Small-molecule inhibition of the uPAR·uPA interaction: Synthesis, biochemical, cellular, in vivo pharmacokinetics and efficacy studies in breast cancer metastasis

Article abstract of DOI:10.1016/j.bmc.2012.12.047

The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration- dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5 h and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10 h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.

Full text of DOI:10.1016/j.bmc.2012.12.047

Bioorganic & Medicinal Chemistry 21 (2013) 2145–2155
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small-molecule inhibition of the uPARꢀuPA interaction: Synthesis,
biochemical, cellular, in vivo pharmacokinetics and efficacy studies
in breast cancer metastasis
Timmy Mani a, Fang Wang a, William Eric Knabe a, Anthony L. Sinn c,d, May Khanna a, Inha Jo a,
George E. Sandusky i,j, George W. Sledge Jr. b,c,e, David R. Jones b, Rajesh Khanna h, Karen E. Pollok c,d
,
Samy O. Meroueh a,e,f,g,h,
⇑
a Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Van Nuys Medical Science Building, MS 4023, 635 Barnhill Drive, Indianapolis,
IN 46202-5122, USA
b Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
c Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA
d In vivo Therapeutics Core, Indiana University School of Medicine, Indianapolis, IN 46202, USA
e Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA
f Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, Indianapolis, IN 46202, USA
g Department of Chemistry and Chemical Biology (IUPUI), Indiana University School of Medicine, Indianapolis, IN 46202, USA
h Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, USA
i Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA
j Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The uPARꢀuPA protein–protein interaction (PPI) is involved in signaling and proteolytic events that pro-
mote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational
screening of a commercial chemical library and shown that the compound inhibited uPARꢀuPA PPI in
competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo
pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show,
using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to
Received 16 November 2012
Revised 13 December 2012
Accepted 20 December 2012
Available online 9 January 2013
Keywords:
uPAR with sub-micromolar affinity of 0.2 lM. We show that 4 blocks invasion of breast MDA-MB-231,
Protein–protein interaction inhibitors
Urokinase receptor
Metastasis
Breast cancer
Cancer
and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives
of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound
4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID
mice revealed a half-life of nearly 5 h and peak concentration of 5 lM. Similar levels of the inhibitor were
Virtual screening
Synthesis
In vivo
detected in tumor tissue up to 10 h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-
231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the trea-
ted mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a
promising template for the development of compounds with enhanced PK parameters and greater
efficacy.
Ó 2013 Elsevier Ltd. All rights reserved.
Abbreviations: uPAR, urokinase receptor; uPA, urokinase-type plasminogen activator; suPAR, soluble urokinase receptor; uPAATF, amino terminal fragment of uPA; MMP,
matrix metalloproteinase; FP, fluorescence polarization; NOD/SCID, non-obese diabetic severe combined immunodeficient; PK, pharmacokinetic; MDA-MB-231, MD
Anderson metastatic breast; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle Medium; SDS, sodium dodecyl sulfate; CBB, coomassie brilliant blue; BSA, bovine
serum albumin; DEAE, diethylaminoethyl cellulose; FITC, fluorescein isothiocyanate; TBST, tris-buffered saline and tween 20; IB, immunoblot; NMR, nuclear magnetic
resonance; DCM, dichloromethane; HRMS, high-resolution mass spectrometry; THF, tetrahydrofuran; PDB, protein databank; DMAP, 4-dimethylaminopyridine; DCC, N,N0-
dicyclohexylcarbodiimide; FAM, 6-carboxyfluorescein; HPLC, high-performance liquid chromatography; GPI, glycosylphosphatidylinositol; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; TMD-MDA-MB-231, tumor-derived MDA-MB-231; AUC, area under the curve; AUMC, area under the moment curve; LARC, laboratory
animal resources center; IACUC, institutional animal care and use committee; HPLC, high-performance liquid chromatography; PPI, protein–protein interaction; FN,
fibronectin.
⇑
Corresponding author.
E-mail address: smeroueh@iupui.edu (S.O. Meroueh).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.12.047

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T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
composed of a repeated 50 ms gauss shaped pulse and 0.1 ms in-
1. Introduction
ter-pulse delay. The STD experiment of the small-molecule with
protein was acquired with 4096 scans. The same experiment was
collected on small-molecule alone using exactly the same acquisi-
tion parameters except with 1024 scans. To ensure that we only
observed magnetization from the small-molecule, the protein
(uPAR) was added at a concentration of 50–100 fold less than the
small-molecule. Compound 4 was dissolved in deuterated DMSO
at a concentration of 50 mM; it was then diluted in phosphate
buffer at a concentration of 0.7 mM for the NMR experiment. The
compound was not fully soluble at 0.7 mM and thus the ratio of
small-molecule to protein may not be exactly 50 fold. NMR experi-
ments were acquired at 298 K on a VNMRS 800 MHz NMR spectrom-
eter operating at a magnetic field strength of 18.8 T and equipped
with a cold probe (Agilent Technologies, Santa Clara CA). Shigemi
NMR tubes were used for the NMR experiments. Water suppression
was done using Varian water sculpting pulse sequence.
Cancer is a set of over 100 diseases that share a number of char-
acteristics. Hanahan and Weinberg proposed several essential
events as hallmarks of cancer,1 including the ability of malignant
cells to invade tissue and spread to distal sites (metastasis). Studies
have shown that protein interactions of the urokinase receptor
(uPAR) are involved in the complex processes that promote inva-
sion and metastasis. uPAR has been implicated in nearly every step
of cancer metastasis including cell migration,2,3 adhesion,4,5 angio-
genesis,6,7 and invasion.5,8–10 This has led to attempts to inhibit its
protein interactions. Most efforts to date have been confined to the
use of biologics consisting of either fusion proteins11–13 or pep-
tides.14–17 Recently, we have reported the first small organic mol-
ecules that inhibit the protein interaction of uPAR with its highest
affinity ligand, the urokinase-type plasminogen activator (uPA).18
11 (IPR-456) was identified using structure-based computational
screening.18 A derivative of 11, namely 4 (IPR-803), emerged from
a substructure search of commercial chemical libraries and showed
higher potency in blocking breast cancer invasion in cellular stud-
ies. A structure–activity study provided valuable insight into the
functional groups required to effectively inhibit the uPARꢀuPA
interaction.19 The effect of these compounds on lung cancer metas-
tasis in cell culture-based studies was investigated in the same
study. The results showed that 4 and other compounds impaired
invasion, migration and adhesion in a panel of non-small cell lung
cancer (NSCLC) cell lines.19
2.3. Cell culture
MDA-MB-231 and TMD-231 were cultured in Dulbecco’s Mod-
ified Eagle Medium (Cellgro, Manassas, VA) supplemented with
10% FBS and 1% penicillin/streptomycin in 5% CO2 atmosphere at
37 °C.
