Probing binding and cellular activity of pyrrolidinone and piperidinone small molecules targeting the urokinase receptor

Article abstract of DOI:10.1002/cmdc.201300340

The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here, we evaluate the binding and biological activity of a new class of

Full text of DOI:10.1002/cmdc.201300340

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DOI: 10.1002/cmdc.201300340
Probing Binding and Cellular Activity of Pyrrolidinone and
Piperidinone Small Molecules Targeting the Urokinase
Receptor
Timmy Mani,[a] Degang Liu,[a] Donghui Zhou,[a] Liwei Li,[a] William Eric Knabe,[a] Fang Wang,[a]
Kyungsoo Oh,[c] and Samy O. Meroueh*[a, b, c, d]
The urokinase receptor (uPAR) is a cell-surface protein that is
part of an intricate web of transient and tight protein inter-
actions that promote cancer cell invasion and metastasis. Here,
we evaluate the binding and biological activity of a new class
of pyrrolidinone and piperidinone compounds, along with de-
rivatives of previously-identified pyrazole and propylamine
compounds. Competition assays revealed that the compounds
displace a fluorescently labeled peptide (AE147-FAM) with in-
hibition constant (Ki) values ranging from 6 to 63 mm. Struc-
ture-based computational pharmacophore analysis followed by
extensive explicit-solvent molecular dynamics (MD) simulations
and free energy calculations suggested the pyrazole-based and
piperidinone-based compounds adopt different binding
modes, despite their similar two-dimensional structures. In
cells, pyrazole-based compounds showed significant inhibition
of breast adenocarcinoma (MDA-MB-231) and pancreatic
ductal adenocarcinoma (PDAC) cell proliferation, but piperidi-
none-containing compounds exhibited no cytotoxicity even at
concentrations of 100 mm. One pyrazole-based compound im-
paired MDA-MB-231 invasion, adhesion, and migration in a con-
centration-dependent manner, while the piperidinone inhibited
only invasion. The pyrazole derivative inhibited matrix metallo-
protease-9 (gelatinase) activity in a concentration-dependent
manner, while the piperidinone showed no effect suggesting
different mechanisms for inhibition of cell invasion. Signaling
studies further highlighted these differences, showing that pyr-
azole compounds completely inhibited ERK phosphorylation
and impaired HIF1a and NF-kB signaling, while pyrrolidinones
and piperidinones had no effect. Annexin V staining suggested
that the effect of the pyrazole-based compound on prolifera-
tion was due to cell killing through an apoptotic mechanism.
The compounds identified represent valuable leads in the
design of further derivatives with higher affinities and potential
probes to unravel the protein–protein interactions of uPAR.
Introduction
A characteristic of malignant tumors is the ability of a small
subpopulation of cells to escape from the primary tumor.[1–3]
Throughout the metastatic cascade and formation of new
blood vessels, there is an intense process of extracellular
matrix (ECM) degradation.[4] With increased motility and more
effective adhesion to the ECM, malignant cells eventually gain
access to the vasculature and spread to distant sites to form
new colonies.[3] These processes require a complex interplay of
various cell-surface-associated proteins.[4,5] Among them, the
glycosyl-phosphatidylinositol (GPI)-anchored urokinase recep-
tor (uPAR) has been implicated in nearly every step of cancer
metastasis including adhesion,[6–8] migration,[8–10] inva-
sion,[8,9,11–14] and angiogenesis.[9,11,12] This is attributed to its
large number of transient and tight protein–protein interac-
tions with soluble and membrane proteins.[15]
[a] Dr. T. Mani,+ Dr. D. Liu,+ Dr. D. Zhou,+ Dr. L. Li, Dr. W. E. Knabe, F. Wang,
Prof. S. O. Meroueh
Department of Biochemistry & Molecular Biology
Indiana University School of Medicine
Indianapolis, IN 46202 (USA)
uPAR contributes to pericellular proteolysis by binding and
sequestering the multidomain protease urokinase-type plasmi-
nogen activator (uPA) to the cell surface.[16,17] Most of its re-
ported interactions are transient, such as binding to vitronec-
tin,[18] integrins,[19] or receptor tyrosine kinases such as the pla-
telet-derived growth factor receptor (PDGFR).[20] Indirect inter-
actions with EGFR and LRP1 have also been reported.[21–23] The
former is expected to occur through integrin, while the latter
is mediated by plasminogen activator inhibitor-1 (PAI-1), which
eventually leads to endocytosis and the recycling of uPAR. The
weak interactions have only been detected in cellular studies
except for the binding of the somatomedin B domain (SMB)
domain of vitronectin to uPAR, which has been extensively
studied through biophysical studies using surface plasmon
E-mail: smeroueh@iupui.edu
[b] Prof. S. O. Meroueh
Center for Computational Biology & Bioinformatics
Indiana University School of Medicine
Indianapolis, IN 46202 (USA)
[c] Prof. K. Oh, Prof. S. O. Meroueh
Department of Chemistry & Chemical Biology
Indiana University-Purdue University Indianapolis (IUPUI)
Indianapolis, IN 46202 (USA)
[d] Prof. S. O. Meroueh
Stark Neurosciences Research Institute
Indiana University School of Medicine
Indianapolis, IN 46202 (USA)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300340.
