Development of Irreversible Diphenyl Phosphonate Inhibitors for Urokinase Plasminogen Activator

Article abstract of DOI:10.1021/jm0499209

In this letter we report the synthesis and biochemical evaluation of selective, irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator (uPA). A diphenyl phosphonate group was introduced on the substratelike peptide Z-D-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A k_app value in the 10³ M ^-1 s^-1 range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.

Full text of DOI:10.1021/jm0499209

J . Med. Chem. 2004, 47, 2411-2413
Sch em e 1. Synthesis of Compounds 1a
2411
Develop m en t of Ir r ever sible Dip h en yl
P h osp h on a te In h ibitor s for Ur ok in a se
P la sm in ogen Activa tor
J . J oossens,† P. Van der Veken,† A.-M. Lambeir,‡
K. Augustyns,† and A. Haemers*,†
Department of Medicinal Chemistry and Department of
Medical Biochemistry, University of Antwerp,
Universiteitsplein 1, B-2610, Antwerp, Belgium
Received J anuary 28, 2004
Abstr a ct: In this letter we report the synthesis and biochemi-
cal evaluation of selective, irreversible diphenyl phosphonate
inhibitors for urokinase plasminogen activator (uPA). A diphe-
nyl phosphonate group was introduced on the substratelike
peptide Z-D-Ser-Ala-Arg, and modification of the guanidine side
chain was investigated. A guanylated benzyl group appeared
the most promising side chain modification. A kapp value in
the 103 M-1 s-1 range for uPA was obtained, together with a
selectivity index higher than 240 toward other trypsin-like
proteases such as tPA, thrombin, plasmin, and FXa.
Angiogenesis is the formation of new blood vessels
from preexisting vasculature and is believed to be
critical for tumor growth and metastasis. Tumors above
a diameter of 2 mm3 need a blood supply to grow. This
blood supply is also important for the metastasis
process. When a tumor switches to the angiogenetic
phenotype, it starts producing growth factors.1-3 These
growth factors activate endothelial cells of the vascu-
lature bed and stimulate them to grow toward the
tumor. The next step requires breakdown of the extra-
cellular matrix (ECM). It was proved that the degrada-
tion of ECM by proteases such as matrix metallopro-
teases (MMP’s) is extremely important in angiogenesis.4,5
MMP’s are activated by plasmin, and this enzyme
degrades several components of ECM, including fibrin,
laminin, and fibronectin. Plasmin is activated from its
zymogen, plasminogen, by urokinase plasminogen ac-
tivator (uPA) and tissue plasminogen activator (tPA),
both of which are serine proteases. It is known that
while tPA is mainly involved in the fibrinolysis process,
uPA is more important in angiogenesis and is activated
by binding to its receptor (uPAR).6-9 The interaction
with uPAR induces proliferation and migration re-
sponses, with both responses being unrelated to the
serine protease activity.
a
(a) (i) Di-tert-butyl dicarbonate, (ii) Dess-Martin oxidation;
(a′) (i) phthalic anhydride, Et3N, toluene, 90 °C; (ii) Swern
oxidation; (b) benzyl carbamate, triphenyl phosphite, Cu(OTf)2,
DCM; (c) (i) TFA, (ii) N,N′-bis(tert-butoxycarbonyl)-1-guanylpy-
razole, MeCN; (c′) (i) NH2NH2‚H2O, THF, (ii) N,N′-bis(tert-butoxy-
carbonyl)-1-guanylpyrazole, MeCN; (d) (i) H2/Pd, (ii) Z-Ala-OH,
TBTU, DMF; (e) (i) H2/Pd, (ii) Z-D-Ser(OtBu), TBTU, DMF; (f) TFA.
(TBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tet-
rafluoroborate).
Two kinds of inhibitors for uPA are reported in the
literature. One group consists of peptides and peptide
derivatives, characterized by a basic function for a
specific ionic interaction with the carboxylate group of
Asp-189 of the S1 site in the deepest pocket of the
substrate binding groove of the enzyme.6 Other inhibi-
tors, such as amiloride, are small molecules with an
arylamidine, arylguanidine, or acylguanidine template,
capable of performing the same ionic interaction as the
peptides.7 The development of very selective inhibitors
for uPA is, however, hampered by its close resemblance
to trypsin-like serine proteases, and several inhibitors
suffer from a poor selectivity toward tPA, plasmin, or
thrombin.
In this paper we describe the development of potent
and selective diphenyl phosphonate peptidic inhibitors
for uPA. The diphenyl phosphonate group is known to
react with the active site serine affording a stable
phosphonylated and irreversibly inhibited enzyme.10,11,13
To the best of our knowledge, these diphenyl phospho-
nate peptides are the first selective and irreversible uPA
inhibitors described in the literature. An irreversible
chloromethyl ketone peptide was reported but lacks
selectivity.14
Research suggests that uPA is important in tumor
development. Experiments with knock-outs of all the
components of the uPA system in mice show a reduction
in tumor growth and metastasis. Moreover, high levels
of uPA are related to poor prognosis in human cancers.7
Experiments also prove that the deletion of the uPA
system in mice has no consequences for viability.6
Hence, the inhibition of uPA and/or the interaction with
its receptor is considered to be an important approach
for finding antiangiogenic agents.8
* Author to whom correspondence should be addressed. E-mail:
achiel.haemers@ua.ac.be.
† Department of Medicinal Chemistry.
‡ Department of Medical Biochemistry.
10.1021/jm0499209 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/06/2004