2.4. Reagents
Biotinylated anti-human uPAR antibody (BAF807) was pur-
chased from R&D Systems (Minneapolis, MN). Rabbit anti-human
uPA antibody 389 was purchased from American Diagnostica Inc.
(Stamford, CT).
In an effort to develop uPAR antagonists with in vivo efficacy,
we focus on breast cancer metastasis. We synthesize 4, for the first
time, and assess it in a breast-to-lung orthotopic model. Previously,
competition studies of 4 using surface plasmon resonance (SPR)
established that it inhibited uPAATF binding to uPAR.18 Here, we
probe the direct binding of the compound to uPAR by performing
binding studies using fluorescence polarization and saturation
transfer difference (STD) NMR. These studies afforded, for the first
time, an equilibrium constant for binding to uPAR. Prior to per-
forming in vivo studies, the effects of 4 on breast MDA-MB-231
invasion, migration and adhesion were explored. Extensive phar-
macokinetic (PK) studies were carried out in SCID mice to generate
PK parameters and measure the levels of the compound in plasma
and tumor tissue. Finally, NSG mice were implanted with TMD-
MDA-MB-231 (TMD-231) breast cancer cells in the mammary fat
pads and dosed with 4 on a daily basis to assess its effect on breast
cancer metastasis to the lungs.
2.5. Western blot analysis for MAPK signaling
Six well plates were coated with 30 ng l
Lꢂ1 of fibronectin at
4 °C overnight. 1.5 ꢁ 106 serum starved MDA-MB-231 cells were
plated onto each well in the presence of 1% DMSO (control) or
50 lM of 4 or IPR-69, a previously described positive control for
30 min as previously described.19 Total cell lysates were prepared
in standard RIPA extraction buffer containing protease and phos-
phatase inhibitors (Sigma). Thirty microgram of protein was sepa-
rated by 10% SDS–PAGE and transferred to nitrocellulose
membrane (Amersham, Arlington Heights, IL) and then blocked
for 1 h at room temperature in PBS-1% fish gelatin buffer. The
membrane was immunoprobed with phospho-p44/42 MAPK
(Thr202/Tyr204) rabbit monoclonal antibody (1:2000) and p44/
42 MAPK mouse monoclonal antibody (1:2000) at 4 °C overnight.
Then, the membrane was incubated with IRDye 800-conjugated
goat anti-mouse IgG (Rockland) and Alexa Fluor 680 goat anti-rab-
bit IgG (Invitrogen) as secondary antibodies (1:10,000). Bands were
detected using Li-Cor Odyssey Imaging System (Li-Cor, Lincoln,
NE).
2. Materials and methods
2.1. Fluorescence polarization
Varying concentrations of suPAR277 protein were titrated
against intrinsically fluorescent 4 at a final concentration of 1 lM
in 1 ꢁ PBS with 0.01% Triton X-100. The inhibitor-protein solution
was incubated for 15 min. at room temperature. Polarized fluores-
cence intensities were measured using EnVisionÒ Multilabel Plate
Readers (PerkinElmer, Waltham, MA) with excitation and emission
wavelengths of 531 and 595 nm, respectively.
2.6. Immunofluorescence imaging
MDA-MB-231 cells were grown on laminin- and poly-d-lysine-
coated glass coverslips, exposed to 100 lM of 4 or 100 lM of 11 for
30 min at 37 °C, washed with PBS, fixed with 4% paraformaldehyde
in PBS for 20 min, and permeabilized with 1% Triton X-100 in PBS
for 10 min. Non-specific binding was blocked with 5% normal goat
serum, 5% BSA, 0.01% Triton X-100 in PBS at RT for 1 h. The cells
were incubated overnight at 4 °C with rabbit polyclonal antihuman
uPA antibody 389 (dilution of 1:50 in 1% BSA/PBS). After 3-fold
washing with PBS, the cells were incubated with anti-rabbit IgG
conjugated with Alexa 594 Texas Red (Invitrogen Grand Island,
NY), at a dilution of 1:500 in 1% BSA/PBS at RT for 1 h in the dark.
2.2. STD NMR
To further characterize and confirm the binding of 4 to uPAR,
saturation transfer difference (STD) nuclear magnetic resonance
(NMR) was used.20 Selective pulses were applied at 0.8 ppm to
irradiate the protein methyl groups (where no ligand peaks ap-
pear) while the off-resonance frequency was positioned at
30 ppm. Saturation was carried out with a total 2 s pulse train

T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
2147
Images were acquired using
microscope.
a
Nikon swept-field confocal
concentrated by Amicon Ultra centrifugal filter units (Millipore,
#UFC 501024), and proteins were normalized and electrophoresed
on 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gels contain-
ing 1 mg/mL gelatin. After electrophoresis, the gel was washed
twice in 2.5% Triton X-100 for 30 min at room temperature and
incubated in buffer that contained 50 mM Tris–HCl (pH 7.6),
200 mM NaCl, 10 mM CaCl2, and 0.02% Brij 35 at 37 °C for 36 h.
Then, the gels were stained with 0.05% Coomassie brilliant blue
(CBB) and de-stained with 30% methanol in 10% acetic acid. Areas
of gelatinolytic degradation appeared as transparent bands on the
blue stained background of the gel. Data were quantified using Li-
Cor Odyssey Imaging System (Li-Cor, Lincoln, NE).
