Inhibition of trypsin and urokinase by Cbz-amino(4-guanidinophenyl)methanephosphonate aromatic ester derivatives: The influence of the ester group on their biological activity

Article abstract of DOI:10.1016/j.bmcl.2006.03.002

The urokinase plasminogen activator is a trypsin-like serine protease, important in tumor development. Here, we report the synthesis and biochemical evaluation of selective and potent diaryl esters of phosphonic-type inhibitors for urokinase. We have found that the substituted phenyl ester ring has a strong influence on the inhibitory activity of these compounds. This led to the most potent phosphonic inhibitor for uPA synthesized to date.

Full text of DOI:10.1016/j.bmcl.2006.03.002

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2886–2890
Inhibition of trypsin and urokinase by Cbz-amino(4-guanidino-
phenyl)methanephosphonate aromatic ester derivatives:
The influence of the ester group on their biological activity
*
´
´
Marcin Sienczyk and Jozef Oleksyszyn
Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology,
Wybrzez_e Wyspian´skiego 27, 50-370 Wrocław, Poland
Received 31 January 2006; revised 1 March 2006; accepted 2 March 2006
Available online 20 March 2006
Abstract—The urokinase plasminogen activator is a trypsin-like serine protease, important in tumor development. Here, we report
the synthesis and biochemical evaluation of selective and potent diaryl esters of phosphonic-type inhibitors for urokinase. We have
found that the substituted phenyl ester ring has a strong influence on the inhibitory activity of these compounds. This led to the most
potent phosphonic inhibitor for uPA synthesized to date.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The urokinase plasminogen activator (urokinase, uPA)
is a trypsin-like serine protease, important in many cel-
lular processes such as tumorigenesis, cell proliferation
and migration, cell adhesion, angiogenesis, intravasa-
tion, and metastasis.1–3 The primary role of uPA is to
convert plasminogen into its active form plasmin, a
broad spectrum serine protease which in turn activates
matrix metalloproteases, and also degrades several
extracellular matrix components, including laminin, col-
lagen type IV, fibrin, and fibronectin.4 It facilitates the
detachment of cancer cells from the primary tumor
and their migration within the surrounding tissue into
blood and lymph vessels leading to the formation of
metastases at distant sites.5
the large group of such molecules, only a few potent and
selective low-molecular weight uPA inhibitors are pres-
ently known.9,10
a-Aminoalkylphosphonate diphenyl esters, the phos-
phonic analogues of naturally occurring amino acids,
and their peptidyl-analogues comprise a group of
irreversible, slow binding, competitive inhibitors for
chymotrypsin-like and trypsin-like serine proteases such
as chymase, trypsin, elastase or urokinase.11–13 Howev-
er, almost no attention has been paid to the possible
enhancement of their activity through the substitution
at the phenyl ester ring.14 Here we describe the synthesis
of new Cbz-N-protected a-aminoalkylphosphonate
diphenyl ester derivatives, aromatic analogues of argi-
nine, as potent and selective inhibitors for urokinase
plasminogen activator. The main purpose of this study
was to determine how simple modifications in the
aromatic ester ring would change the potency of synthe-
sized inhibitors. We have also examined how these
modifications influence its selectivity for trypsin.
Since the urokinase plasminogen activator is involved at
major steps in tumor progression, several molecules able
to inhibit uPA activity have been synthesized. The com-
mon structural motif found in uPA inhibitors is an aro-
matic moiety substituted by an amidino or guanidino
function, which mimics the arginine side chain. Some
of these compounds inhibit uPA-mediated processes
such as angiogenesis, extracellular matrix degradation,
tumor cell adhesion, migration, and invasion.6–8 Despite
The synthetic approach is outlined in Scheme 1.15 In the
first step, different aromatic phosphites 1a–i were syn-
thesized using PCl3 and substituted phenols in refluxing
acetonitrile. The crude phosphites 1a–i were used in the
next step in the amidoalkylation reaction with benzyl
carbamate and 4-nitrobenzaldehyde affording Cbz-N-
Keywords: a-Aminoalkylphosphonates; Arginine mimetics; Trypsin-
like serine protease inhibitors; Urokinase
.
