Small molecule antagonists of the urokinase (uPA): Urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand

Article abstract of DOI:10.1016/j.bmc.2011.03.016

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of th

Full text of DOI:10.1016/j.bmc.2011.03.016

Bioorganic & Medicinal Chemistry 19 (2011) 2549–2556
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR)
interaction with high reported potencies show only weak effects in cell-based
competition assays employing the native uPAR ligand
Melissa De Souza a, , Hayden Matthews b, , Jodi A. Lee a, Marie Ranson a, Michael J. Kelso b,
⇑
a School of Biological Sciences, University of Wollongong, NSW 2522, Australia
b School of Chemistry, University of Wollongong, NSW 2522, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and
upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor
prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-
tumour/metastasis agents and several approaches have been explored for this purpose, including the
Received 27 January 2011
Revised 2 March 2011
Accepted 7 March 2011
Available online 12 March 2011
use of small molecule antagonists. Two highly potent non-peptidic antagonists
1 and 2 (IC50
1 = 0.8 nM, IC50 2 = 33 nM) from the patent literature were reportedly identified using competition assays
employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for
studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their
potencies in physiologically relevant cell-based competition assays employing uPAR’s native binding
partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis
of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative
competition flow cytometry assays and quantitative cell-based uPA activity assays that employed
HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations
Keywords:
uPA
uPAR
Antagonist
Plasminogen activation
of 2 (10–100
onist effects at 100
inhibitors of cell surface-bound HMW-uPA activity (IC50 >100
l
M) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antag-
M. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor
M for 1 and 2). The report highlights
l
l
the dangers of identifying false-positive lead uPAR antagonists from competition assays employing
labelled competing ligands other than the native HMW-uPA.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
for developing novel anti-cancer/metastasis therapeutics.7–9 In its
simplest form, the PAS comprises the serine protease urokinase-
The plasminogen activation system (PAS) plays a central role in
a variety of tissue remodelling processes including tumour inva-
sion and metastasis.1,2 Metastasis has been clinically correlated
with upregulation of the PAS in multiple tumour types3–6 and at
least two PAS components are considered as promising targets
type plasminogen activator (uPA), its cognate GPI-anchored cell
surface-bound receptor (uPAR) and two endogenous serpins; plas-
minogen activator inhibitor 1 (PAI-1) and plasminogen activator
inhibitor 2 (PAI-2). Activation of uPA upon binding to uPAR leads
to cleavage of cell surface-associated plasminogen to reveal the
broad spectrum serine protease plasmin. Localisation of plasmin
activity on cell surfaces at the leading edge of migrating cells pro-
vides focused activation of downstream extracellular zymogens
(e.g., proelastases, procollagenases and prostromelysins) and latent
growth factors and together these lead to directional pericellular
proteolysis and remodelling of the local extracellular environ-
ment—key events required during metastasis.10 A secondary func-
tion of the PAS is to activate further cell migration and proliferation
signalling via multimeric complexes involving uPAR, transmem-
brane integrins and vitronectin.11–13
Abbreviations: ATF, amino-terminal fragment of uPA; Boc, tert-butyloxycar-
bonyl; DCC, N,N0-dicyclohexylcarbodiimide; DIPEA, diisopropylethylamine; DMAP,
4-(N,N-dimethylamino)pyridine; EGF, epidermal growth factor; GPI, glycophos-
phatidylinositol; HBTU, O-benzotriazole-N,N,N0,N0-tetramethyluroniumhexafluoro-
phosphate; HMW-uPA, high molecular weight urokinase-type plasminogen
activator; MAb, monoclonal antibody; NMP, N-methylpyrrolidone; PAS, plasmino-
gen activation system; PMA, phorbol 12-myristate-13-acetate; rp-HPLC, reverse
phase high performance liquid chromatography; siRNA, small interfering ribonu-
cleic acid; SMB, vitronectin somatomedin B domain; uPA, urokinase-type plasmin-
ogen activator; uPAR, urokinase-type plasminogen activator receptor.
The two principle strategies that have been explored for damp-
ening PAS activity with the goal of producing new anti-cancer
leads include: (1) inhibitors of the serine protease domain of uPA
⇑
Corresponding author. Tel.: +61 2 4221 5085; fax: +61 2 4221 4287.
E-mail address: mkelso@uow.edu.au (M.J. Kelso).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.016

2550
M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
and (2) antagonists of the uPA:uPAR interaction. Several structural
classes of uPA inhibitors have been identified and selected ana-
logues have undergone preclinical evaluation as non-cytotoxic
anti-tumour/metastasis drugs.14 An orally active uPA inhibitor
(MESUPRONÒ) being developed by Wilex AG (Germany) is
currently undergoing phase II trials in patients with breast and
pancreatic cancers (http://www.wilex.de/R&D/Mesupron.htm).
The uPA-binding site presented by uPAR is a relatively large
hydrophobic cavity (25 Å across, 14 Å deep).15 Residues
Asn22-Trp31 located on a b-hairpin loop of the amino-terminal
fragment (ATF) of uPA bury deeply into the cavity giving rise to a
high affinity interaction (KD ꢀ0.2 nM).16,17 Tyr24 is a major contrib-
utor to high affinity as it is located at the tip of the b-hairpin and
interacts with all three uPAR domains (DI–DIII).17,18
Many of the known uPAR antagonists specifically target the
hydrophobic cavity, with reported examples including linear19
and cyclic20 peptides and fusion proteins bearing the uPAR binding
fragment of uPA.21 A limited number (five in total) of small mole-
cule non-peptidic uPAR antagonists have also been described.22
Other anti-uPAR strategies that have been explored include the
use of monoclonal antibodies23 and siRNA.24
ered as anti-cancer/metastasis leads since inhibition of uPA activa-
tion and downstream pericellular proteolysis is ultimately the
effect required for reducing tumour invasion and metastasis in
vivo. This paper describes two new syntheses for 1 and 2 and re-
ports the important finding that in physiologically relevant compe-
tition cell-based assays using HMW-uPA both compounds are poor
uPAR antagonists and show only minimal inhibition of uPA activity
at high (lM) concentrations.
2. Chemistry
Syntheses of 1 and 2 are described in the patent literature but
few experimental details and no compound characterisations were
provided.25,26 The reported method for preparing 1 and various
analogues made use of Fmoc-based solid phase synthesis protocols
on Sasrin resin. We now report the synthesis and characterisation
of 1 using a solution phase peptide synthesis strategy starting from
the commercially available 30-aminobiphenyl-4-carboxylic acid 3
(Scheme 1).