2.7. Invasion assay
Invasion transwell chambers were purchased from BD biosci-
ences (San Jose, CA).21 The undersurface of the inserts was coated
with 30 ng l
Lꢂ1 of fibronectin at 4 °C overnight. The filters were
equilibrated with 0.5 mL of serum-free medium for 2 h at 37 °C.
After 4 h of serum starvation, cells were harvested and 5 ꢁ 104
cells in 500 lL medium containing 0.1% FBS and the indicated com-
pounds or 1% DMSO control were plated onto the upper chamber
of a transwell filter. Five hundred microlitre of 10% FBS medium
containing the same amount of compounds or DMSO control was
added to the lower chamber. After a 16 h incubation at 37 °C in
5% CO2, the filters were fixed and the number of cells that had in-
vaded was determined as described below in the migration assay.
2.12. Cloning, expression and purification of uPAR
suPAR was obtained by a one-step purification process as previ-
ously described.24
2.8. Transwell migration assay
2.13. Western blot analysis
24-well transwell plates (Costar, Corning, NY) were coated with
Total cell lysates were prepared in standard RIPA extraction
buffer containing protease and phosphatase inhibitors (Sigma).
Thirty lg of protein was separated by 10% SDS–PAGE and trans-
30 ng l
Lꢂ1 of fibronectin at 4 °C overnight for migration assays as
described previously.18,19 After 4 h of serum starvation, 5 ꢁ 104
cells in 250
lL of 0.1% FBS medium containing the indicated com-
ferred to nitrocellulose membranes (Amersham, Arlington Heights,
IL). The membranes were immunoprobed with uPAR (10G7), uPA
(H-140) or Actin (C-2) antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA) at 4 °C overnight. Next, membranes were incubated with
IRDye 800-conjugated goat anti-mouse IgG (Rockland) and Alexa
Fluor 680 goat anti-rabbit IgG (Invitrogen) as secondary antibodies.
Bands were detected using Li-Cor Odyssey Imaging System (Li-Cor,
Lincoln, NE).
pounds or 1% DMSO control were added to the upper chamber and
incubated at 37 °C for 16 h. 500 L of 10% FBS medium containing
l
the same amount of compounds or 1% DMSO control was simulta-
neously added to the lower chamber. Non-migrated cells on the
top of the transwell were scrapped off with a cotton swab, and
the cells that migrated through the filter were fixed in methanol
for 30 min at room temperature and stained with Hematoxylin
Stain (Fisher Scientific) for 1 h at room temperature. The filters
were washed with water 3 times. Filters were air-dried, and the
number of migrated cells was counted in ten separate fields and
averaged across two independent experiments with each concen-
tration in triplicate.
2.14. Apoptosis assay
MDA-MB-231 cells were treated with indicated concentrations
of 4 or 1% DMSO control for 24 h and flow cytometry analysis
was performed as described previously.19
2.9. Adhesion assay
2.15. Tissue preparation
96-well plates were coated with 15 ng l
Lꢂ1 fibronectin (Sigma–
Aldrich, St. Louis, Missouri) at 4 °C overnight, then blocked with 2%
BSA in PBS for 1 h, as described previously.14,18,19,22,23 After starv-
ing with serum-free medium for 4 h, MDA-MB-231 cells
All lung tissues were collected and fixed in 10% neutral buffered
formalin within 30 min of removal during surgery. The tissues
were fixed overnight in neutral buffered formalin and then trans-
ferred to 70 percent ethanol prior to processing to a paraffin block.
The slides are then baked overnight at 59 °C in an oven before
staining with H&E.
(2.5 ꢁ 105 cells mlꢂ1) were trypsinized and suspended in 100
lL
of 0.1% FBS DMEM medium with various concentrations of com-
pounds or DMSO control at 37 °C for 90 min. Medium was then
carefully suctioned out from each well. Each well was washed
three times with PBS. The wells were washed, and the number of
adherent cells was quantified by MTT assay at 570 and 630 nm. Re-
sults are representative of three independent experiments with
each concentration in quadruplicate. Similar results were obtained
following harvesting the cells with trypsin or 2 mM EDTA for the
control compound IPR-69 (data not shown).
2.16. Slide evaluation
Three investigators examined the slides by light microscopy to
examine for metastatic carcinoma in the lungs. Each lung lobe was
analyzed for tumor cells and large tumor metastases. The number
of lung metastases were counted in each lobe, and the size of the
tumor foci were scored on a scale of 0 to 3+ (0, no staining; 1, mild:
tumor foci composed of cells from 2 to 25; 2, moderate: tumor foci
composed of cells from 25 to 150; 3, severe tumor foci composed of
cells from 150 to 600 cells, and 4, marked: large coalescing tumor
foci with 600 plus tumor cells). Each lobe was counted in its
entirety.
2.10. Cell viability assay
104 mL-1 MDA-MB-231 cells were plated overnight in 100
lL/
well of 96 well plates. Cells were treated with DMSO (control) or
compounds at different concentrations for 3 days. Viable cells were
quantified by MTT assay at absorbance of 570 with 630 nm for ref-
erence background as previously described.19
2.17. Synthesis
2.11. Gelatin zymography
All chemicals were purchased from commercially available
sources and used as received. Column chromatography was carried
MDA-MB-231 cells were treated with compounds in serum free
medium for 24 h. The conditioned medium was collected and
out with silica gel (25–63
l). High-Res Mass Spectra were mea-
sured on an Agilent 6520 Accurate Mass Q-TOF instrument. 1H

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T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
NMR was recorded in CDCl3 or DMSO on a Bruker 500 MHz spec-
trometer. RP-LCMS was carried out on a Agilent 1100 LC/MSD fit-
ted with an Eclipse XBD-C18 (4.6 ꢁ 150 mm) column eluting at
1.0 ml/min employing a gradient of (acetonitrile:methanol):water
(each containing 5 mM NH4OAc) from 70% to 100% acetoni-
trile:methanol over 15 min and holding at 100% acetonitrile:meth-
anol for 2 min. Chemical shifts are reported in ppm using either
residual CHCl3 or DMSO as internal references. Unless otherwise
stated, the following procedures were modified or adopted from
the literature.
