Diphenyl Phosphonate Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5791
100%; 254 nm, tr 19.56 min, 100%. LC/MS: tr 13.3 min, 93.5%.
MS (ESI): m/z 723 [M + Na].
Yield: 92%. 1H NMR (CDCl3): δ 1.26 (dd, J ) 2.4 and J ) 7.2,
1H), 1.28 (dd, J ) 2.4 and J ) 7.2), 2.4 (s, 3H), 3.2 (m, 1H), 3.4
(m, 1H), 3.8 (m, 2H), 4.25 (m, 1H), 4.4 (m, 1H), 5.1 (m, 1H),
7.0-7.9 (m, 18H). HPLC: 214 nm, tr 18.56 min, 100%. HPLC:
254 nm, tr 18.56 min, 46.32%, tr 19.00 min, 46.32%. LC/MS: tr
13.5 min, 100%. MS (ESI): m/z 687 (M+1).
Diphenyl 1-[(N-o-Methylbenzoyl-D-seryl)-L-alanyl]amino-2-
(4-guanidinophenyl)ethanephosphonate Trifluoroacetate (3n).
Yield: 91%. 1H NMR (CDCl3): δ 1.28 (dd, J ) 4.4 and J ) 7.2,
1H), 1.30 (dd, J ) 4.4 and J ) 7.2), 2.4 (m, 3H), 3.25 (m, 1H),
3.45 (m, 1H), 3.85 (dd, J ) 5.2 and J ) 21.2, 1H), 3.92 (dd, J )
5.2 and J ) 21.2, 1H), 4.3 (m, 1H), 4.45 (m, 0.5H), 4.52 (m, 0.5H),
5.1 (m, 1H), 7.0-7.9 (m, 18H). HPLC: 214 nm, tr 18.46 min,
43.41%; tr 18.82 min, 43.81%; 254 nm, tr 18.80 min, 40.74%; tr
19.04 min, 39.77%. LC/MS: tr 13.6 min, 100%. MS (ESI): m/z
687 (M+1).
Diphenyl 1-[(N-o,o-Dimethylbenzoyl-D-seryl)-L-alanyl]amino-
2-(4-guanidinophenyl)ethanephosphonate Trifluoroacetate (3o).
Yield: 94%. 1H NMR (CDCl3): δ 1.28 (dd, J ) 7.2 and J ) 12.4,
1H), 1.30 (dd, J ) 7.2 and J ) 12.4), 2.28 (s, 3H), 2.31 (s, 3H),
3.25 (m, 1H), 3.45 (m, 1H), 3.82 (dd, J ) 6.4 and J ) 21.2, 1H),
3.88 (dd, J ) 6.4 and J ) 21.2, 1H), 4.3 (m, 1H), 4.55 (m, 0.5H),
4.58 (m, 0.5H), 5.1 (m, 1H), 7.0-7.9 (m, 17H). HPLC: 214 nm, tr
18.97 min, 39.36%; tr 19.13 min, 39.54%; 254 nm, tr 19.00 min,
39.78%; tr 19.20 min, 38.64%. LC/MS: tr 13.7 min, 100%. MS
(ESI): m/z 701 (M+1).
Di-(4-acetamidophenyl) 1-[(N-Benzyloxycarbonyl-D-seryl)-L-
alanyl]amino-2-[4-(guanidino)phenyl]- ethanephosphonate Tri-
fluoroacetate (4). Yield: 93%. 1H NMR (CDCl3): δ 1.3 (m, 3H),
2.0 (s, 6H), 3.0-3.4 (m, 2H), 3.7 (m, 2H), 4.1-4.4 (m, 2H), 5.0
(m, 1H), 5.1 (m, 2H), 7.1-7.6 (m, 17H). HPLC: 214 nm, tr 14.3
min, 42.1%; tr 14.0 min, 44.3%. HPLC: 254 nm, tr 13.8 min,
45.1%; tr 14.0 min, 49.0%. LC/MS: tr 12.0 min. Purity: 92.0%.