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resonance (SPR).[24] A crystal structure of uPAR in complex with
the N-terminal fragment of ATF (uPAATF) shows the presence of
a large pocket with hydrophobic characteristic that is located
at the uPAR·uPA interface.[25,26]
Small molecules that bind to uPAR are expected to modu-
late its function. We recently reported three classes of com-
pounds that target uPAR.[8,13] An anthraquinone-based com-
pound (IPR-803) was shown to bind with sub-micromolar affin-
ity (Ki =0.2 mm)[27] and inhibited the uPAR·uPA interaction with
an IC50 value of 10 mm.[13] Pyrazole-based compounds displaced
a fluorescently labeled peptide with an IC50 value of 30 mm.
Cellular studies on a breast adenocarcinoma cell line (MDA-
MB-231) of these compounds revealed inhibition of adhesion,
migration, invasion, and angiogenesis.[8] This range of cellular
activity, particularly inhibition of cell growth, suggests that the
compound is likely to inhibit other targets in addition to uPAR,
making it a potentially useful multitargeted agent. In vivo, the
compound had promising pharmacokinetics and little toxici-
ty.[8]
ieved using commercially available 3,4-dimethylphenyl hydra-
zine to give 10. With the pyrazole core formed, the ester
group of 10 was subsequently hydrolyzed to give 11, and the
free carboxylic acid was then reacted with various anilines to
form the corresponding amide (12). Removal of the Boc group
gave desired N-1-substituted pyrazoles 1e–i.
The synthesis of substituted pyrrolidin-2-one 3 and piperi-
din-2-one 4 are summarized in Scheme 2 and 3, respectively.
First, Schiff bases 13 and 15 were obtained with the appropri-
ate aldehyde and amine in the presence of anhydrous magne-
sium sulfate.[30] The subsequent condensation of the Schiff
Starting with the structure of the pyrazole- and propyl-
amine-based compounds (1 and 2, respectively), we identify
a series of derivatives from commercial sources that included
molecules with pyrrolidinone (3) and piperidinone (4) core
structures. Additional derivatives of the pyrazole-based com-
pounds were designed and synthesized. The four classes of
compounds were evaluated in MDA-MB-231 cells and a pancre-
atic ductal adenocarcinoma cell line (PANC-1), and the results
revealed distinct biological activity despite their two-dimen-
sional structural similarity. Finally,
gel zymography, immunoblot-
ting, and flow cytometry studies
were carried out to gain further
insight into their mechanism of
action.
Results
Synthesis
The synthesis of N-1-substituted
pyrazoles 1e–i followed the
route
reported
previously
(Scheme 1).[28] The pyrazole core
was prepared by condensation
of a 1,3-dicarbonyl enol ether
with a hydrazine. Thus, the pi-
peridine ring of commercially
available isonipecotic acid (5)
was protected with tert-butylox-
ycarbonyl (Boc) to give 6, which
was converted to known b-keto
ester 8 through a simple two-
step sequence (coupling of Mel-
drum’s acid followed by ethanol-
ysis).[29] With the requisite b-keto
ester (8) in hand, the formation
Scheme 1. Reagents and conditions: a) K2CO3, BOC2O, THF/H2O (1:1), 08C!RT, o/n; b) DMAP, DCC, 2,2-dimethyl-
1,3-dioxane-4,6-dione, CH2Cl2, 08C!RT, o/n; c) abs EtOH, reflux, 48 h; d) triethylorthoformate, Ac2O, reflux, 48 h;
e) EtOH, 3,4-dimethylphenyl hydrazine, reflux, 48 h; f) aq NaOH (2.0m), EtOH, reflux, 20 h; g) DMAP, DCC, aniline,
of the pyrazole core was ach- CH2Cl2, 08C!RT, 48 h; h) TFA/CH2Cl2, 08C!RT, 1 h.
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base with succinic anhydride or glutaric anhydride in refluxing
xylenes gave pyrrolidin-2-one 14 and piperidin-2-one 16 car-
boxylic acids, respectively.[31] Amide formation was carried out
by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cou-
pling in the case of 16 and N,N’-dicyclohexylcarbodiimide
(DCC) coupling for 14 to give the final substituted pyrrolidin-2-
one (3) and piperidin-2-one (4).
first time, we characterize the uPAR binding and cellular activi-
ty of these compounds along with their derivatives.