J = 8 Hz, 1H-C6), 7.58–7.50 (m, 2H), 6.18 (s, 1H), 4.51–3.40 (vbr s,
4H), 3.94 (s, 3H-OMe), 1.91 (br s, 4H), 1.65 (br s, 4H). 13C NMR
(126 MHz, CDCl3) d 176.0, 166.3, 154.7, 153.6, 148.0, 146.7,
139.1, 133.5, 131.4, 130.6, 129.7, 127.9, 127.7, 126.2, 124.2,
121.8, 118.9, 96.2, 91.9, 52.3, 26.6, 18.9. HRMS Calcd for
C
28H25N3O4 (MꢂH)ꢂ 466.1775, found 466.1772.
2.21. 3-((3-(Azepan-1-yl)-6-oxo-6H-anthra[1,9-cd]isoxazol-5-
yl)amino)benzoic acid (4)
Compound 3 (433 mg, 0.93 mmol) was hydrolyzed in a metha-
nol (4 mL) and 2 M aq NaOH (2 mL) solution at 80 °C with stirring
overnight. The residual methanol was removed in vacuo, and the
resulting residue was acidified to pH 2. The precipitate was filtered
off and washed with cold water to give 4 (314 mg, 74%) as a red-
dish-brown solid. 1H NMR (500 MHz, DMSO) d 11.79 (s, 1H-NH),
8.44 (app d, J = 8 Hz, 1H-C5), 8.13 (app d, J = 8 Hz, 1H-C8), 8.01 (s,
1H), 7.84–7.78 (m, 2H), 7.72–7.67 (m, 2H), 7.64–7.59 (m, 1H),
6.12 (s, 1H), 4.51–3.97 (br s, 4H), 1.80 (br s, 4H), 1.55 (br s, 4H);
13C NMR (126 MHz, DMSO) d 174.7, 166.8, 153.6, 152.8, 147.6,
146.0, 138.5, 132.9, 132.4, 131.2, 130.2, 128.3, 127.5, 127.4,
126.0, 123.4, 121.8, 118.5, 99.5, 95.0, 91.7, 56.0, 26.0, 18.6. HRMS
Calcd for C27H22N3O4 (MꢂH)ꢂ 452.1610, found 452.1601.
2.18. 3,5-Dibromo-6H-anthra[1,9-cd]isoxazol-6-one (1)
Sodium nitrite (993 mg, 14.4 mmol) was added with stirring to
concd H2SO4 (25 mL) at 30–40 °C over 10 min then stirred for an
additional 30 min. Next 1-amino-2,4-dibromoanthraquinone
(5.0 g, 13.1 mmol) was added over 15 min and the mixture was
stirred overnight (16 h) at 50–55 °C. The heated solution was
poured directly over ice and the resulting yellow precipitate was
filtered, washed with cold water, and a 1:1 mixture of ethanol-
ether. The moist anthraquinonediazonium hydrogensulfate was
added to a solution of NaN3 (1.37 g, 21.0 mmol) in water (25 mL)
and stirred overnight (16 h). The light orange solid was filtered
off and washed with water followed by a 9:1 mixture of acetone-
water. The moist azide was suspended in toluene (40 mL) and
heated to 70 °C with stirring. Water and acetone were slowly dis-
tilled (using a Dean–Stark apparatus) over a 12 h period. The yel-
low-orange crystals were filtered and washed with methanol to
give 3.78 g (76%) of 3,5-dibromo-6H-anthra[1,9-cd]isoxazol-6-
one.25 1H NMR (500 MHz, CDCl3) d 8.44 (d, J = 7.8 Hz, 1H-C5),
8.07 (d, J = 7.4 Hz, 1H-C8), 7.97 (s, 1H), 7.80 (t, J = 7.0 Hz, 1H-C7),
7.71 (t, J = 7.4 Hz, 1H-C6); 13C NMR (126 MHz, CDCl3) d 179.9,
153.6, 141.8, 134.0, 131.9, 131.4, 130.2, 125.0, 123.5, 123.5,
3. Results
3.1. Synthesis of 4
Despite its availability in a commercial database, the synthesis
of 4 has not been reported. To enable cellular and in vivo studies
of the compound, we conceived a synthesis route that successfully
led to the preparation of the compound in gram-scale (Scheme 1).
It consisted of first preparing 3,5-dibromo-6H-anthra[1,9-cd]iso-
xazol-6-one by the diazotation of 1-amino-2,4-dibromoanthraqui-
none. This was accomplished by adding sodium nitrite to a solution
of the amine in concentrated sulfuric acid to give the 1-anthra-
quinonediazonium hydrogen sulfate, which was subsequently con-
verted to the 1-azidoanthraquinone by reacting with an aqueous
solution of sodium azide. The azidoanthraquinone was azeotropi-
cally refluxed in toluene to provide, by evolution of nitrogen, the
required isoxazole ring in 1.25 Arylamination of 1 at the 5-position
was accomplished with anhydrous AlCl3 in nitrobenzene at ambi-
ent temperature to give a moderate yield of the 6-arylamino
substituted.26 This occurs by an increase in electrophilicity at the
4-bromo position via conjugation with the keto group after its
complexation with AlCl3. A second amination was carried out at
the 3-position using refluxing acetonitrile to give a moderate yield
of 3.27 Next, hydrolysis of the benzoate ester 3 to the carboxylic
acid 4 occurred under conditions of heating an aqueous solution
of sodium hydroxide and methanol.
122.8, 121.6, 115.6. HRMS Calcd for
379.8742, found 379.8740.