Corresponding author. Tel.: +48 71 320 40 27; fax: +48 71 328 40
64; e-mail: jozef.oleksyszyn@pwr.wroc.pl
*
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.002

´
M. Sienczyk, J. Oleksyszyn / Bioorg. Med. Chem. Lett. 16 (2006) 2886–2890
2887
NO2
R
O
P
i
ii
R OH
PCl
+
3
O
O
O
R
R
O
O
R
R
O
N
H
P
O
1a-i
2a-i
iii
Boc
NH2
HN NH
HN
HN
*TFA
Boc
N
NH2
O
O
O
O
R
R
R
R
R
O
O
O
O
O
O
O
N
H
P
O
O
N
P
O
N
P
O
H
H
R
5a-i
3a-i
4a-i
Scheme 1. Preparation of compounds 5a–i. Reagents and conditions: (i) acetonitrile, reflux; (ii) benzyl carbamate, 4-nitrobenzaldehyde, AcOH,
80–90 ꢁC; (iii) SnCl2/H2O, AcOEt, reflux; (iv) S-ethyl-N,N0-di(Boc)-isothiourea, Et3N, HgCl2, CHCl3; (v) TFA, CH2Cl2.
1
protected amino(4-nitrophenyl)methanephosphonate de-
rivatives 2a–i as the racemic mixtures in good yields.16
characterized by H, 13C, 31P NMR, and melting point
analyses.17
For the reduction of nitro derivatives 2a–i, SnCl2 dis-
solved in water, as a reductive agent in refluxing ethyl
acetate, was applied. The desired aromatic amines 3a-i
were obtained in quantitative yields. The crucial, guany-
lating step was achieved using S-ethyl-N,N0-di(Boc)iso-
thiourea in chloroform, in the presence of Et3N and
HgCl2. Subsequent flash chromatography on Silica gel
using CHCl3/ethyl acetate (4:1) as an eluent gave the
guanidines 4a–i.
The TFA salts of Cbz-N-amino (4-guanidinophenyl) meth-
anephosphonate aromatic esters 5a–j were evaluated for
their ability to inhibit the proteolytic activity of uroki-
nase and trypsin using a chromogenic assay.18 The
enzyme and the inhibitor were incubated for 15 min at
25 ꢁC before the addition of the substrate. Longer incu-
bation times did not change the IC50 values.
The effects resulting from the substitution of simple
aliphatic chain in the phenyl ring at para position of
Cbz-(4-GuPhg)P(OPh)2 are noticeable, as seen in
Table 1. In general, a comparison of the IC50 values
clearly shows that the substitution at position 4 has
a strong influence on the inhibitory activity of these
derivatives, but in most cases it lacks the selectivity.
Compound 5h is the most potent phosphonic uPA
inhibitor synthesized to date (8.7 nM toward trypsin
and 26 nM toward uPA). The highest selectivity was
obtained for 5j and 5f, and total selectivity for 5i.
These results are in agreement with the 3D-QSAR
studies and the model which predict the existence of
The oxidation of the S-methyl groups in compound 4h
into the methylsulfonyl groups was performed in the
absolute THF using 30% hydrogen peroxide as an
oxidizing agent, followed by the addition of TFA in
CH2Cl2 (Scheme 2) without isolation of the intermediate
product 4j.
Boc-deprotection of the guanidine group was achieved
by using trifluoroacetic acid (50% solution in methylene
chloride) to yield the TFA salts of 5a–j. The structures
of all final compounds, as well as the intermediates, were
Boc
HN
Boc
HN
HN
Boc
NH2
HN
N
Boc
*TFA
N
HN NH
O
S
O
S
O
O
S
i
O
ii
O
O
O
O
O
N
P
O
O
O
O
N
P
O
H
O
O
O
N
P
O
H
H
S
S
S
O
O
O
O
5j
4j
4h
Scheme 2. Reagents: (i) THF, 30% H2O2 (this step should be handled carefully, as highly explosive peroxides may be generated); (ii) TFA, CH2Cl2.

´
M. Sienczyk, J. Oleksyszyn / Bioorg. Med. Chem. Lett. 16 (2006) 2886–2890
2888
Table 1. The inhibitory activity of different aromatic esters of a-aminoalkylphosphonates
NH2
*TFA
HN NH
O
R
O
O
N
H
P
O
O
R
Compound
R
IC50 (lM)
kobsd/[I]b [Mꢀ1 sꢀ1
]
Selectivity
0.21
uPA
Trypsin
uPA
1450
Trypsin
6870
5a
5b
5c
5d
5e
5f
0.532
0.112
0.370
7.80
0.176
0.017
0.061
0.088
2.31
2080
4370
0.48
100
>10,000
>10,000
8750
0.002
0.29
0.208
0.098
0.204
2.60
3700
7850
0.90
3800
330
11.3
O
S
5g
5h
5i
0.251
0.0087
NIa
300
3070
0.10
0.026
0.455
0.053
>10,000
1700
>10,000
NI
0.33
—
O
S
O
5j
23
>10,000
30
434
a NI, no inhibition after 25 min of incubation with enzyme.