A similarity search of the ZINC chemical library was carried
out using chemical structure 1–4. Derivatives were acquired
and tested for activity at an initial concentration of 50 mm
using a fluorescence polarization (FP) assay (Figure 1a). The FP
assay probe consisted of labeling an a-helical peptide (AE147)
with fluorescein (AE147-FAM). The labeled peptide was shown
to bind to uPAR with a KD value of 499 nm. A concentration-de-
pendent study was performed for several derivatives that were
active (Figure 1b). The structures of all derivatives are shown
in Tables 1–4.
For compound 1, four derivatives were selected, namely 1a–
d, and their Ki values were found to range from 10 to 27 mm
(Table 1). Replacement of the para-isopropopyl group of the
parent compound with a dimethyl substituted phenyl ring (1a
and 1b) enhanced the affinity of the compound compared
with that of the parent compound, suggesting a better fit into
the binding pockets of uPAR. This is reflected in 1c and 1d
whereby a hydrogen or a chlorine at the meta position of the
R3 group also led to inhibition constants that were higher than
parent compound 1. Starting with the structure of 1a, a struc-
Scheme 2. Reagents and conditions: a) anhyd MgSO4, CH2Cl2, RT, 72 h; b) suc-
cinic anhydride, xylenes, reflux, 24 h; c) DMAP, DCC, 08C!RT, 3 d.
Scheme 3. Reagents and conditions: a) anhyd MgSO4, CH2Cl2, RT, 72 h; b) glu-
taric anhydride, xylenes, reflux, 72 h; c) EDC, HOBt, 2,4-dimethoxyaniline,
CH2Cl2, 08C!RT, 2 d.
uPAR binding activity
Previously, we reported the use of virtual screening to identify
compounds that bind to uPAR.[8] Docking of 300000 com-
pounds to the uPAR crystal structure (PDB code: 1YWH) was
focused to a pocket located at the interface of the protein–
protein complex between uPAR and its serine protease ligand
uPA. Among the active compounds that emerged were pyra-
zole 1 (IPR-69) and propylamine 2. Among the top ranking
candidates, two compounds shared structural similarity to 1.
These include pyrrolidinone 3 and piperidinone 4. Here, for the
Figure 1. Fluorescence polarization: a) Fluorescence polarization screening
of 393 compounds that are derivatives of 1–4. Compounds (50 mm) were
screened against uPAR (320 nm) and AE147-FAM peptide (100 nm). b) Con-
centration-dependent fluorescence polarization study of the top-ranking 18
compounds.
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Table 1. Structure and uPAR binding affinities of compound 1 deriva-
Table 2. Structure and uPAR binding affinities of compound 2 deriva-
tives.
tives.
Compd
R1
R2
R3
Ki [mm]
Compd R1
R2
R3
R4
Ki [mm]
IPR-8 (2a)
19.3
IPR-81
(1a)
21.9
IPR-21 (2b)
IPR-22 (2c)
33.8
13.9
IPR-82
(1b)
18.9
27.0
IPR-361
(1c)
IPR-380
(1d)
10.3
51.2
Table 3. Structure and uPAR binding affinities of compound 3 derivatives.
IPR-735
(1e)
IPR-737
(1 f)
17.2
Compd R1
R2
R3
IC50 [mm] Ki [mm]
IPR-743
(1g)
28.3
63.3
31.9
IPR-99
(3)
ND
ND
IPR-744
(1h)
IPR-979
(3a)
18
20
10.3
11.4
IPR-745
(1i)
IPR-994
(3b)
ture-based approach was followed to design and synthesize
additional derivatives (1 f–i). Four compounds were more
active than the parent at a single concentration and were stud-
ied in a concentration-dependent manner (Table 1). The high-
est affinity derivative was 1 f (Ki =17 mm), whereas the lowest
affinity derivative was 1h (Ki =63.3 mm) (Table 1).
responsible for the decreased affinity since 4e and 4 f have
a methoxy moiety at the ortho and para positions with Ki
values of 16 and 12.6 mm, respectively (Table 4).