C
14H5Br2NO2 (M+H)+
2.19. Methyl 3-((3-bromo-6-oxo-6H-anthra[1,9-cd]isoxazol-5-
yl)amino)benzoate (2)
To a solution of methyl 3-aminobenzoate (481 mg, 3.18 mmol)
in nitrobenzene (3 mL), anhydrous AlCl3 (353 mg, 2.65 mmol) was
added with vigorous stirring. After 5 min, 1 was added and the
reaction mixture stirred for 3 h at ambient temperature. The reac-
tion mixture was poured into an ice-water slurry that precipitated
a reddish solid which was filtered off. The reddish solid was recrys-
tallized from toluene to give 144 mg (61%) of 2.26 1H NMR
(500 MHz, CDCl3) d 11.40 (s, 1H-NH), 8.55 (d, J = 8 Hz, 1H-C5),
8.15 (d, J = 8 Hz, 1H-C8), 8.07–8.01 (m, 2H), 7.81 (t, J = 7.5 Hz,
1H-C7), 7.72-7.66 (m, 2H), 7.63-7.53 (m, 2H), 3.97 (s, 3H-OMe).
13C NMR (126 MHz, CDCl3) d 181.0, 166.0, 157.9, 151.2, 148.7,
137.5, 132.7, 132.5, 132.2, 130.2, 129.3, 128.7, 128.6, 128.0,
126.5, 125.4, 123.5, 122.5, 119.9, 117.1, 101.9, 52.5. HRMS Calcd
for C22H13Br N2O4 (M+H)+ 449.0135, found 449.0141.
3.2. Direct binding and inhibition studies
In a previous study, we had reported the use of SPR to show that
4 inhibited uPA binding to uPAR in a concentration-dependent
manner.18 Here, we sought to provide evidence that 4 inhibited
the protein interaction by direct binding to uPAR. We also use this
opportunity to determine a binding constant for the compound.
The red-shifted fluorescence of 4 was exploited to measure its di-
rect binding to uPAR using fluorescence polarization (FP).18 When
exposed to a larger binding partner, a small-molecule is expected
to adopt a slower tumbling rate leading to an increase in light
polarization. Increasing the concentration of uPAR in the presence
2.20. Methyl 3-((3-(azepan-1-yl)-6-oxo-6H-anthra[1,9-
cd]isoxazol-5-yl)amino)benzoate (3)
Hexamethyleneimine (62 lL, 0.55 mmol) and 2 (100 mg,
0.22 mmol) were dissolved in acetonitrile (4 mL). The reaction
solution was heated to 70–80 °C. As determined by TLC the reac-
tion was complete after 3 h afterwards the reaction mixture was
cooled to ambient temperature then to ꢂ15 °C for 1 h. The reddish
solid was filtered off and washed with cold acetonitrile to give
52 mg (50%) of 3.27 1H NMR (500 MHz, CDCl3) d 11.94 (s, 1H-
NH), 8.63 (d, J = 8 Hz, 1H-C5), 8.19–8.14 (m, 2H), 7.91 (d, J = 7 Hz,
1H, H para to amino group), 7.73 (t, J = 7 Hz, 1H-C7), 7.64 (t,
of 4 at a fixed concentration of 1
lM led to a corresponding in-
crease in fluorescence polarization (Fig. 1A). This confirmed the

T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
2149
Scheme 1. Reagents and conditions: (a) NaNO2/H2SO4, NaN3, toluene, reflux; (b) AlCl3, nitrobenzene, rt; (c) acetonitrile, 80 °C; (d) aq NaOH/MeOH 80 °C.
direct binding of 4 to uPAR. A fit of the data resulted in a dissocia-
tion constant KD of 0.19 M. When the titration of 4 with uPAR was
repeated in the presence of excess uPAATF at 50 M concentration,
no increase in FP was detected (Fig. 1A). This suggests that 4 and
uPAATF compete for the same site on the receptor.
To further confirm the binding of 4 to uPAR and gain additional
insight into the recognition process, we resorted to the use of sat-
uration transfer difference (STD) NMR. STD NMR is an entirely li-
gand-based method, such that measurement of the binding of a
small-molecule to a protein is done by monitoring the transfer of
magnetization by fast chemical exchange. First, a standard 1D
spectrum of 4 was collected alone (Fig. 1B) and in the presence
of uPAR (Fig. 1C). Comparison of the two spectra reveals that two
NH peaks for the compound alone (inset of Fig. 1B). These peaks
disappear when uPAR is added (inset of Fig. 1C). This observation
is evidence that 4 binds to uPAR. STD spectra for 4 alone
l
(Fig. 1D) and in the presence of uPAR (Fig. 1E) were subsequently
collected. In the presence of uPAR, there are distinctive peaks that
are attributed to 4 (Fig. 1E). These peaks occur only when spin dif-
fusion signal (originating from the protein) is transferred to the
small-molecule. This indicates that the small-molecule is binding.
In addition, comparison of Figure 1D and E shows distinct peaks
for 4, further confirming binding of the compound to uPAR.
l
3.3. Effect of 4 on uPARꢀuPA binding in cells
Immunofluorescence confocal microscopy was used to probe
the effect of 4 on the uPARꢀuPA interaction at the cell surface of
breast MDA-MB-231 cancer cells. Endogenous uPA in a breast can-
cer cell line MDA-MB-231 was immunostained with a selective
Figure 1. Biochemical characterization of 4: (A) Direct binding of 4 to uPAR probed with fluorescence polarization with increasing concentration of uPAR; (B–E) STD NMR
experiments were used to probe direct binding of 4 to uPAR; (B) 1D NMR spectrum of 4 only, the NH peaks of 4 highlighted by arrows in the inset, which shows region inside
the box magnified, are broadened in the presence of uPAR as shown in the inset of the spectrum below; (C) 1D NMR spectrum of 4 in the presence of uPAR; (D) STD NMR
spectrum of 4 only showing no ligand peaks arising; (E) STD NMR spectrum of 4 in the presence of uPAR where peaks highlighted with asterisks represent peaks arising from
binding of 4 to uPAR. Saturation of the protein peaks was set at 0.8 ppm.