b The apparent second-order inhibition rate constant kobsd/[I] was calculated using incubation method under pseudo-first-order reaction conditions
(inhibitor concentration P10· enzyme concentration).19
large hydrophobic subsite in the uPA structure on the
leaving group site.20 It is possible that one of the
substituted aromatic ester rings could attach to this
site. This region seems to play a pivotal role in the
selectivity of these molecules.
peptidyl-derivatives. We are currently at advanced stag-
es in the synthesis of peptides which contain such phos-
phonic motif at the C-terminal as selective toward
several other trypsin-like serine proteases. Irreversible
inhibition of u-PA by phosphonate type inhibitors
may be advantageous for in vivo activity.21 However,
this needs to be established in the animal model, if
u-PA gene knockout mice remain healthy.22
In conclusion, we describe herein the synthesis and the
inhibitory activity of new Cbz-N-protected derivatives
of a-aminoalkylphosphonates with different aromatic
ester rings. The compounds obtained are potent inhibi-
tors of urokinase and trypsin even as the racemic
mixtures. This leads to the conclusion that such modifi-
cations have a strong influence on the inhibitory activity
of these derivatives. Further investigation in this field
may lead to more potent and selective inhibitors. The
next challenge in this area is the synthesis of pure
enantiomers of the most potent inhibitors and their
Acknowledgments
This work was supported by State Committee for Scien-
tific Research Grant 2P05A 039 28. We also thank
Katarzyna Sołowska, Ewa Pietrusewicz, and Maciej
Kliszczak for their assistance during the synthetic and
enzymatic experiments.

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M. Sienczyk, J. Oleksyszyn / Bioorg. Med. Chem. Lett. 16 (2006) 2886–2890
2889
(DMSO): 20.72, 52.88 (d, J = 158.0 Hz), 66.69, 120.44,
120.47, 120.58, 124.46, 128.50, 128.86, 130.25, 130.63,
132.95, 134.91 (d, J = 10.6 Hz), 135.95, 137.09, 148.13,
156.21.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.
bmcl.2006.03.002.
Compound 5c: White solid, mp 163–170 ꢁC; 31P NMR
(DMSO): 14.39 (s); 1H NMR (DMSO): 1.15 (t, J = 7.8 Hz,
6H), 2.57 (d, J = 7.2, 15.3 Hz, 4H), 5.07 (d, J = 12.6 Hz,
1H), 5.15 (d, J = 12.6 Hz, 1H), 5.61 (dd, J = 10.2, 22.8 Hz,
1H), 6.93–7.70 (m, Ar-H, NH, 20H), 8.94 (d, J = 10.2 Hz,
1H), 10.08 (s, 1H); 13C NMR (DMSO): 16.12, 27.88, 52.80
(d, J = 158.6 Hz), 66.70, 120.60 (dd, J = 3.0, 8.3 Hz),
124.43, 128.51, 128.86, 129.43 (d, J = 6.8 Hz), 130.26,
132.92, 135.98, 137.09, 141.26 (d, J = 9.8 Hz), 148.23 (d,
J = 10.6 Hz), 156.25, 156.46 (d, J = 9.0 Hz), 159.53,
159.74.
References and notes
1. Duffy, M. J. Curr. Pharm. Des. 2004, 10, 39.
2. Danø, K.; Andreasen, P. A.; Grøndahl-Hansen, J.;
Kristensen, P.; Nielsen, L. S.; Skriver, L. Adv. Cancer
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3. Andreasen, P. A.; Egelund, R.; Petersen, H. H. Cell. Mol.
Life Sci. 2000, 57, 25.
4. (a) Ramos-DeSimone, N.; Hahn-Dantona, E.; Spiley, J.;
Nagase, H.; French, D. L.; Quigley, J. P. J. Biol. Chem.
1999, 274, 13066; (b) DeClerck, Y. A.; Laug, W. E.
Enzyme Protein 1996, 49, 72; (c) Johnsen, M.; Lund, L. R.;
Rømer, J.; Almholt, K.; Danø, K. Curr. Opin. Cell Biol.