Initial attempts to produce N-Boc-30-aminobiphenyl-4-carbox-
ylic acid 4 from 3 under standard conditions using Boc-anhydride
(Boc2O) proved surprisingly difficult (yields <70%) but it was found
that very high yields (>99%) of 4 could be obtained when catalytic
I2 was added to reactions.30 The carboxylic acid 4 was coupled to
O
H
N
A
L
-Phe-OMeꢁHCl using O-benzotriazole-N,N,N0,N0-tetramethyl-uro-
N
H
O
H
N
B
O
nium-hexafluoro-phosphate (HBTU) and diisopropylethylamine
(DIPEA) in DMF to produce 5 in 79–88% yield. The N-Boc group
was removed from 5 by stirring with neat trifluoroacetic acid
(TFA) in the presence of p-cresol as cation scavenger. The deprotec-
OH
Cl
O
N
H
1
ted crude product was coupled to Boc-L-Trp-OH using HBTU and
NH2
O
O
MeO
DIPEA in DMF to afford 6 in 53–65% yield. Boc-deprotection of 6
(neat TFA/p-cresol) followed by acetylation with acetic anhydride
and hydrolysis of the methyl ester (LiOH/THF/H2O) provided 1 in
90–95% yield over the final three steps.
H
H2N
N
N
N
O
O
The patented method for preparing the putative antagonist
CHIR11509 226 made use of the solid phase submonomer strategy
reported by Zuckermann et al.31 for producing oligo-N-substituted
glycine peptoids. The method involves coupling bromoacetic acid
to Wang resin followed by halogen substitution with N-(4,40-dim-
ethoxybenzhydryl)glycinamide. Further manipulations followed
by resin cleavage with 90% TFA/H2O apparently afforded com-
pound 2. It is clear, however, that this procedure would not yield
2 due to loss of the 4,40-dimethoxybenzhydryl group during resin
cleavage with TFA. A new unambiguous mixed solid/solution phase
synthesis of 2 based on the submonomer method is now reported
(Scheme 2).
Rink amide AM resin bearing an Fmoc-protected amine was
deprotected using 20% piperidine in CH2Cl2. Bromoacetic acid
was coupled to the amino-functionalised resin using DCC and
4-(N,N-dimethylamino)pyridine (DMAP) in a mixture of N-methyl-
pyrrolidone (NMP) and CH2Cl2. Displacement of the bromide with
H2N-Gly-OtBuꢁHCl in DMSO at 45 °C afforded a secondary amine
which was acylated with bromoacetic acid using the above
method. Displacement of the newly introduced bromide with
b-naphthylamine in DMSO at 45 °C, followed by coupling of the
OMe
2
Of the few small molecule uPA:uPAR antagonists known two
compounds show very high potencies: Schering’s peptidomimetic
derivative
1
(IC50 = 0.8 nM)25 and Chiron’s CHIR11509
2
(IC50 = 33 nM).26 Due to their high potencies, small size and syn-
thetic accessibility compounds 1 and 2 appear to represent useful
pharmacological tools for probing the role of the PAS in metastasis.
The IC50 value for 1 was reportedly determined in a cell-based
radioligand competition binding assay using human prostate can-
cer DU-145 cells, which express high levels of unoccupied uPAR
receptors, and an 125I-labelled (125I-Tyr24) cyclic version of the
minimal receptor-binding sequence of uPA (residues 12–32,
Ala19).25,27 The reported IC50 value for 2 was measured using a
modified version of a radioligand binding assay reported by Rosen-
berg and co-workers,28 where biotinylated soluble uPAR is immo-
bilised onto streptavidin-coated plates and the competitive
binding of 125I-labelled uPA ATF measured. The modified assay
used in the IC50 determination for 2 substituted 125I-labelled uPA
ATF with a tagged version of the epidermal growth factor (EGF)-
like domain of uPA.26,29
secondary amine to bromoacetic acid provided an a-bromocarbon-
yl which was subsequently displaced with propargylamine (DMSO
at 45 °C). The resin was then cleaved and the tert-butyl ester simul-
While both 1 and 2 are clearly potent antagonists of labelled
uPA fragments it was noted that neither compound had to date
been tested for their ability to compete with the native uPAR ligand
(high molecular weight uPA: HMW-uPA) for uPAR binding, or for
their functional effects on plasminogen activation in physiologi-
cally relevant cellular settings where the native ligand is present.
Such experiments are essential for uPAR antagonists being consid-
taneously removed using a mixture of TFA/triisopropylsilane
(TIPS)/H2O to afford the free acid 7 (17%) after purification by
preparative rp-HPLC (Scheme 2). Solution phase amide coupling
of 7 with 4,40-dimethoxybenzhydrylamine using HBTU and DIPEA
in DMF gave the target peptoid 2 (28%) after rp-HPLC. Interestingly,
two-dimensional NMR experiments in DMSO-d6 revealed that 2
exists as a slowly interconverting 1:1 mixture of cis/trans rotomers

M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
2551
O
Boc-HN
Boc-HN
O
N
H
O
H
N
H
N
R
b
c
d, e
OMe
OMe
N
H
1
O
O
OH
N
H
O
R = H
= Boc
3
4
5
6
a
Scheme 1. Synthesis of putative uPAR antagonist 1. Reagents and conditions: (a) Boc2O, I2, DIPEA, 99%; (b)
L
-Phe-OMeꢁHCl, HBTU, DIPEA, 79–88%; (c) (i) TFA, p-cresol; (ii) Boc-
L
-Trp-OH, HBTU, DIPEA, 53–65%; (d) (i) TFA, p-cresol; (ii) Ac2O, DIPEA; (e) LiOH, THF/H2O, 90–95% (over 3 steps).
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
d
a, b
Br
c
b
H
N
Fmoc
HN
N
N
Rink amide
AM Resin
O
O
Br
NH
O
O
O
HO
O
O
N
O
O
H
N
H
N
NH2
O
O
N
N
N
b
e
f
g
O
O
O
H2
2
H
N
O
O
N
Br
TFA
N
N
7
Scheme 2. Synthesis of putative uPAR antagonist 2. Reagents and conditions: (a) 20% piperidine, CH2Cl2; (b) BrCH2CO2H, DCC, DMAP, NMP/CH2Cl2; (c) H2N-Gly-OtBuꢁHCl,
DIPEA, DMSO; (d) b-naphthylamine, DMSO, 45 °C; (e) propargylamine, DMSO, 45 °C; (f) TFA/H2O/TIPS 95:2.5:2.5, rp-HPLC, 17%; (g) 4,40-dimethoxybenzhydrylamine, HBTU,
DIPEA, rp-HPLC, 28%.
about the C(O)-N(b-naphthyl) tertiary amide, a 1:0.87 mixture of
cis/trans rotomers about the sterically hindered secondary amide
bond and a 1:0.95 mixture of cis/trans rotomers about the remain-
ing tertiary amide.
ing HMW-uPA as the competing ligand. The flow cytometry exper-
iments showed that binding of exogenous Alexa-HMW-uPA to
uPAR receptors on PMA-stimulated U-937 cells occurs in a dose
dependent manner (Fig. 1A, closed circles) and that saturation of
ꢀ1 ꢂ 106 cells is achieved in the presence of 40 nM Alexa-
HMW-uPA or greater. Unstimulated U-937 cells showed no in-
creases in fluorescence upon addition of increasing concentrations
of Alexa-HMW-uPA (Fig. 1A, closed circles) suggesting that the
lower number of uPAR receptors present on these cells were pre-
saturated by endogenous uPA prior to the addition of Alexa-
HMW-uPA. Accordingly, 1 ꢂ 106 PMA-stimulated U-937 cells and
40 nM Alexa-HMW-uPA were chosen for use when the competi-
tion flow cytometry assays were carried out in the presence of
antagonists (see Fig. 2).