2412 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Ta ble 1. Inhibition of Trypsin-Like Serine Proteasesa
Letters
a
(*) standard error on the fit; (**) selectivity index: IC50/IC50 uPA.
Tamura et al. reported the biological activity of Z-D-
Ser-Ala-Arginal as an uPA inhibitor with an IC50 value
of 19 nM and a selectivity ratio of more than 130 toward
tPA.15 The pharmacokinetic properties of this compound
revealed a short plasma half-life following oral or
subcutaneous administration.7,15 Our experience with
the diphenyl phosphonate group as an inhibitory moiety
in peptide inhibitors for dipeptidylpeptidases prompted
us to substitute the aldehyde group of Z-D-Ser-Ala-
Arginal for an irreversibly inhibiting diphenyl phospho-
nate group.10 The use of irreversible inhibitors could be
very useful for further investigation of the usefulness
of uPa inhibitors in antiangiogenetic settings. In addi-
tion, we optimized the arginine moiety by incorporating
various linear or cyclic guanylated side chains at the
P1 position.12
The synthetic pathway is shown in Scheme 1. tert-
Butyl carbamate (Boc) amino alcohols 2d -g were used
as the starting materials for cyclic side chain compounds
1d -g. When these amino alcohols were not com-
mercially available, they were prepared from the cor-
responding amino acids by reduction with LiAlH4 after
Boc protection. A Swern16 or Dess-Martin17 oxidation
to aldehydes 3d -g followed by an amidoalkylation with
benzyl carbamate and triphenyl phosphite afforded
protected diphenyl phosphonate amino acids 4d -g as
racemic mixtures.11,13,18-21 The amidoalkylation reac-
tion, as originally described by Oleksyszin,21 was slightly
modified: higher yields were obtained when the usual
solvent AcOH was substituted with dichloromethane,
and copper triflate was employed as a catalyst. Treat-
ment with TFA followed by conversion of the amine into
a Boc-protected guanidine with N,N′-di(Boc)guanylpy-
razole furnished the guanylated phosphonates 5d -g.22
The Z-protecting group was removed by hydrogenolysis,
and the resultant free amine was coupled by the use of
TBTU with Z-Ala-OH to obtain 6d -g.23 The Z-protect-
ing group was again removed, and the protected pep-
tides 7d -g were obtained by coupling with Z-D-Ser-
(tBu)-OH. The Boc protecting groups were removed with
TFA. Final compounds 1d -g were isolated as the
corresponding TFA salt. The Boc protected amino alco-
hols could not be used as starting materials for com-
pounds 1a -c. Aldehydes 3a -c cyclized easily, and the
amidoalkylation gave low yields and/or afforded pyrro-
lidine phosphonate esters or piperidine phosphonate
esters.10,24 To avoid this reaction, the synthetic pathway
was slightly modified, and a phthalimide protection was

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2413
used for compounds 2a -c. After amidoalkylation in
AcOH, the phthalimide moiety was removed with hy-
drazine. This deprotection unfortunately gave low yields
due to the instability of the diphenyl phosphonate group.
Aldehyde 3b can also be prepared through acid hydroly-
sis of the N-phthalimido-protected 4-aminobutyralde-
hyde diethylacetal.
The diphenyl phosphonate tripeptides 1a -g were
evaluated for their ability to inhibit various trypsin-like
serine proteases.15 The IC50 values were calculated for
uPA and four other trypsin-like serine proteases in-
volved in the blood coagulation cascade (tPA, thrombin,
plasmin, FXa). Potent and selective irreversible inhibi-
tors of uPA were discovered. Compounds 1b and 1f were
the most potent inhibitors for uPA with IC50 values
around 60 nM. The results are summarized in Table 1.
Amiloride was used as the standard uPA inhibitor.
Compound 1b containing the substratelike arginine
side chain showed poor selectivity toward thrombin.
Compound 1f with a guanylated benzyl side chain
showed a selectivity ratio (IC50 enzyme/IC50 uPA) of
more than 200 for all enzymes tested.
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solutions, no rise in activity of uPA was observed, and
it was concluded that these inhibitors were irreversible
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Ack n ow led gm en t. This work received support from
The Fund for Scientific Research-Flanders (Belgium)
(FWO). J . J oossens and P. Van der Veken are fellows
of the Institute of Promotion of Innovation in Science
and Technology of Flanders (IWT). The excellent techni-
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Su p p or tin g In for m a tion Ava ila ble: Experimental, bio-
chemical, and analytical data. This material is available free
of charge via the Internet at http://pubs.acs.org.
J M0499209