmol) in anhydrous THF (50 mL) were added NMM (0.55 mL,
5 mmol) and isobutyl chloroformate (0.31 mL, 2.4 mmol) at 0 °C.
Stirring was continued for 45 min. Then H-Arg(Z)2-CH2Cl (10),
H-Lys(Z)-CH2Cl (11), or H-Phe-Lys(Z)-CH2Cl hydrochloridate (2
mmol, obtained as described above for method A) suspended in
anhydrous THF (30 mL) was added. After 24 h of stirring, the
mixture was worked up and purified the same manner as described
above.
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measured under pseudo-first-order conditions: the concentration
of an inhibitor exceeded the protease concentration by at least 10
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s, and diluted at least 15 times with the reaction buffer containing
substrate. The final concentration of Tos-Gly-Pro-Arg-AMC and
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