Protease Inhibitors
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XNHC6H4SO2), 149.1 (s, NHCONH), 175.1 (s, SO2NdC). Anal.
(C25H27N5O7S2, 573.65) C, H, N, S.
p Ka Deter m in a tion . The half-neutralization point was
measured by titrating the organic acids/bases with 0.05 N
NaOH and 0.05 N HCl in EtOH-water (30%, v/v), using a
glass electrode, as described by Bell and Roblin56 for the
structurally related antibacterial sulfonamides.
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W. C. Design, synthesis, and evolution of a novel selective, and
orally bioavailable class of thrombin inhibitors: P1-argininal
derivatives incorporating P3-P4 lactam sulfonamide moieties.
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J . Enyme Inhib. 1998, 13, 267-284.
En zym e Assa ys: KI Deter m in a tion s. Human thrombin
and human trypsin were purchased from Sigma Chemical Co.
(St. Louis, MO); their concentrations were determined from
the absorbance at 280 nm and the extinction coefficients
furnished by the supplier. The activity of such preparations
was in the range of 2500-3000 NIH units/mg. The potency of
standard and newly obtained inhibitors was determined from
the inhibition of the enzymatic (amidolytic) activity of these
serine proteases, at 21 °C, using Ts-Gly-Pro-Arg-pNA (Chro-
mozym TH) from Sigma as substrate, by the method of
Lottenberg et al.23 The substrate was reconstituted as 4 mM
stock in ultrapure water and brought to pH 4 with hydrochloric
acid. Substrate concentrations were determined from absor-
bance at the isosbestic wavelength for the peptide-p-nitro-
anilide-p-nitroaniline mixtures. Extinction coefficients of 8270
L‚mol-1‚cm-1 in the used buffer (0.01 M Hepes-0.01 M Tris-
0.1 M NaCl-0.1% poly(ethylene glycol) 6000) were employed.
The rate of p-nitroanilide hydrolysis was determined from the
change in absorbance at 405 nm using an extinction coefficient
for p-nitroaniline of 9920 L‚mol-1‚cm-1 for the above-men-
tioned reaction buffer. Measurements were made using a Cart
3 spectrophotometer interfaced with a PC. Initial velocities
were thus estimated using the direct linear plot-based proce-
dure as reported by Lottenberg et al.23 KI’s were then deter-
mined according to Dixon, using a linear regression program.57
The KI values determined are the means of at least three
determinations.
Ack n ow led gm en t. This research was financed in
part by the EU Grant ERB CIPDCT 940051.
Su p p or tin g In for m a tion Ava ila ble: Tables S1-S6 giv-
ing the calculated descriptors for the 99 drugs considered in
the QSAR section. This material is available free of charge
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