Preliminary fluorometric investigations of cell surface-bound
HMW-uPA activity in PMA stimulated U-937 cells were conducted
using the fluorogenic uPA-selective substrate Z-Glu-Gly-
Arg-AMCꢁHCl33 in the presence/absence of added exogenous
HMW-uPA. Cell number-dependent fluorescence increases were
observed in both cases confirming cell-bound uPA activity. The
low but measurable uPA activity observed in the absence of exog-
enous HMW-uPA (Fig. 1B, unshaded squares) confirmed that PMA
stimulated U-937 cells express low levels of endogenous receptor-
bound uPA. The rate of increase in fluorescence was found to be
significantly higher (indicating higher cell-bound uPA activity) in
the presence of 40 nM exogenous HMW-uPA (Fig. 1B, shaded
squares). The assay was subsequently extended to a competition
format to determine the IC50 values of uPA inhibition by antago-
nists (see Fig. 3) and in all assays 40 nM exogenous HMW-uPA
was added to enhance cell surface-bound uPA activity and increase
sensitivity.
3. Results
Phorbol 12-myristate-13-acetate (PMA) is known to induce high
expression levels of unoccupied cell surface uPAR receptors in
monocyte-like U-937 leukemia cells without a corresponding in-
crease in uPA expression.32 Accordingly, PMA-stimulated U-937
cells were used in a semi-quantitative competition flow cytometry
assay where the binding of HMW-uPA labelled with the fluorescent
probe Alexa-488 (Alexa-HMW-uPA) to cell surface uPAR receptors
was measured in the presence/absence of antagonists. PMA-
stimulated U-937 cells were similarly used in a sensitive and
quantitative cell-based fluorometric uPA activity assay wherein
the rate of conversion of a fluorogenic uPA-selective substrate by
uPAR-bound HMW-uPA was measured in the presence/absence
of uPAR antagonists. Antagonists which compete with HMW-uPA
for uPAR binding indirectly inhibit conversion of uPA substrate
thereby allowing for the determination of IC50’s of uPA inhibition
by antagonists.
3.1. Characterisation of Alexa-HMW-uPA binding to cell surface
uPAR receptors and uPAR-bound HMW-uPA activity in U-937
cells
Preliminary flow cytometry and uPA activity investigations
were carried out to confirm that PMA stimulated U-937 cells were
suitable for use in the cell-based uPAR antagonist assays employ-

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M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
Figure 1. (A) Characterisation of Alexa-HMW-uPA binding to uPAR receptors on U-
937 cells by flow cytometry. Unstimulated (unshaded circles) and PMA stimulated
(shaded circles) U-937 cells were incubated in the presence of increasing concen-
trations of Alexa-HMW-uPA and the geometric mean of cell-associated fluorescence
measured using dual colour flow cytometry. (B) Characterisation of uPAR-bound
HMW-uPA activity in U-937 cells. Increasing numbers of PMA stimulated U-937
cells were pre-incubated in the absence (unshaded squares) or presence (shaded
squares) of 40 nM HMW-uPA and analysed for cell surface HMW-uPA activity using
a fluorogenic uPA-selective substrate. Initial rates of increase in fluorescence per
min were measured. Values shown represent the mean SD (n = 3) from represen-
tative experiments.
Figure 3. Fluorescence assays of cell surface-bound HMW-uPA activity in PMA-
stimulated U-937 cells. Cells were pre-incubated for 30 min with range of
concentrations of (A) i-uPA, (B) 1 or (C) 2 before adding 40 nM HMW-uPA and
incubating for further 30 min. Cells were washed, fluorogenic uPA-specific
a
a
substrate added and fluorescence measurements recorded. Initial rates of change in
fluorescence after subtraction of background fluorescence (cells only) are presented
as a percentage of control (no test compound added). Values represent means SD
(n = 3) obtained from representative experiments.
3.2. Evaluation of 1 and 2 as antagonists of the HMW-uPA:uPAR
interaction in U-937 cells by flow cytometry
Semi-quantitative competition flow cytometry experiments
were used to assess compounds 1 and 2 as antagonists of
HMW-uPA. Two positive control antagonists were included for
assay validation. These were: (1) HMW-uPA which had been inac-
tivated with Glu-Gly-Arg-chloromethyl ketone (i-uPA)34 and (2)
mouse anti-human uPAR monoclonal antibody (MAb #3936,
American Diagnostica Inc. CT, USA).35 Briefly, 1 ꢂ 105 PMA-
stimulated U-937 cells were incubated with 1 and 2 or positive
controls before addition of Alexa-HMW-uPA (40 nM). After a
further short incubation cell surface fluorescence was measured
by dual colour flow cytometry.
Figure 2. Competition flow cytometry analysis of antagonists of the HMW-
uPA:uPAR interaction in U-937 cells. PMA stimulated U-937 cells were
pre-incubated with 10 or 100 lM 1 or 2 prior to incubating with 40 nM Alexa-
HMW-uPA for 15 min. Binding of Alexa-HMW-uPA to cells was then assessed using
dual colour flow cytometry. The observed fluorescence is expressed as a percentage
of the fluorescence observed in the presence of 40 nM Alexa-HMW-uPA alone (i.e.,
no compound added). Fluorescence decreases observed after pre-incubation with
positive controls (1) HMW-uPA inactivated with Glu-Gly-Arg-chloromethyl ketone
(i-uPA; 10 and 100 nM) and (2) mouse anti-human uPAR monoclonal antibody
(MAb #3936; 100 nM) are shown for comparison. Values represent means SD
(n = 3) from representative experiments.
The i-uPA positive control strongly antagonised Alexa-HMW-
uPA binding at concentrations of 10 nM (80% reduction in binding)
and 100 nM (90% reduction in binding). The anti-uPAR MAb pro-
duced a 70% reduction in binding when administered at 100 nM,

M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
2553
which is in reasonable agreement with a previous finding that
cell-surface uPA activity in the fluorescence assay (IC50 >100
Compound 2 was shown to be a weak uPAR antagonist at concen-
trations of 10 and 100 M and a weak inhibitor of cell-surface uPA
activity (IC50 >100 M). Nanomolar concentrations of positive
controls (anti-uPAR MAb and chloromethylketone-inactivated
HMW-uPA) antagonised HMW-uPA binding and inhibited cell sur-
face uPA activity under the same assay conditions. These results
are in direct contrast to the high antagonist potencies reported
for 1 and 2 from the competition assays using labelled uPA
fragments.25,26
One possible explanation for the conflicting results arises from
the fact that the uPA fragments employed in previous measure-
ments contained 125I-labelled Tyr24 residues. As stated previously,
Tyr24 is located at the tip of uPA’s b-hairpin loop and is crucial for
high affinity uPAR binding. Evidence shows that modification of
Tyr24 with bulky groups (e.g., iodine) greatly reduces uPA’s affinity
for uPAR.41,42 Given this and the fact that data comparing the uPAR
binding affinity of the 125I-Tyr24-uPA fragments to their unlabelled
counterparts appears not to have been reported suggests that the
previously reported IC50 values for 1 and 2 are not meaningful.