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T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
antibody and subsequently visualized by staining with a fluores-
cently conjugated secondary antibody. Representative decon-
volved confocal immunofluorescence images are presented in
Figure 2A and B. To get a quantitative estimate of the effect of 4
on the uPA level, we quantified its surface staining intensity
(Fig. 2C). The pixel immunodensities for uPA were determined
using Nikon elements software. In the absence of any compound
(DMSO; vehicle control), uPA was detected in intracellular clusters
as well as on surface membranes of the MDA-MB-231 cells (Fig. 2A,
arrows). In the presence of 4, added 30 min prior to immunostain-
ing, there was a significant reduction of uPA staining at the cell sur-
face (Fig. 2C). Surface staining was reduced by ꢃ95% and ꢃ80% at
synthesized compound showed similar inhibition of invasion as
the purchased compound reported earlier (Fig. S1).18 To gain fur-
ther insight into the mechanism by which these compounds inhibit
invasion, we study the effect of the compound on matrix metallo-
proteinase (MMP) activity using gelatin zymography. MMPs are
well-known proteases that degrade components of the ECM, such
as collagen, laminin, fibronectin and vitronectin. Activation of
MMPs is promoted by uPA binding to uPAR. We found that 4 effec-
tively inhibited matrix metalloproteinase (MMP-9) mediated deg-
radation of gelatin in a concentration-dependent manner (Fig. 3A).
At 25 lM nearly 40 percent inhibition is achieved. Cell viability
studies using MTT were carried out on 4 to evaluate its effects on
MDA-MB-231 cell growth. A concentration-dependent curve was
constructed over a period of 3 days. It resulted in an IC50 of
100 lM of 4 and at 100 lM of 11, respectively. These results sug-
gest that 4 can effectively prevent uPA from binding to uPAR, sup-
porting the findings from our biochemical assays that 4 acts as a
direct inhibitor of the PPI.
58
over a significantly shorter time, and given the more than 90 per-
cent blockage of invasion that is observed for 4 at 50 M, it is
lM for 4 (Fig. 3B). Since the invasion studies are carried out
l
3.4. Effect of 4 on invasion, migration and adhesion of MDA-
MB-231 cells
therefore expected that most of the inhibition of cell invasion is
unlikely due to cytotoxicity of the compound.
For cells to effectively metastasize, they must acquire additional
phenotypes, such as adhesion and migration. The Boyden chamber
apparatus without the Matrigel layer can be used to assess the ef-
Previously, we had shown that siRNA knockdown of uPAR in
MDA-MB-231 cells resulted in more than 60 percent impairment
of cell invasion.18 We had also shown that 4, which was purchased
from a commercial library, inhibited MDA-MB-231 invasion using
the same Boyden chamber apparatus. Here, we confirm that our
fect of 4 on cell migration (Fig. 3C). At 40
inhibition of MDA-MB-231 migration is observed, and at 80
nearly 70 percent inhibition is measured. The effect of 4 on
lM, nearly 30 percent
l
M
Figure 2. Immunofluorescence imaging to probe inhibition in cells: Representative deconvolved confocal immunofluorescence images of MDA-MB-231 cells incubated with
DMSO (control, A) or 100 M of 4 (B) and visualized by staining with a polyclonal antibody against uPA (red). Arrows indicate examples of two cells with clear surface staining
l
of uPA; (C) quantification of the surface expression levels of uPA in MDA-MB-231 cells exposed to the indicated conditions. 100 lM of the previously reported 11 was used a
positive control.18,19 The pixel immunodensity of the surface uPA compared to the total staining of uPA was calculated. Only cells where the cell membrane was clearly
discernible were included in the analyses. Each value represents the mean SE from 17 to 38 cells taken from six different fields from two independent samples.

T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
2151
Figure 3. Invasion, migration and adhesion studies: (A) Matrix metalloproteinase (MMP-9) activity using gelatin zymography. Results are representative of three independent
experiments; (B) effect of 4 on MDA-MB-231 cell viability as assessed by MTT assay; (C) cell migration studies with the Boyden chamber apparatus to characterize the role of
4 in chemotaxis-mediated cell migration; Error bars represent means S.D. Representative experimental cells from control and in the presence of 4 were photographed
(ꢁ400) to illustrate the effect of 4 on migration, as shown below; (D) effect of 4 on MDA-MB-231 cell attachment to fibronectin; error bars represent means S.D.
adhesion was studied using MDA-MB-231 cells that were added to
wells pre-coated with fibronectin as we have done previously.18 In
the presence of 4, a concentration-dependent impairment of cell
To determine whether 4 cause apoptosis, a flow cytometry anal-
ysis was performed with Annexin V-FITC and PI staining. The level
of apoptosis and necrosis in MDA-MB-231 cells was assessed after
exposure to increasing concentrations of 4 for 24 h as a percentage
of Annexin V-positive/PI-negative cells (apoptotic) and Annexin V-
positive/PI-positive cells (necrotic) respectively (Fig. S3). Control
DMSO treated cells showed 3 percent apoptotic and 6 percent ne-
crotic cells (Fig. S3A). At concentrations of 1 (Fig. S3B), 10
adhesion is observed with an IC50 of approximately 30 lM
(Fig. 3D). The effect on adhesion, albeit weaker than those observed
in invasion, may be attributed to the effect of the compound on
other interactions mediated by uPAR.
A limited structure–activity study is conducted on six deriva-
tives of 4 (Scheme 2). Their effect on ECM degradation was deter-
mined with gelatin zymography (Fig. 4). The binding affinity of
these compounds to uPAR was recently assessed in a comprehen-
sive structure–activity (SAR) study.28 A range of anti-invasion
activity was observed for the compounds. At a concentration of
(Fig. S3C), 25 (Fig. S3D) and 50 lM (Fig. S3E), 4 showed 3, 2, 2
and 0 percent apoptotic cells and 7, 5, 6 and 3 percent necrotic
cells, respectively. These results indicate that 4 does not have a sig-
nificant effect on apoptosis or necrosis.