1998, 10, 667.
5. Schweinitz, A.; Steinmetzer, T.; Banke, I. J.; Arlt, M. J.;
Sturzebecher, A.; Schuster, O.; Geissler, A.; Giersiefen,
H.; Zeslawska, E.; Jacob, U.; Kruger, A.; Sturzebecher, J.
J. Biol. Chem. 2004, 279, 33613.
Compound 5d: White solid, mp 135 ꢁC; 31P NMR
(DMSO): 14.37 (s); 1H NMR (DMSO): 1.17 (d,
J = 7.2 Hz, 12H), 2.85–2.89 (m, 2H), 5.06 (d,
J = 12.6 Hz, 1H), 5.15 (d, J = 12.6 Hz, 1H), 5.61 (dd,
J = 10.8, 22.5 Hz, 1H), 6.93–7.70 (m, Ar-H, NH, 20H),
8.93 (d, J = 10.2 Hz, 1H), 10.01 (s, 1H);13C NMR
(DMSO): 24.23, 33.25, 52.95 (d, J = 167.6 Hz), 66.71,
120.53, 120.55, 120.66, 124.44, 127.93, 128.03, 128.52 (d,
J = 6.0 Hz), 128.86, 130.27, 132.94, 135.96, 137.08, 145.82.
145.95, 148.23 (d, J = 9.1 Hz), 148.53 (d, J = 9.0 Hz),
156.21, 156.49.
Compound 5e: White solid, mp 105–108 ꢁC; 31P NMR
(DMSO): 16.08 (s); 1H NMR (DMSO): 1.22 (s, 18H), 5.03
(d, J = 12.4 Hz, 1H), 5.13 (d, J = 12.4 Hz, 1H), 5.58 (dd,
J = 10.3, 22.7 Hz, 1H), 6.90–7.68 (m, Ar-H, NH, 20H),
8.92 (d, J = 10.2 Hz, 1H), 9.92 (s, 1H); 13C NMR
(DMSO): 31.62, 34.62, 52.90 (d, J = 157.6 Hz), 66.73,
120.20 (d, J = 4.1 Hz), 120.29 (d, J = 3.8 Hz), 124.47,
126.97 (d, J = 7.7 Hz), 128.55, 128.87, 130.30 (d,
J = 5.7 Hz), 133.02, 135.94, 137.09, 147.94, 148.10,
148.24, 148.31, 148.44, 156.22, 156.48 (d, J = 8.6 Hz).
Compound 5f: White solid, mp 106–109 ꢁC; 31P NMR
(DMSO): 14.31 (s); 1H NMR (DMSO): 0.68 (s, 18H), 1.30
(d, J = 3.6 Hz, 12H), 1.70 (s, 4H), 5.08 (d, J = 12.6 Hz,
1H), 5.17 (d, J = 12.6 Hz, 1H), 5.62 (dd, J = 10.2, 22.2 Hz,
1H), 6.91–7.71 (m, Ar-H, NH, 20H), 8.95 (d, J = 10.2 Hz,
1H), 10.10 (s, 1H); 13C NMR (DMSO): 31.85, 32.04,
32.47, 38.42, 52.96 (d, J = 158.6 Hz), 56.63, 66.68, 119.96,
119.99, 120.10, 124.36, 127.67, 127.78, 128.46, 128.52,
128.85, 135.27 (d, J = 6.0 Hz), 132.92, 136.00, 137.14,
147.00, 147.13, 147.84 (d, J = 9.1 Hz), 148.15 (d,
J = 9.1 Hz), 156.24, 156.52.
6. Ertongur, S.; Lang, S.; Mack, B.; Wosikowski, K.;
Muehlenweg, B.; Gires, O. Int. J. Cancer 2004, 110, 815.
7. Rabbani, S. A.; Harakidas, P.; Davidson, D. J.; Henkin,
J.; Mazar, A. P. Int. J. Cancer 1995, 63, 840.
8. Xing, R. H.; Mazar, A. P.; Henkin, J.; Rabbani, S. A.
Curr. Pharm. Des. 2002, 8, 2541.
9. Magdolen, V.; de Prada, N. A.; Sperl, S.; Muehlenweg, B.;
Luther, T.; Wilhelm, O. G.; Magdolen, U.; Graeff, H.;
Reuning, U.; Schmitt, M. Adv. Exp. Med. Biol. 2000, 477,
331.