In conclusion, compounds 1 and 2 are poor antagonists of the
uPA:uPAR interaction in physiologically relevant cellular systems
and are therefore not useful as small molecule probes for investiga-
tions of the PAS. The report highlights the importance of using
HMW-uPA as the competing ligand in competition experiments
aimed at identifying and/or measuring the potency of small mole-
cule uPAR antagonists as pharmacological tools or anti-cancer drug
leads.
lM).
133 nM concentrations of the antibody produce 50% reductions
in binding of HMW-uPA to uPAR receptors on glioblastoma cells.35
Compound 1 produced no reduction of fluorescence relative to
the negative control (no test compound) indicating that it does
not antagonise HMW-uPA binding, even at the high concentrations
l
l
used (10 and 100
reduced the observed fluorescence relative to the negative control
by approximately 60% (10 M) and 50% (100 M) (Fig. 3). This
indicated that while 2 antagonises HMW-uPA binding its effects
are relatively weak and require M concentrations to effectively
lM) (Fig. 3). The same concentrations of 2
l
l
l
compete with the native uPAR ligand.
3.3. Quantitative analysis of 1 and 2 as inhibitors of cell surface-
bound HMW-uPA activity in U-937 cells
Fluorescence enzyme activity assays using PMA-stimulated
U-937 cells and the uPA-selective fluorogenic HMW-uPA substrate
were used to quantitate the effects of 1 and 2 on cell-surface-
bound HMW-uPA activity. i-uPA34 was included as a control.
Briefly, 5 ꢂ 104 PMA-stimulated U-937 cells were incubated with
varying concentrations of 1, 2 or i-uPA for 30 min before adding
HMW-uPA. Cells were further incubated for 30 min before being
washed twice by centrifugation with buffer, transferred to a fluor
plate containing fluorogenic substrate and fluorescence measure-
ments recorded. Changes in fluorescence per minute relative to
controls (no test compound added) were plotted as a function of
the concentration of added compounds (Fig. 3). The control com-
pound i-uPA showed potent inhibition of cell-bound HMW-uPA
activity exhibiting an IC50 value of 2.9 nM. In contrast, compounds
1 and 2 showed only very weak inhibitory effects with each show-
5. Experimental section
5.1. General
ing IC50 values greater than 100 lM.
4. Discussion and conclusions
Rink amide AM resin (200–400 mesh) was purchased from
Novabiochem. Peptide synthesis-grade CH2Cl2 and DMF were pur-
chased from Auspep. 4,40-dimethoxybenzhydrylamine was pur-
chased from TCI Japan and 30-aminobiphenyl-4-carboxylic acid
from Parkway Scientific. Propargylamine and b-naphthylamine
were purchased from Sigma Aldrich. RPMI 1640 powder and foetal
calf serum (FCS) used for cell culture experiments were purchased
from Trace Bioscientific (NSW, Australia). Phorbol 12-myristate
13-acetate (PMA) and protease free bovine serum albumin (BSA)
were purchased from Sigma-Aldrich (USA). Mouse anti-human
uPAR (# 3936) monoclonal antibody and Spectrozyme uPA were
obtained from American Diagnostica Inc. (CT, USA). HMW-uPA
fluorogenic substrate III (Z-Glu-Gly-Arg-AMCꢁHCl; Z = benzyloxy-
carbonyl, AMC = amino-4-methylcoumarin) was purchased from
Calbiochem, (USA). Alexa-uPA conjugate was constructed using
the Alexa FluorÒ 488 labelling kit purchased from Molecular
Probes Inc., USA. Incubation and wash buffers for all cell experi-
ments consisted of Dulbecco’s phosphate buffered saline (PBS,
1 mM CaCl2, MgCl2) and 0.1% BSA (pH 7.4). Previously described
radio-ligand binding assays reporting nanomolar potencies for 1
and 2 included 0.1% BSA in buffers.25–29 High Resolution Mass
Spectra were obtained using a Waters QTOF Ultima ESI Mass
Spectrometer and were within 0.4% of the theoretical values.
Compounds 1 and 2 were purified to greater than 95% purity using
In addition to the hydrophobic cavity used to engage uPA there
exists a second well characterised protein–protein interaction site
on uPAR which binds to the somatomedin B (SMB) domain of
matrix embedded vitronectin.36 The uPAR:vitronectin interaction
is implicated in cell migration processes such as, for example,
cytoskeletal rearrangements37 and remodelling of focal adhesion
sites.38 Importantly, occupancy of the hydrophobic binding site
on uPAR by uPA leads to a 5-fold increase in the affinity of uPAR
for vitronectin (KD ꢀ 1.9
lM?KD ꢀ 0.4
l
M),39,40 suggesting that
pre-formation of the uPA:uPAR complex may enhance uPAR:vitro-
nectin adhesion in vivo. It would therefore be of considerable inter-
est to examine the effects of small molecule uPA:uPAR antagonists
on the uPAR:vitronectin interaction. Compounds 1 and 2 were re-
ported in the patent literature as highly potent uPAR receptor
antagonists and seemingly represented excellent pharmacological
tools for such investigations.
It was noted that the potencies of 1 and 2 had only previously
been measured using radioligand binding assays employing la-
belled uPAR-binding uPA fragments as the competing ligands in
place of uPAR’s native ligand HMW-uPA.25,26 As a precautionary
step before proceeding to further pharmacological investigations
with 1 and 2 we sought to confirm that both compounds retained
their high potencies in physiologically relevant cell-based compe-
tition assays with the native ligand HMW-uPA. A semi-quantitative
competition flow cytometry assay using uPAR-expressing U-937
cells and a quantitative U-937 cell-based fluorescence assay were
used for this purpose. Access to 1 and 2 was afforded through
two simple new syntheses.
a 19 ꢂ 150 mm 5
lM Sunfire™ PREP C18 OBD™ column on a
Waters LC150 Preparative HPLC System run at 20 mL/min with
UV detection at 254 nm. 1 and 2 were analysed for purity using a
4.6 ꢂ 250 mm 5
lM Phenomenex Luna C18 column on a Shimadzu
CLASS-LC10 VP HPLC system run at 1 mL/min with UV detection at
254 nm. Manual solid-phase peptide synthesis was performed in a
Peptides International water-jacketed reaction vessel using a St.