MS (ESI): m/z 817 (M+1).
uPA Inhibition: in Vitro Evaluation. Enzymatic activity was
measured at 37 °C in a Spectramax 340 (Molecular Devices)
microtiter plate reader using the chromogenic substrate S-2444 (L-
pyroGlu-Gly-L-Arg-p-NA‚HCl), with a Km of 80 µM. The substrate
was obtained from Chromogenix. The human enzyme was obtained
from Sigma-Aldrich and the mouse uPA was obtained from
Molecular Innovations, Inc. (U.S.A.). The reaction was monitored
at 405 nm, and the initial rate was determined between 0 and 0.25
absorbance units in 20 min. The reaction mixture contained 250
µM substrate and approximately 1 mU of enzyme in 145 µL of
buffer in a final volume of 200 µL. A 50 mM Tris buffer, pH 8.8,
was used. From each inhibitor concentration, 5 µL was added,
obtaining a final concentration from 0 to 250 µM in a total volume
of 0.2 mL. Activity measurements were routinely performed in
duplicate. The IC50 value is defined as the concentration of inhibitor
required to reduce the enzyme activity to 50% after a 15-min
preincubation with the enzyme at 37 °C before addition of the
substrate. IC50 values were obtained by fitting the data with the
four-parameter logistics equation using Grafit 5.
Diphenyl 1-[(N-Benzyl-D-seryl)-L-alanyl]amino-2-(4-guanidi-
nophenyl)ethanephosphonate Trifluoroacetate (3d). Yield: 46%.
1H NMR (CD3OD): δ 1.3 (d, 3H), 3.3 (d, 1H), 3.5 (m, 1H), 4 (m,
2H), 4.2 (2m, 1H), 4.5 (dm, 1H), 7.0-7.6 (m, 19H). HPLC: 214
nm, tr 18.12 min, 46.90%; tr 17.72 min, 43.45%; 254 nm, tr 17.99
min, 48.69%; tr 17.59 min, 42.39%. LC/MS: tr 12.7 min, 99.9%.
MS (ESI): m/z 695 [M + Na].
Diphenyl 1-[(N-Naftalenesulfonyl-D-seryl)-L-alanyl]amino-2-
(4-guanidinophenyl)ethanephosphonate Trifluoroacetate (3e).
1
Yield: 55%. H NMR (CD3OD): δ 1.0 (d, 3H), 3.2 (m, 1H), 3.5
(m, 2H), 3.6-3.8 (dm, 2H), 4.1 (dm, 1H), 5.0 (m, 1H), 7.0-7.7
(m, 18 H), 8.0 (m, 1H), 8.1 (m, 1H), 8.3 (d, 1H), 8.7 (m, 1H).
HPLC: 214 nm, tr 20.20 min, 100%; 254 nm, tr 20.13 min, 100%.
LC/MS: tr 14.4 min, 97.7%. MS (ESI): m/z 781 [M + Na].
Diphenyl 1-[(N-2-Thiophenesulfonyl-D-seryl)-L-alanyl]amino-
2-(4-guanidinophenyl)ethanephosphonate Trifluoroacetate (3f).
1
Yield: 35%. H NMR (CDCl3): δ 1.3 (m, 3H), 3.1 (m, 1H), 3.4
(m, 1H), 3.6 (m, 2H), 3.9 (m, 1H), 4.25 (m, 1H), 4.4 (m, 1H),
7.1-7.4 (m, 15H), 7.6 (m, 1H), 7.7 (m, 1H). HPLC: 214 nm, tr
18.62 min, 100%. HPLC: 254 nm, tr 18.23 min, 100%. LC/MS: tr
12.9 min, 94.9%. MS (ESI): m/z 715 (M+1).
Diphenyl 1-[(N-2-Thienyl-D-seryl)-L-alanyl]amino-2-(4-guani-
dinophenyl)ethanephosphonate Trifluoroacetate (3g). Yield:
55%. 1H NMR (CDCl3): δ 1.3 (m, 3H), 3.4 (m, 2H), 3.9 (m, 2H),
4.2-4.3 (m, 2H), 5.1 (m, 1H), 7.1-7.8 (m, 17H). HPLC: 214 nm,
tr 17.02 min, 42.13%; 17.50 min, 57.87%. HPLC: 254 nm, tr 16.89
min, 41.55%; 17.45 min, 58.45%. LC/MS: tr 12.9 min, 94.9%. MS
(ESI): m/z 679 (M+1).
Diphenyl 1-[(N-1-Adamantanyl-D-seryl)-L-alanyl])amino-2-(4-
guanidinophenyl)ethanephosphonate Trifluoroacetate (3h).
1
Yield: 67%. H NMR (CDCl3): δ 1.28 (dd, J ) 8.4 and J ) 16,
1H), 1.30 (dd, J ) 8.4 and J ) 16), 1.7 (m, 6H), 1.9 (m, 5H), 2.0
(m, 4H), 3.25 (m, 1H), 3.45 (m, 1H), 3.75 (m, 2H), 4.1 (m, 1H),
4.3 (m, 1H), 5.1 (m, 1H), 7.0-7.9 (m, 14H). HPLC: 214 nm, tr
19.96 min, 77.42%; 254 nm, tr 20.89 min, 35.77%; 21.22 min,
38.96%. LC/MS: tr 13.9 min, 98.8%. MS (ESI): m/z 731 (M+1).