10. Sperl, S.; Jacob, U.; de Prada, A. N.; Sturzebecher, J.;
Wilhelm, O. G.; Bode, W.; Magdolen, V.; Huber, R.;
Moroder, L. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5113.
11. Oleksyszyn, J.; Powers, J. C. Biochemistry 1991, 30, 485.
12. Oleksyszyn, J.; Boduszek, B.; Kam, C.-M.; Powers, J.
J. Med. Chem. 1994, 37, 226.
13. Joossens, J.; Van der Veken, P.; Lambeir, A.-M.; Augus-
tyns, K.; Haemers, A. J. Med. Chem. 2004, 47, 2411.
14. Boduszek, B.; Oleksyszyn, J.; Kam, C.-M.; Selzler, J.;
Smith, R.; Powers, J. J. Med. Chem. 1994, 37, 3969.
´
15. Sienczyk, M.; Oleksyszyn, J. Tetrahedron Lett. 2004, 45,
7251.
Compound 5g: White solid, mp 80–85 ꢁC; 31P NMR
(DMSO): 14.81 (s); 1H NMR (DMSO): 3.72 (d,
J = 3.0 Hz, 6H), 5.07 (d, J = 12.6 Hz, 1H), 5.16 (d,
J = 12.6 Hz, 1H), 5.58 (d, J = 10.2, 22.5 Hz, 1H), 6.75–
7.69 (m, Ar-H, NH, 20H), 8.92 (d, J = 10.2 Hz, 1H), 9.98
(s, 1H); 13C NMR (DMSO): 52.63 (d, J = 158.6 Hz),
55.93, 66.70, 115.16 (d, J = 12.1 Hz), 121.62, 121.64,
121.79, 124.48, 128.54, 128.87, 130.24, 133.02, 135.94,
137.10, 143.66 (d, J = 4.5 Hz), 144.02 (d, J = 4.5 Hz),
156.21, 156.46, 156.85, 156.94.
16. Oleksyszyn, J.; Subotkowska, L.; Mastalerz, P. Synthesis
1979, 985.
17. 1H, 13C, and 31P NMR spectra were recorded at 300.13,
75.47, and 121.50 MHz, respectively. Spectroscopic data
of all synthesized compounds can be found in Supple-
mentary material. Compound 5a: White solid, mp 97–
100 ꢁC; 31P NMR (DMSO): 14.36 (s); 1H NMR (DMSO):
5.07 (d, J = 12.6 Hz, 1H), 5.15 (d, J = 12.0 Hz, 1H), 5.65
(dd, J = 10.2, 22.2 Hz, 1H), 7.37–7.72 (m, Ar-H, NH,
22H), 8.97 (d, J = 10.2 Hz, 1H), 10.03 (s, 1H); 13C NMR
(DMSO): 52.84 (d, J = 158.6 Hz), 66.72, 120.82 (d,
J = 3.0 Hz), 120.88 (d, J = 4.5 Hz), 124.46, 125.82 (d,
J = 9.1 Hz), 128.51, 128.87, 130.35 (d, J = 6.0 Hz), 132.79,
136.03, 137.08, 150.25 (d, J = 5.3 Hz), 150.56 (d,
J = 5.3 Hz), 156.22, 156.46 (d, J = 2.3 Hz), 159.21 (d,
J = 15.8 Hz).
Compound 5h: White solid, mp 90–93 ꢁC; 31P NMR
(DMSO): 16.37 (s); 1H NMR (CDCl3): 2.43 (d, J = 1.1 Hz,
6H), 5.03 (d, J = 12.4 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H),
5.60 (dd, J = 10.2, 22.7 Hz, 1H), 6.95–7.68 (m, Ar-H, NH,
20H), 8.89 (d,J = 9.7 Hz, 1H), 9.75 (s, 1H); 13C NMR
(DMSO): 15.78 (d, J = 3.7 Hz), 52.78 (d, J = 156.4 Hz),
66.78, 121.31, 121.36, 121.49, 124.64, 128.08, 128.55,
128.89, 130.32, 132.84, 137.06, 148.20, 156.15.