John Associates 180° variable rate shaker. 1H and 13C NMR spectra
Compound 1 was found to show no antagonist effects at
100 lM in the flow cytometry assay and only weak inhibition of

2554
M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
were recorded on a Varian Premium Shielded 500 MHz NMR spec-
trometer at 25 °C. Chemical shifts are reported as d (ppm) relative
to TMS (0 ppm). The abbreviations s (singlet), d (doublet), t (trip-
let), q (quadruplet), m (multiplet) and br s (broad singlet) are used
throughout.
concentration in vacuo. The residue was taken up into CH2Cl2
(15 mL) and washed with 0.1 M HCl (2 ꢂ 10 mL), saturated NaH-
CO3 (2 ꢂ 10 mL) and brine (2 ꢂ 10 mL). The organic layer was con-
centrated and the crude product purified by column
chromatography using a 1:1 mixture of petroleum spirit (40–
60 °C) and EtOAc to yield 6 (181 mg, 65%) as a white solid: mp
168–170 °C; 1H NMR (500 MHz, CDCl3) d 8.59 (s, 1H), 8.13 (br s,
1H), 7.70 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.41 (d,
J = 7.5 Hz, 2H), 7.38 (s, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.29 (m, 2H),
7.27 (m, 1H), 7.24 (t, J = 4.0 Hz, 1H), 7.21 (m, 1H), 7.19 (d,
J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.17 (m, 1H), 7.09 (t,
J = 7.5 Hz, 1H), 7.01 (s, 1H), 5.46 (s, 1H), 5.10 (dd, J = 6.0, 13.5 Hz,
1H), 4.70 (br s, 1H), 3.76 (s, 3H), 3.33 (br s, 1H), 3.30 (dd, J = 6.0,
13.5 Hz, 1H), 3.23 (br s, 1H), 3.23 (dd, J = 6.0, 13.5 Hz, 1H), 1.44
(s, 9H); 13C NMR (125 MHz, CDCl3) d 172.6, 170.5, 166.6, 155.9,
143.6, 140.4, 137.8, 136.3, 136.1, 132.2, 130.5, 129.2, 128.6,
127.4, 127.2, 127.1, 127.0, 123.4, 123.0, 122.3, 119.9, 119.8,
118.7, 118.7, 111.4, 110.3, 80.4, 55.7, 53.8, 52.5, 37.7, 28.7, 28.3;
5.2. 30-[[(1,1-Dimethylethoxy)carbonyl]amino][1,10-biphenyl]-
4-carboxylic acid (4)
30-Aminobiphenyl-4-carboxylic acid 3 (1.7 g, 6.8 mmol) was
covered with dry MeOH (6.8 mL) and stirred. A solution of I2
(170 mg, 0.68 mmol) and di-tert-butyl dicarbonate (1.48 g,
6.8 mmol) was formed in a separate vessel using a minimal volume
of dry MeOH and added to the stirred mixture. DIPEA (1.186 mL,
6.8 mmol) was added dropwise to the stirred mixture, producing
a solution that was stirred for 5 h and then concentrated. The res-
idue was diluted in EtOAc (80 mL) and washed with a 1 M HCl
solution (40 mL) and saturated Na2S2O3 solution (3 ꢂ 40 mL). The
washes were repeated, and all aqueous layers were combined
and extracted with EtOAc (3 ꢂ 30 mL). After separation the com-
bined organic layers were dried over MgSO4 and concentrated,
yielding the N-Boc amino acid 4 (2.471 g, 99%) as an off-white so-
lid: mp 210–212 °C (dec); 1H NMR (CD3OD) d 8.09 (d, J = 8.5 Hz,
2H), 7.77 (t, J = 1.5 Hz, 1H), 7.71 (d, J = 8.5 Hz, 2H), 7.43 (dt,
J = 1.5, 8.0 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.32 (dt, J = 1.5, 8.0 Hz,
1H), 1.54 (s, 9H); 13C NMR (CD3OD) d 175.4, 155.4, 144.1, 142.6,
141.0, 137.9, 130.8, 130.2, 127.3, 122.4, 119.0, 118.4, 81.0, 28.7;
HRMS (ESI) for C18H18NO4: Expected 312.1236 [MꢃH]. Found
312.1242.
[
a
]
D = ꢃ12.7 (c 3.0, MeOH); HPLC (254 nm) tr 29.0 min; HRMS
(ESI) for C39H41N4O6: Expected 661.3041 [M+H]. Found 661.3026.
L
5.5. 30-(N-Acetyl- -tryptophyl-amino)(1,10-biphenyl)-4-carbonyl-
L-phenylalanine (1)
Compound
6
(88 mg, 0.13 mmol) and p-cresol (30 mg,
0.28 mmol) were stirred with neat TFA (2 mL) under N2 for 1 h.
The TFA was removed under a stream of N2 and the residue was
triturated with diethyl ether (3 ꢂ 5 mL). The TFA salt was then dis-
solved in DMF (5 mL) and to the stirring solution was added acetic
anhydride (26 lL, 0.28 mmol) and DIPEA (70 lL, 0.4 mmol). The
5.3. 30-[[(1,1-Dimethylethoxy)carbonyl]amino]-[1,10-biphenyl]-
4-carbonyl-L-phenylalanine methyl ester (5)
reaction was stirred at rt under N2 for 30 min before being
quenched by the addition of saturated NaHCO3 solution (40 mL).