Diphenyl 1-[(N-p-Methoxybenzenesulfonyl-D-seryl)-L-alanyl]-
amino-2-(4-guanidinophenyl)ethane-phosphonate Trifluoroac-
1
etate (3i). Yield: 23%. H NMR (CD3OD): δ 1.2 (m, 3H), 3.1
(m, 1H), 3.4 (m, 3H), 3.5-3.6 (m, 2H), 3.8 (m, 2H), 4.0 (m, 1H),
4.2 (m, 1H), 5.1 (m, 1H), 7.0-8.0 (m, 18H). HPLC: 214 nm, tr
19.62 min, 100%; 254 nm, tr 19.23 min, 100%. LC/MS: tr 13.8
min, 94.9%. MS (ESI): m/z 761 [M + Na].
Diphenyl 1-[(N-p-Bromobenzenesulfonyl-D-seryl)-L-alanyl]-
amino-2-(4-guanidinophenyl)ethane-phosphonate Trifluoroac-
etate (3j). Yield: 55%. 1H NMR (CDCl3): δ 1.2 (m, 3H), 3.1 (m,
1H), 3.2 (s, 2H), 3.5 (m, 1H), 3.6-3.7 (dm, 2H), 4.2 (m, 2H), 7.0-
8.0 (m, 18H). HPLC: 214 nm, tr 21.09 min, 94.07%; 254 nm, tr
20.10 min, 94.61%. LC/MS: tr 14.1 min, 96.4%. MS (ESI): m/z
809 [M + Na].
Diphenyl 1-[(N-p-Cyanobenzenesulfonyl-D-seryl)-L-alanyl]-
amino-2-(4-guanidinophenyl)ethane-phosphonate Trifluoroac-
1
etate (3k). Yield: 43%. H NMR (CD3OD): δ 1.3 (d, 3H), 3.2
V ) (V range)/(1 + exp(s × ln(abs(I0/IC50)))) + background
(m, 1H), 3.5 (m, 2H), 3.6-3.9 (dm, 2H), 4.2 (m, 1H), 5.1 (m, 1H),
7.1-7.4 (dm, 14H), 7.8-8.0 (dm, 4H). HPLC: 214 nm, tr 18.86
min, 92.64%; 254 nm, tr 18.79 min, 95.59%. LC/MS: tr 13.1 min,
100%. MS (ESI): m/z 757 [M + Na].
where s ) slope factor, V ) rate, I0 ) inhibitor concentration, and
range ) the fitted uninhibited value minus the background. The
equation assumes the y falls with increasing x.
Diphenyl 1-[(N-p-Methylbenzoyl-D-seryl)-L-alanyl]amino-2-
(4-guanidinophenyl)ethanephosphonate Trifluoroacetate (3l).
Yield: 93%. 1H NMR (CDCl3): δ 1.28 (dd, J ) 2.8 and J ) 7.2,
1H), 1.30 (dd, J ) 2.4 and J ) 7.2), 2.38 (d, 3H), 3.25 (m, 1H),
3.45 (m, 1H), 3.85 (dd, J ) 5.6 and J ) 14.8, 1H), 3.92 (dd, J )
5.6 and J ) 14.8, 1H), 4.2 (m, 0.5H), 4.35 (m, 0.5H), 4.45 (m,
0.5H), 4.53 (m, 0.5H), 5.1 (m, 1H), 7.0-7.9 (m, 18H). HPLC: 214
nm, tr 18.46 min, 47.58%; tr 18.82 min, 44.80%. HPLC: 254 nm,
tr 18.59 min, 47.64%; tr 19.04 min, 43.96%. LC/MS: tr 13.4 min,
100%. MS (ESI): m/z 687 (M+1).
Inhibitor stock solutions were prepared in DMSO and stored at
-20 °C. Because the compounds described in this paper completely
inactivate uPA following pseudo-first-order kinetics, the IC50 value
is inversely correlated with the second-order rate constant of
inactivation. For a simple pseudo-first-order inactivation process,
the activity after incubation with inhibitor (Vi) varies with the
inhibitor concentration (i), as described in the following equation:
Vi ) V0‚e-kit, where V0 is the activity in absence of inhibitor, k is
the second-order rate constant of inactivation, and t is the time.
The inactivation rate constant was determined from the time
course of inhibition.
Diphenyl 1-[(N-o-Methylbenzoyl-D-seryl)-L-alanyl]amino-2-
(4-guanidinophenyl)ethanephosphonate Trifluoroacetate (3m).