Compound 5b: White solid, mp 95 ꢁC; 31P NMR
(DMSO): 14.36 (s); 1H NMR (DMSO): 2.27 (s, 6H),
5.06 (d, J= 12.0 Hz, 1H), 5.16 (d, J = 12.0 Hz, 1H), 5.59
(dd, J = 10.2, 22.2 Hz, 1H), 6.90–7.69 (m, Ar-H, NH,
20H), 8.92 (d, J = 10.2 Hz, 1H), 9.99 (s, 1H); 13C NMR
Compound 5i: White solid, mp 130 ꢁC; 31P NMR
(DMSO): 14.81 (s); 1H NMR (DMSO): 5.07 (d,
J = 12.6 Hz, 1H), 5.12 (d, J = 12.0 Hz, 1H), 5.79 (dd,
J = 10.2, 22.8 Hz, 1H), 7.31–7.95 (m, Ar-H, NH, 26H),

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M. Sienczyk, J. Oleksyszyn / Bioorg. Med. Chem. Lett. 16 (2006) 2886–2890
2890
9.06 (d, J = 10.2 Hz, 1H), 9.91 (s, 1H); 13C NMR
200 lM for its substrate, 10 U/ml for trypsin and
100 lM for its substrate. Typical kinetic analysis
procedure: 50 ll (for trypsin) or 10 ll (for uPA) of
the inhibitor solution in DMSO was added to the
buffered enzyme solution (2.05 ml for trypsin and
0.58 ml for urokinase) to initiate the inactivation
reaction. After incubation at 25 ꢁC for 15 min, 50 ll
(for trypsin) or 10 ll (for uPA) of the substrate
solution was added. The change of absorbance was
measured for 20 min.
(DMSO): 51.86 (d, J = 148.0 Hz), 66.79, 117.25 (d,
J = 15.1 Hz), 120.82 (d, J = 6.0 Hz), 124.59, 126.26 (d,
J = 7.5 Hz), 127.48, 127.82 (d, J = 4.5 Hz), 128.18, 128.51,
128.85, 130.43 (d, J = 12.1 Hz), 131.03 (d, J = 6.0 Hz),
132.89, 133.76, 136.05, 137.01, 147.90, 147.96, 148.31,
156.17.
Compound 5j: Pale yellow solid, mp 240 ꢁC; 31P NMR
1
(CDCl3): 14.23 (s); H NMR (CDCl3): 3.06 (s, 6H), 4.71–
4.81 (m, 1H), 4.85 (d, J = 12.6 Hz, 1H), 4.92 (d,
J = 12.6 Hz, 1H), 6.80 (d, J = 8.4 Hz, 2H), 7.08–7.38 (m,
Ar-H, 17H), 7.72 (d, J = 8.7 Hz, 2H), 9.77 (s, 1H); 13C
NMR (CDCl3): 44.32, 54.05 (d, J = 140.5 Hz), 65.97,
121.36, 122.72, 128.16, 128.23, 128.79, 129.05, 129.81,
131.33, 134.60, 135.06, 137.55, 156.01, 156.22, 162.41.
19. Oleksyszyn, J.; Powers, J. C.. In Methods in Enzymology;
Academic, 1994; Vol. 244, pp 423–441.
20. Bhongade, B. A.; Gadad, A. K. Bioorg. Med. Chem. Lett.
2004, 12, 2797.
21. Initial experiments on the stability of the enzyme–inhibitor
complex were performed for compound 5a and trypsin.
First, trypsin was inhibited by compound 5a and the
excess of inhibitor was removed by centrifugation using a
Millipore Centriconꢂ 10kDa cut-off filter. The enzyme was
then incubated in HEPES buffer at pH 7.5 for 7 days.
After this time, the enzyme regained 5% of its original
activity as compared to the control, which retained 95% of
its original activity after 7 days.
18. The inhibitory effects of inhibitors on the enzymatic
activity of urokinase-type plasminogen activator (LMW
uPA from human urine, Calbiochem) and trypsin (from
bovine pancreas, Sigma–Aldrich) were evaluated using
Cbz-Val-Gly-Arg-pNA as a chromogenic substrate for
uPA and Bz-L-Arg-pNA for trypsin. The change of
absorbance was measured at 410 nm at 25 ꢁC (Bio-
chrom 4060). The assay buffer used was Tris–HCl (pH
8.8) containing 0.1 M NaCl (for urokinase) and HEPES
0.1 M (pH 7.5) containing 0.01 M CaCl2 (for trypsin).
The final concentration was 30 IU/ml for urokinase and
22. Carmeliet, P.; Schoonjans, L.; Kieckens, L.; Ream, B.;
Degen, J.; Bronson, R.; De Vos, R.; van denOord, J. J.;
Collen, D.; Mulligan, R. C. Nature 1994, 368, 379.