We also tested the effects of 4 on MAPK signaling following
plating of serum starved cells onto fibronectin-coated culture
50 lM, 4 showed highest potency with nearly 90% inhibition of
gelatinase activity. The compound was more active that the parent
11, which showed about 40% inhibition. 5 (IPR-632) revealed no
inhibition, consistent with findings from a previous study that
plates for 30 min in the presence of 1% DMSO control, 50
lM of 4
or 50 M of IPR-69, a previously described compound that was
l
used as a positive control. Western immunoblotting shows that
as compared to DMSO control, IPR-69 significantly impaired MAPK
phosphorylation as previously shown.19 4 also showed inhibition
of MAPK phosphorylation, as compared to DMSO control but the
effect was weaker than that IPR-69 (Fig. S4).
the compound did not inhibit the tight uPARꢀuPA interaction.28
6
(IPR-658) and 8 (IPR-664) inhibit the uPARꢀuPA more weakly,28
consistent with their reduced efficacy in blocking ECM degradation
(Fig. 4). Compound 10 (IPR-824) is more potent than 7 (IPR-661)
consistent with our previous observation that m-carboxylate is
more effective than p-carboxylate in blocking binding of uPA to
uPAR. Compound 9 (IPR-809) also inhibited gelatinase activity de-
spite its significantly weaker inhibition of uPARꢀuPA.28 We had pre-
viously argued that the combination of a para-carboxylate on the
benzoic acid moiety with a piperidinone may have contributed to
its weaker activity and potential off-target effects. Invasion studies
using the Boyden chamber apparatus confirmed the gelatin
zymography results. For example, compound 7 inhibited MDA-
MB-231 cell invasion in a concentration-dependent manner with
3.5. In vivo PK studies
Compound 4 was administered to mice via a single PO delivery at
200 mg/kg using a formulation of saline. Blood (20 lL) was taken
from the mice at their tails at 1, 2, 4, 6, 8 and 10 h (three time points
from each of the 18 mice29) post injection. Plasma was separated
from blood and 4 was quantified by internal standardization, protein
precipitation, and HPLC–MS/MS (Fig. 5A). The lower limit of quanti-
fication was 100 ng/mL using 20
detected in plasma reaching a maximum concentration (Cmax) of
M after 1 h (tmax) (Fig. 5C). The stability of the compound in plas-
ma is reflected by its half-life (t1/2) that was estimated at 5 h. To gain
lL of plasma. The compound was
an IC50 of approximately 30 lM (Fig. S2A). Cell viability studies
using MTT over a period of three days revealed an IC50 of 33
lM
5 l
for 7 (Fig. S2B).

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T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
Scheme 2. Structure of derivatives of 4.
concentrations near 10 lM. The levels of the compound in tumor tis-
sue remained high even after 10 h.
3.6. Effect on breast cancer metastasis
An evaluation of the efficacy of 4 on metatsasis was conducted
in vivo using an orthotopic model that we have previously imple-
mented.19 TMD-231 cells were inoculated into the mammary fat
pads of female NSG mice. Like MDA-MB-231, TMD-231 overex-
press uPAR as shown by immublotting analysis in Figure 6A. Dos-
ing was initiated at day 15 post implantation. Animals were
randomized and treated with vehicle or with 4 by daily oral gavage
at a dose of 200 mg/kg (n = 13). Tumor volumes were determined
by caliper measurements on a twice weekly basis, and calculated
according to the formula (a2 ꢁ b)/2, where
a is the shorter and b
is the longer of the two dimensions. The study was conducted over
a period of 33 days. The primary tumor in both control and treated
mice grew over the course of the study (Fig. 6B). Tumor volumes
reached nearly 693 and 785 mm3 for treated and untreated mice,
respectively. The small effect (ꢃ10%) on tumor size is fully consis-
tent with previous studies that have shown that uPAR is not a
lethal target. In addition, these studies further support our cellular
Figure 4. Invasion and cytotoxicity studies of 4 and its derivatives: Gelatin zymogram
(below) showing the effects of 4 derivatives at 50 lM concentration on matrix
metalloproteinase (MMP-9) activity as quantified above. Error bars represent
means S.D.
insight into its absorption following oral dosing, intravenous bio-
availability of 4 was determined. The compound was injected intra-
venously (IV) into the tail vein. Blood samples were obtained at 5, 10,
20, 45 and 120 min (three mice per time point) post injection. Plas-
ma was separated from blood and 4 was quantified by internal stan-
dardization, protein precipitation, and HPLC–MS/MS (Fig. 5B). Based
on these data, the oral bioavailability was 4 percent. Despite the low
bioavailability in plasma, HPLC–MS/MS quantification of 4 in the
primary tumor at 1, 4, and 10 h intervals detected the inhibitor at
studies that have found that 4 is not cytotoxic with IC50 of 58 lM.
There was no statistical significance to the differences in body
weight between treated and untreated, suggesting that the com-
pound may be well tolerated by the mice (Fig. 6C).
To assess the effect of 4 on metastasis, control and experimental
animals were sacrificed and organs (lungs) were removed and
evaluated for the presence of tumors. The analysis of tumor metas-
tasis lesions showed that they ranged roughly from 2 to 25 cells, 25

T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
2153
Figure 5. In vivo PK parameters: (A) PK analysis of 4 in female NOD/SCID mice (n = 3 per time point) dosed by oral gavage (200 mg/Kg); (B) plasma levels of 4 as a result of
intravenous injection; (C) PK parameters.
