Tri-Ring P3 Benzamide-Containing Aminonitriles
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 24 7533
39n. Synthesized via general procedure E. 1H NMR
(DMSO-d6) (270 MHz): δ 8.19 (1H, t, J ) 5.4 Hz), 7.97 (1H,
s), 7.92 (2H, d, J ) 8.8 Hz), 7.88 (2H, d, J ) 8.8 Hz), 7.42 (1H,
s), 4.04 (2H, d, J ) 5.7 Hz), 3.46 (6H, m), 3.30 (3H, s), 2.55
(4H, m), 2.10 (2H, m), 1.74 (2H, m), 1.52 (5H, m), 1.25 (3H,
m). LC/MS: M + 1: 511.6.
Acknowledgment. The authors thank Joanne Fa-
nucchi for help with preparation of this manuscript and
Drs. Jochen Knolle and Ann Welton for helpful discus-
sions.
Supporting Information Available: Elemental analysis
data for compounds 1-3, 5, 11-14, 18b,c,e-i,n,o, 19j, and
39n. This material is available free of charge via the Internet
39p. Synthesized via general procedure E: 1H NMR
(DMSO-d6) (270 MHz): δ 8.20 (1H, t), 8.00 (1H, s), 7.90 (4H,
s), 7.39 (1H, s), 4.01 (4H, d, m), 3.02 (3H, t), 2.09 (2H, m), 1.74
(5H, m), 1.49 (17H, m). MS: M + H+: 535.4.
General Procedure F, Illustrated for 39q. A 1:1 mixture
of tert-butylpiperazine13a (23, R ) t-Bu) and 2,4-dibromothia-
zole (35) in DMF (in the presence of 2 equiv of triethylamine)
was heated for 3 h, cooled, and purified by flash chromatog-
raphy (10% MeOH/CH2Cl2) to give 36 (R ) t-Bu) in 50% yield.
This material was coupled with 37 using the palladium-
assisted cross-coupling procedure as described for the biphe-
nylamide derivatives 19a-m, above.
Enzyme Assays. All enzymes used in these studies, with
the exception of human cathepsin B, were produced by Celera
Genomics. Cathepsin B was from human liver and was
purchased from Athens Research and Technology (Athens,
GA). The substrates used in these studies were purchased from
the following vendors: Z-Phe-Arg-AMC, Boc-Leu-Lys-Arg-
AMC, and Z-Val-Val-Arg-AMC were from Bachem (Torrance,
CA) and Z-Leu-Arg-AMC was from Calbiochem-Novabiochem
(San Diego, CA). Bovine serum albumin was purchased from
the Sigma Chemical Company (St. Louis, MO). Routine buffer
components and all other chemicals used in these experiments
were of the highest available quality.
Methods. Enzyme inhibition studies were performed under
several sets of conditions, and each set was tailored to provide
the optimal activity of the given cathepsin being assayed. Each
cathepsin was incubated with the inhibitor, present at variable
concentrations, under the conditions specified below. Enzyme
and inhibitor were incubated together for 30 min at room
temperature (21 to 24 °C) in 96-well U-bottom, microtiter
plates (Falcon, from Becton Dickinson, Franklin Lakes, NJ).
After the preincubation phase, reactions were initiated with
the addition of the 7-amino-4-methylcoumarin (AMC) sub-
strate specified below. The hydrolysis of these substrates yields
7-amino-4-methylcoumarin which was monitored fluorometri-
cally (excitation at 355 nm and emission at 460 nm) using an
FMAX 96-well plate reader from Molecular Devices (Sunny-
vale, CA) interfaced with a Macintosh computer. Under these
experimental conditions, 1 µM of product produces a fluores-
cence signal of approximately 125 units. The velocity of the
enzyme-catalyzed reaction was obtained from the linear por-
tion of the progress curves (usually the first 5 min after
addition of substrate).
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Specific assay conditions for each cathepsin tested are
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Human Cathepsin K. The buffer used to assay this
enzyme consisted of 50 mM MES (pH 5.5), 2.5 mM D,L-
dithiothreitol (DTT), 2.5 mM ethylenediaminetetraacetic acid
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Human Cathepsin B. The buffer used to assay this enzyme
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Human Cathepsin L. The buffer used to assay this enzyme
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EDTA, and 10% DMSO. Recombinant human cathepsin L was
supplied at 500 pM. Substrate, Z-Phe-Arg-AMC, was supplied
at 10 µM.
Human Cathepsin S. The buffer used to assay this enzyme
consisted of 50 mM MES (pH 6.5), 2.5 mM â-mercaptoethanol
(BME), 2.5 mM EDTA, 100 mM NaCl, 0.001% bovine serum
albumin (BSA), and 10% DMSO. Recombinant human cathe-
psin S was supplied at 500 pM. Substrate, Z-Val-Val-Arg-AMC,
was supplied at 60 µM.
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