Stirring was continued for a further 30 min before the resulting
suspension was extracted with EtOAc (4 ꢂ 20 mL) and the com-
bined organic layers washed with 0.1 M HCl (3 ꢂ 20 mL) before
drying over MgSO4. The solution was filtered and concentrated in
vacuo to yield a light brown solid. The solid (76 mg) was sus-
pended in H2O (1 mL) and stirred while THF was added dropwise
until a solution was obtained. LiOHꢁH2O (32 mg, 0.76 mmol) was
then added and the solution stirred for 4 h at rt. 2 M HCl (10 mL)
was added and the acidic suspension extracted with EtOAc
(3 ꢂ 10 mL). The combined organic extracts were dried over
MgSO4, filtered, concentrated in vacuo and the residue taken up
in DMSO (2 mL) and purified by preparative rp-HPLC using a gradi-
ent from 100% solvent A (100% H2O, 0.1% TFA) to 10% solvent A:90%
N-Boc amino acid 4 (2.18 g, 6.96 mmol) was stirred in dry DMF
(80 mL) along with HBTU (2.64 g, 6.96 mmol) and DIPEA (4.85 mL,
27.84 mmol). To the stirred solution was added L-phenylalanine
methyl ester hydrochloride (3.0 g, 13.91 mmol). After stirring un-
der N2 for 24 h the solution was concentrated before diluting with
CH2Cl2 (50 mL). The organic layer was washed with saturated NaH-
CO3 solution (2 ꢂ 40 mL) and brine (2 ꢂ 40 mL) and dried over
MgSO4. The crude product was purified by silica gel column chro-
matography using a 3:1 mixture of petroleum spirit (40–60 °C) and
EtOAc to yield 5 (2.914 g, 88%) as pale yellow crystals: mp 104–
106 °C; 1H NMR (CDCl3) d 7.75 (d, J = 8.0 Hz, 2H), 7.69 (s, 1H),
7.58 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.33 (t, J = 8.0 Hz,
1H), 7.28 (d, J = 7.5 Hz, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.23 (d,
J = 8.0 Hz, 1H), 7.15 (d, J = 7.5 Hz, 2H), 6.99 (s, 1H), 6.75 (d,
J = 7.5 Hz, 1H), 5.11 (dd, J = 5.5, 14.0 Hz, 1H), 3.75 (s, 3H), 3.30
(dd, J = 5.5, 14.0 Hz, 1H), 3.22 (dd, J = 5.5, 14.0 Hz, 1H), 1.52 (s,
1H); 13C NMR (CDCl3) d 172.0, 166.6, 152.8, 144.1, 140.4, 139.1,
135.8, 132.2, 129.2, 129.1, 128.4, 127.3, 127.0, 126.9, 121.4,
solvent
B (90% CH3CN:10% H2O, 0.1% TFA) over 30 min
(tr = 14 min). Pure fractions were combined and lyophilized to
yield 1 (71 mg, 95%) as a light brown solid: mp 186–188 °C; 1H
NMR (500 MHz, (CD3)2SO) d 10.83 (s, 1H, biphenyl NH), 10.24 (s,
1H, Trp-NH), 8.77 (d, J = 7.5 Hz, 1H, Phe-NH), 8.25 (d, J = 7.5 Hz,
1H, Trp-NH), 7.93 (s, 1H, Ar-H Ring A), 7.91 (d, J = 8.5 Hz, 2H,
2 ꢂ Ar-H Ring B), 7.68 (d, J = 8.5 Hz, 2H, 2 ꢂ Ar-H Ring B), 7.65
(m, 1H, ArH Trp), 7.64 (m, 1H, Ar-H Ring A), 7.41 (m, 2H, 2 ꢂ Ar-
118.0, 117.1, 80.2, 53.5, 52.2, 37.6, 28.1; [
a
]
D
= -24.5 (c. 1.0,
MeOH); HPLC (254 nm) tr 29.5 min; HRMS (ESI) for C28H31N2O5:
Expected 475.2233 [M+H]. Found 475.2240.
H
Ring A), 7.33 (m, 3H, Ar-H Trp, 2 ꢂ Ar-H Phe), 7.28 (t,
J = 7.0 Hz, 2H, 2 ꢂ Ar-H Phe), 7.19 (m, 2H, Ar-H Phe and Ar-H
5.4. 30-[N-[(1,1-Dimethylethoxy)carbonyl]-
[1,10-biphenyl]-4-carbonyl-
-phenylalanine methyl ester (6)
L-tryptophylamino]-
Trp), 7.05 (t, J = 7.5 Hz, 1H, Ar-H Trp), 6.97 (t, J = 7.5 Hz, 1H, Ar-H
L
Trp), 4.72 (dd, J = 14.0, 7.5 Hz, 1H, Phe-H ), 4.65 (br s, 1H, Trp-
a
H ), 3.21 (dd, J = 8.5, 13.0 Hz, 1H, Phe-Hb), 3.19 (dd, J = 5.5,
a
Compound
5
(200 mg, 0.42 mmol) and p-cresol (91 mg,
14.5 Hz, 1H, Trp-Hb), 3.08 (dd, J = 11.0, 13.0 Hz, 1H, Phe-Hb), 3.02
(dd, J = 8.5, 14.5 Hz, 1H, Trp-Hb), 1.84 (s, 3H, Ac-CH3); 13C NMR
(125 MHz, (CD3)2SO) d 173.1, 170.9, 169.2, 165.9, 142.8, 139.6,
139.5, 138.2, 136.0, 132.8, 129.3, 129.0, 128.2, 128.0, 127.2,
126.4, 126.3, 123.6, 121.9, 120.8, 119.1, 118.5, 118.2, 117.8,
0.84 mmol) were stirred with neat TFA (2 mL) under N2 for 1 h.
The TFA was removed under a stream of N2 and the remaining res-
idue was triturated with diethyl ether (3 ꢂ 5 mL). The TFA salt was
then dissolved in DMF (10 mL) and to the stirring solution was
added a solution of HBTU (175 mg, 0.46 mmol), Boc-
(320 mg, 1.05 mmol) and DIPEA (366 L, 2.1 mmol) in a minimal
volume of DMF. The solution was stirred for 24 h at rt before
L
-Trp-OH
111.2, 109.9, 54.3, 54.2, 36.2, 27.9, 22.5; [
MeOH); HPLC (254 nm) tr 24.2 min, 100%; HRMS (ESI) for
35H33N4O5: Expected 589.2451 [M+H]. Found 589.2467.
a
]
D = ꢃ14.1 (c. 1.0,
l
C

M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
2555
5.6. 2-[N-[2-Amino-2-oxoethyl]-2-[N-(naphthalen-2-yl)-2-(prop-
(94 mg, 386 lmol) were dissolved in DMF (1 mL) and stirred under
2-ynylamino)acetamido]-acetamido]acetic acid trifluoroacetate
(7)
N2 for 24 h. The solvent was removed in vacuo and the residue ta-
ken up into DMSO (2 mL) and purified by preparative rp-HPLC
using a gradient from 100% solvent A (100% H2O, 0.1% concd HCl)
to 10% solvent A:90% solvent B (90% CH3CN:10% H2O, 0.1% concd
HCl) over 30 min (tr = 15 min). Pure fractions were combined and
lyophilized to yield 2 (32 mg, 28%) as a light brown solid: mp
>300 °C (dec); 1H NMR (500 MHz, (CD3)2SO) d 9.65 and 9.10
(rotomer, d, J = 8.0 Hz, 1H, NH); 8.16 (s, 1H, NH2), 8.02 (m, 1H,
Ar-Naphthyl), 8.01 (m, 1H, Ar-H Naphthyl), 7.98 (m, 2H, 2 ꢂ Ar-H
Naphthyl), 7.91 (m, 1H, Ar-H Naphthyl), 7.70 (s, 1H, NH2þ), 7.59
(m, 1H, Ar-H Naphthyl), 7.54 and 7.50 (rotomer, d, J = 8.5 Hz, Ar-
H Naphthyl), 7.37 (s, 1H, NH2), 7.20 (s, 1H, NH2þ), 7.13 and 7.11
(rotomer, d, J = 8.5 Hz, 4H, 2 ꢂ Ar-H), 6.81 and 6.77 (rotomer, d,
J = 8.5 Hz, 4H, 2 ꢂ Ar-H), 5.95 (m, 1H, Benzhydryl-H), 4.60 and
4.59 (rotomer, s, 2H, Gly2-CH2), 4.21 and 4.03 (rotomer, s, 2H,
Gly4-CH2), 4.11 and 3.90 (rotomer, s, 2H, Gly3-CH2), 3.80 (s, 2H,
Propargyl-CH2), 3.77 (s, 2H, Gly1-CH2), 3.69 and 3.66 (rotomer, s,
6H, OCH3), 3.58 (s, 1H, Alkyne-H); 13C NMR (125 MHz, (CD3)2SO)
d 171.0 and 170.9 (rotomer), 168.1 and 168.0 (rotomer), 167.9
and 167.6 (rotomer), 165.0, 158.1, 137.7, 134.8 and 134.5 (rotom-
er), 133.0, 132.5, 129.8 and 129.8 (rotomer), 128.3 and 128.2
(rotomer), 128.2, 127.8, 127.2, 127.0, 126.9 and 126.8 (rotomer),
125.6 and 125.5 (rotomer), 113.7, 79.6, 74.7, 55.2 and 54.9 (rotom-
er), 55.1 and 55.1 (rotomer), 52.1 and 52.0 (rotomer), 51.9 and 51.5
(rotomer), 51.0 and 51.0 (rotomer), 46.6, 35.5; HPLC (254 nm) tr
24.7 min, 100%; HRMS (ESI) for C36H38N5O6: Expected 636.2822
M+. Found 636.2853.