Figure 6. In vivo efficacy studies: (A) Western immunoblot showing the expression levels of uPAR and uPA in MDA-MB-231 and TMD-231 cell lines; (B) effect of 4 on TMD-231
tumor growth. TMD-231 cells were inoculated in the mammary fat pads of female NOD/SCID mice. Once the tumor volume reached 30–50 mm3, animals were randomized
and treated with vehicle alone as control or 200 mg/kg of compound 4 three times a week for 5 weeks by oral gavage. Tumor volumes were determined by caliper
measurements obtained weekly; (C) body weight change upon completion of the study after 33 days; (D) representative H&E staining images that illustrate metastasis in the
lungs of animals; (E) scoring system was used to quantify the level of metastasis in the lungs. The number of lung tumor nuclei/cells were counted in each lobe, and their size
were scored on a scale of 0–4 (0, few; 1, mild; 2, moderate; 3, severe; and 4: marked).
to 150 cells, and about 150 to 600 cells. There were a few large
clusters of coalescing tumor balls seen. A few vehicle control lungs
had tumor metastases that were coalescing into large tumor balls
(greater than 1000 cells). The metastases were characterized by
very large bizarre cells, which had large undifferentiated nuclei
with large nucleoli. These were easy to discern by routine H&E
staining and count. The number and size of metastasis in two to
five fields per sample were calculated. A score of 4+ was given to
a sample with highest metastasis index and relative metastasis
in other samples are calculated (i.e., 1+, 2+, 3+) by a sample-
blinded pathologist (Fig. 6E). The extent of metastasis in treated
versus untreated was significantly different. For example, 10 out
of 13 untreated mice developed severe (score = 3) or marked
(score = 4) metastasis to the lung. This is compared to only 4 out

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T. Mani et al. / Bioorg. Med. Chem. 21 (2013) 2145–2155
of 12 in the treated mice (2 severe and 2 marked). Among the trea-
ted mice, 4 developed moderate (score = 2) metastasis. Another
four were scored at 1 or lower, showing mild metastasis to the
lungs. Representative H&E staining images for control and treated
mice are shown in Figure 6D.
suggests that small improvements in absorption could results in
significant increases in plasma and tumor concentrations.
In vivo, compound 4 was tested using an orthotopic breast-
to-lung metastasis model that we had previously implemented.19
TMD-231 cells are highly aggressive breast cancer cell lines de-
rived from MDA-MB-231.39 Just like MDA-MB-231, TMD-231 cells
overexpress uPAR. Analysis of the tumor growth reveals that 4 had
little effect on tumor growth, in agreement with the weak cytotox-
icity that was found in our proliferation assay study. The lack of
cytotoxicity is consistent with the fact that uPAR is not required
for normal biological function, as it is not even expressed in normal
cells except during embryogenesis.40,41 It is highly expressed dur-
ing inflammation42,43 or metastasis.44,45 The confined effect of 4 on
metastasis and the lack of cytotoxicity suggest selectivity. Com-
pound 4 and its analogs are the first small-molecules that have
been identified to inhibit the uPARꢀuPA PPI. These results are an
excellent starting point, as 4 provides a lead molecule for the de-
sign and synthesis of derivatives with higher affinity and better
PK properties.
4. Discussion
Recently, we reported the discovery of 11, a small-molecule
inhibits the tight PPI between uPAR and its ligand uPA.18 A deriv-
ative, namely 4, was also found to inhibit the PPI.28 A recent study
showed that 4 inhibited invasion in a panel of non-small cell lung
cancer (NSCLC) cells.28 Here, we extend on these studies and focus
on probing uPAR with 4 in breast cancer metastasis and assess its
efficacy in animal studies. The compound, which was previously
purchased, is prepared in gram-scale to enable in vivo studies. In
a departure from previous work that was confined to competition
studies, direct binding studies are carried out using fluorescence
polarization and STD NMR. These showed that the compound binds
directly to uPAR with a sub-micromolar affinity of 0.2 lM, which is
Acknowledgments
nearly an order of magnitude higher than previously-measured
IC50 in competition assays. The nearly 10-fold difference between
KD and IC50 is not unexpected. The binding interface of the uP-
ARꢀuPA PPI (estimated at 1200 Å2) is significantly larger than the
surface occupied by the small-molecule. Hence, a higher concen-
tration of the compound is required to shift the equilibrium to-
wards dissociation of the PPI complex. STD NMR studies not only
provided further confirmation that the compounds binds to uPAR
directly, but also generated data that supports the computationally
predicted binding mode of 4.
Compound 4 and its derivatives were used to probe the role of
the uPARꢀuPA interaction in breast cancer cell invasion and metas-
tasis. Gelatin zymography revealed that MMP-9 activity is im-
paired by 4, suggesting that blocking uPA binding to uPAR likely
affects activation of uPA and the subsequent proteolytic cascade
that has been shown to lead to MMP activation.30–32 The effects
The research was supported by the National Institutes of Health
(CA135380) (SOM), the INGEN grant from the Lilly Endowment, Inc
(SOM), by The Indiana University Melvin and Bren Simon Cancer
Center Translational Research Acceleration Collaboration (ITRAC)
(SOM), the Showalter Trust (SOM), by the Indiana University Bio-
medical Research Fund (SOM) and by the American Cancer Society
Research Scholar Grant RSG-12-092-01-CDD (SOM). We acknowl-
edge the analytical work performed by the Clinical Pharmacology
Analytical Core laboratory, a core laboratory of the Indiana Univer-
sity Melvin and Bren Simon Cancer Center supported by the
National Cancer Institute grant P30 CA082709.
Supplementary data
of 4 on cell adhesion (IC50 = 30
l
M) and its weaker effect on migra-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.047.
tion (IC50 = 50 M) may be attributed to inhibition of uPAR binding
l
to integrins. It is well-known that uPAR engages integrins, which
are responsible for attachment of cells to ECM components and
migration.22,33,34 Although the integrin binding pocket is located
away from the uPA binding site where 4 was targeted,33 studies
have shown cooperativity between uPA and vitronectin binding.35
Extensive biophysical studies have shown that uPA binding to
uPAR stabilizes the receptor and promotes binding to vitronec-
tin.36,37 Hence, blocking of uPA binding by 4 likely alters the
dynamics of uPAR resulting in impaired vitronectin binding and
therefore less interaction with integrins. However, recent FRET-
based cellular studies have shown that the uPARꢀintegrin interac-
tion can occur even in the absence of uPA.38 Since uPA is highly
expressed in malignant cells, the effects on adhesion of our
compounds could be attributed to destabilization of the uPAR–
vitronectin complex. It is interesting that 4 inhibited signal
transduction of the MAPK pathway, consistent with previous
studies that have reported a role for uPAR in MAPK signaling.
The inhibition of the uPARꢀuPA interaction and the effect of 4 on
invasion prompted us to probe the compound for its effect on
metastasis in vivo. When administered orally, 4 reached a concen-
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