Rink amide AM resin (2 g, 0.63 mmol/g) was shaken in a 45 mL
solid-phase peptide synthesis vessel with 20% piperidine in CH2Cl2
(10 mL) for 20 min before draining and washing with CH2Cl2
(3 ꢂ 15 mL) and DMF (2 ꢂ 15 mL). The resin was reswollen with
CH2Cl2 (15 mL). Bromoacetic acid (700 mg, 5 mmol) was combined
with DCC (1.032 g, 5 mmol) in NMP (5 mL) and CH2Cl2 (10 mL).
DMAP (36.6 mg, 0.3 mmol) was added to the drained resin fol-
lowed by the bromoacetic acid/DCC solution. The mixture was sha-
ken for 1 h at rt before draining and washing with CH2Cl2
(3 ꢂ 15 mL), DMF (2 ꢂ 15 mL) and isopropanol (2 ꢂ 15 mL). The re-
sin was then dried in vacuo. The dry resin was reswollen in DMSO
(24 mL) and drained before
a
solution of H2N-Gly-OtBuꢁHCl
(832 mg, 4.96 mmol) and DIPEA (1.728 mL, 9.92 mmol) in DMSO
(24 mL) was added. The mixture was shaken at 45 °C for 4 h before
draining and washing with DMF (6 ꢂ 36 mL) and CH2Cl2
(6 ꢂ 36 mL). The resin was reswollen in CH2Cl2 (36 mL). Bromoace-
tic acid (700 mg, 5 mmol) was combined with DCC (1.032 g,
5 mmol) in NMP (5 mL) and CH2Cl2 (10 mL). DMAP (36.6 mg,
0.3 mmol) was added to the drained resin followed by the bromo-
acetic acid solution. The mixture was shaken for 1 h at rt before
draining and washing with CH2Cl2 (6 ꢂ 36 mL) and DMF
(6 ꢂ 36 mL). The resin was reswollen with DMSO (24 mL) and
drained before a solution of b-naphthylamine (716 mg, 5 mmol)
in DMSO (30 mL) was added. The mixture was shaken at 45 °C
for 4 h before draining and washing with DMF (6 ꢂ 36 mL) and
CH2Cl2 (6 ꢂ 36 mL). The resin was reswollen with CH2Cl2
(36 mL). Bromoacetic acid (700 mg, 5 mmol) was combined with
DCC (1.032 g, 5 mmol) in NMP (5 mL) and CH2Cl2 (10 mL). DMAP
(36.6 mg, 0.3 mmol) was added to the drained resin followed by
the bromoacetic acid solution. The mixture was shaken for 1 h at
rt before draining and washing with CH2Cl2 (6 ꢂ 36 mL) and DMF
(6 ꢂ 36 mL). The resin was reswollen with DMSO (24 mL) and
drained before a solution of propargylamine (272 mg, 4.96 mmol)
in DMSO (30 mL) was added. The mixture was shaken at 45 °C
for 4 h before draining and washing with DMF (6 ꢂ 36 mL) and
CH2Cl2 (6 ꢂ 36 mL). The resin was then dried in vacuo before treat-
ing with a mixture of 95% TFA:2.5% water:2.5% TIPS (36 mL) and
cleaved for 1 h at rt. After filtering into a collecting vessel the fil-
trate was concentrated in vacuo and the residue taken up in DMSO
(2 mL) and purified by preparative rp-HPLC using a gradient from
100% solvent A (100% H2O, 0.1% TFA) to 10% solvent A:90% solvent
B (90% CH3CN:10% H2O, 0.1% TFA) over 30 min (tr = 9 min). Pure
fractions were combined and lyophilized to yield 7 (112 mg, 17%)
5.8. Cell culture
Human monocyte-like U-937 cells were used for all assays
(American Tissue Culture Centre, USA). Cells were maintained in
culture at 37 °C in a humidified incubator containing 5% CO2 (Ther-
mo Scientific) using pre-warmed RPMI 1640 media supplemented
with 2 mM L-glutamine and 5% foetal calf serum (FCS, heat inacti-
vated). Cells were passaged every 3–4 days to maintain a popula-
tion >1 ꢂ 106 cells mLꢃ1
.
5.9. PMA treatment
Cells were diluted in RPMI 1640 + 5% FCS to 2 ꢂ 105 cells mLꢃ1
and cultured for 7–8 h at 37 °C. Solutions of PMA in DMSO were
added to the cells to provide final PMA concentrations of 100 nM
(final DMSO concentrations <0.01%). Cells were then incubated
for a further 16 h to induce optimal uPAR expression.43
as
a
white powder: mp >300 °C (dec); 1H NMR (500 MHz,
5.10. Preparation of i-uPA
(CD3)2SO) d 9.60 (br s, 1H), 8.05 (s, 1H), 8.03 (m, 2H), 8.01 (m,
2H), 7.96 (m, 1H), 7.73 (s, 1H), 7.59 (t, J = 4.5 Hz, 1H), 7.55 and
7.53 (rotomer, d, J = 1.5 Hz, 1H), 7.34 (s, 1H), 7.235 (s, 1H), 4.64
and 4.63 (rotomer, s, 2H), 4.19 and 3.98 (rotomer, s, 2H), 4.05
and 3.89 (rotomer, s, 2H), 3.80 (s, 2H), 3.78 (s, 2H), 3.57 (s, 1H);
13C NMR (125 MHz, (CD3)2SO) d 170.8 and 170.8 (rotomer), 170.5
and 170.1 (rotomer), 168.0 and 167.8 (rotomer), 165.0, 137.7,
133.0, 132.5, 129.8, 128.1, 127.7, 127.2, 127.0, 126.9, 125.6, 79.5,
74.7, 50.7, 50.2 and 49.8 (rotomer), 49.4, 46.6, 35.4; HPLC
(254 nm) tr 18.5 min, 100%; HRMS (ESI) for C21H23N4O5: Expected
411.1668 M+. Found 411.1605.
One hundred microlitre of HMW-uPA (1 mg mLꢃ1, 19
tilled water) was reacted with Glu-Gly-Arg-chloromethyl ketone
100
L (1 mM in distilled water) for 24 h at 4 °C to form i-uPA.34
Absence of activity was confirmed by treating i-uPA (5 nM) with
Spectrozyme uPA chromogenic substrate (0.125 mM in distilled
water) and monitoring (Spectramax Plus 384: Molecular Devices
plate reader) for absence of colour development at 405 nM.
lM in dis-
l
5.11. Flow cytometry assays
U-937 cells in log phase growth were re-suspended in cold PBS/
BSA at 1 ꢂ 106 cells mLꢃ1 and pre-incubated with varying concen-
trations of antagonists. Compounds 1 and 2 were dissolved in
DMSO and diluted with PBS to varying concentrations such that fi-
nal addition of diluted compounds to cells yielded DMSO concen-
trations of 2% in each sample. Visible inspection of assay wells
showed no evidence of compound precipitation and cell viability
5.7. N-[2-Amino-2-oxoethyl]-N-[2-[bis(4-methoxyphenyl)methyl-
amino]-2-oxoethyl]-2-[N-[naphthalen-2-yl]-2-[prop-2-ynylami-
no]acetamido]acetamide hydrochloride (2)
Compound 7 (88 mg, 168
lmol), HBTU (82 mg, 216 lmol),
DIPEA (137 L, 786
l
l
mol) and 4,40-dimethoxybenzhydrylamine,

2556
M. D. Souza et al. / Bioorg. Med. Chem. 19 (2011) 2549–2556
3. Nekarda, H.; Schlegel, P.; Schmitt, M.; Stark, M.; Mueller, J. D.; Fink, U.; Siewert,
J. R. Clin. Cancer Res. 1998, 4, 1755.
4. Kuhn, W.; Schmalfeldt, B.; Reuning, U.; Pache, L.; Berger, U.; Ulm, K.; Harbeck,
N.; Späthe, K.; Dettmar, P.; Höfler, H.; Jänicke, F.; Schmitt, M.; Graeff, H. Br. J.
Cancer 1999, 79, 1746.
was unaffected by this concentration of DMSO. Control samples
containing 2% DMSO without antagonists were included in all as-
says. Controls for the i-uPA and MAb #3936 assays included an
appropriate dilution of vehicle (PBS). All samples were incubated
on ice for 30 min before adding 40 nM Alexa-HMW-uPA. After
30 min incubations with Alexa-HMW-uPA on ice, cells were
washed twice in ice-cold PBS/BSA by centrifugation (300 ꢂ g,
5 min 4 °C) before final re-suspending in ice cold PBS containing
5. Duffy, M. J. Clin. Chem. 2002, 48, 1194.
6. Look, M. P.; van Putten, W. L. J.; Duffy, M. J.; Harbeck, N.; Christensen, I. J.;
Thomssen, C.; Kates, R.; Spyratos, F.; Ferno, M.; Eppenberger-Castori, S.; Sweep,
C. G. J. F.; Ulm, K.; Peyrat, J.-P.; Martin, P.-M.; Magdelenat, H.; Brunner, N.;
Duggan, C.; Lisboa, B. W.; Bendahl, P.-O.; Quillien, V.; Daver, A.; Ricolleau, G.;
Meijer-van Gelder, M. E.; Manders, P.; Fiets, W. E.; Blankenstein, M. A.; Broet,
P.; Romain, S.; Daxenbichler, G.; Windbichler, G.; Cufer, T.; Borstnar, S.; Kueng,
W.; Beex, L. V. A. M.; Klijn, J. G. M.; O’Higgins, N.; Eppenberger, U.; Janicke, F.;
Schmitt, M.; Foekens, J. A. J. Natl. Cancer Inst. 2002, 94, 116.
7. Rømer, J.; Nielsen, B. S.; Ploug, M. Curr. Pharm. Des. 2004, 10, 2359.
8. Nozaki, S.; Endo, Y.; Nakahara, H.; Yoshizawa, K.; Ohara, T.; Yamamoto, E. Anti
Cancer Drugs 2006, 17, 1109.
5 lg/ml propidium iodide. Viable (i.e., propidium iodide negative)
cells in the samples were analysed by dual colour flow cytometry
as described in Ranson et al.44
5.12. Fluorometric HMW-uPA activity assays
9. Tyndall, J. D. A.; Kelso, M. J.; Clingan, P.; Ranson, M. Recent Pat. Anti Cancer 2008,
3, 1.
Cell surface-bound HMW-uPA activity was measured using the
fluorogenic substrate Z-Gly-Gly-Arg-AMC. Fluorescence observed
in this assay is directly proportional to cell-bound HMW-uPA activ-
ity due to the high specificity of the substrate for HMW-uPA.33 The
excitation wavelength range of the substrate is 365–380 nm and
the emission wavelength range is 430–460 nm. PMA stimulated
cells were prepared as described above and incubated with test
compounds for 30 min at 4 °C, after which HMW-uPA (40 nM)
was added and the cells incubated for a further 30 min at 4 °C. Cells
were then washed twice by centrifugation with PBS at room
temperature, transferred to a fluor plate and overlayed with an
equivalent volume of buffer containing 1 mM Z-Gly-Gly-Arg-AMC
to give a final concentration of 0.5 mM of the fluorogenic substrate.
Fluorescence emission was measured immediately using an
Fluorostar Optima instrument at 37 °C (BMG Labtech, Offenburg,
Germany). Data was recorded at 30 sec intervals over a period of
40–50 min. A sample to indicate background fluorescence contain-
ing cells, fluorogenic substrate and buffer was included in assay
plates. A control sample with no antagonist added included both
HMW-uPA and fluorogenic substrate to designate a fluorescence
value at full receptor occupancy in the presence of fluorogenic sub-
strate. The background fluorescence was subtracted from each
reading before statistical analysis. Calculation of the rate of change
in fluorescence minꢃ1 allowed quantitative interpretation of fluo-
rescence data and was generated using the linear region of a graph
where fluorescence was plotted against time.
10. Ploug, M. Curr. Pharm. Des. 2003, 9, 1499.
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Acknowledgements
This work was partly funded by a University of Wollongong
URC Small Grant awarded to M.R. and M.J.K. Australian Post-
graduate Awards to H. Matthews and J. Lee are gratefully
acknowledged.
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