Computational studies
The process was repeated for compounds 2–4. Similarity
search identified derivatives that were tested at a single con-
centration and follow-up studies in a concentration-dependent
manner were done for compounds that were more active than
the parent. Three derivatives were identified for 2, namely 2a,
2b, and 2c, which had Ki values of 19.3, 33.8, and 13.9 mm, re-
spectively (Table 2). For 3, two compounds were identified
with higher affinity for uPAR than the parent compound,
namely 3a and 3b, with Ki values of 10.3 and 11.4 mm, respec-
tively (Table 3). For 4, we found seven active derivatives (4a–g)
with binding affinities (Ki) ranging from 5.7 to 24.1 mm. The
highest affinity compound was 4c, which has a phenyl rather
than a benzyl moiety at the R3 position. When two methoxy
groups are introduced at R2 to generate 4b, the affinity is sig-
nificantly decreased. A methoxy group at the meta position is
Two binding modes were selected from the pharmacophore
and QSAR modeling studies (Figure 2a,b). We refer to these
two modes as hypothesis HS1119 and hypothesis HS1135. In
HS1119, the R2 group of the pyrazole, piperidinone, and pyrroli-
dinone compounds are ensconced in a pocket in the uPA bind-
ing hydrophobic pocket on uPAR (Figure 2c). In HS1135, the R2
group is instead directed away from the protein toward the
solvent (Figure 2d). Rigorous computational studies were con-
ducted to determine the stability and binding strength of the
compounds in both binding modes. Explicit-solvent molecular
dynamics (MD) simulations for each binding mode were
carried out for three compounds, 1b, 3a, and 4c. Analysis of
the 12 ns trajectories revealed that 1b is highly unstable in
binding mode HS1119 as evidenced by the root-mean-square
deviation (RMSD) value of the compound that increases to 4 ꢁ
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pose, with an RMSD value that remained within 3 ꢁ (Fig-
ure 3e,g) in contrast to the HS1135 binding pose that exhibit-
ed an RMSD value greater than 6 ꢁ for two of the trajectories
(Figure 3 f,h). A similar observation was made for piperidinone-
based compound 4c, which remained remarkably stable over
the course of the simulations, showing RMSD values within
1.5 ꢁ for binding pose HS1119 (Figure 3i,k) compared with the
significantly larger RMSD values observed for HS1135 (Fig-
ure 3j,l).
Table 4. Structure and uPAR binding affinities of compound 4 deriva-
tives.
Compd
R1
R2
R3
Ki [mm]
Free-energy calculations using protein–compound structures
collected from the MD simulations were carried out using the
molecular mechanics/Poisson–Boltzmann surface area (MM/
PBSA) approach (Table 5). The MM-PBSA free energy (DGPBSA) is
obtained by subtracting the free energy of the protein–com-
pound complex from protein and compound for each snap-
shot collected from the MD simulation. The free energy con-
sists of an internal energy term consisting of van der Waals
and Coulomb potential energy (DEPBELE), a solvation energy
term consists of electrostatic and nonpolar components
(DEPBTOT), and finally an entropy term (TDSNM) (Table 5). For
compound 1a, the DGPBSA value was only determined for
HS1135, as the compound was unstable in the HS1119 binding
mode. In contrast, piperidinone- and pyrrolidinone-based com-
pounds 3a and 4c trajectories were stable in the HS1119 bind-
ing mode and resulted in DGPBSA values of ꢀ20 and ꢀ25 kcal
molꢀ1, respectively. The trajectories for the HS1135 binding
mode resulted in less stable binding, with DGPBSA values of
ꢀ13, ꢀ11, and ꢀ21 kcalmolꢀ1 for 1a, 3a and 4c, respectively.
The binding free energy for compounds 3a and 4c was signifi-
cantly more stable for the HS1119 binding mode, in contrast to
the pyrazole-based compound 1a, which prefers the HS1135
binding mode.
IPR-84 (4)
18.9
IPR-89 (4a)
18.9
IPR-108 (4b)
IPR-949 (4c)
IPR-968 (4d)
IPR-974 (4e)
24.1
5.7
19.5
16.0
IPR-1036 (4 f)
IPR-1046 (4g)
12.6
10.9
Effect of compounds on cell proliferation
We evaluated the cytotoxicity of several compounds using an
MTT assay as previously described.[8,32] A concentration-depen-
dent MTT study in MDA-MB-231 cells gave IC50 values of 31, 13,
and 25 mm for 1b, 2c, and 1 f, respectively (Figure 4). Com-
pound 1 f had IC50 values of 14 and 22 mm in human pancreat-
ic adenocarcinoma (AsPC-1) and PANC-1 cell lines, respectively.
for three of the 12 ns trajectories and 8 ꢁ for one of the trajec-
tories (Figure 3a,c). Compound 1b was more stable in binding
mode HS1135 (Figure 3b,d). Compound 3a on the other hand
showed significantly greater stability in the HS1119 binding
Table 5. Binding free energy and its components from computational studies.
[a]
[b]
[c]
[d]
[e]
[f]
[g]
[h]
[i]
Compd
Hypothesis
DEVDW
DEPBELE
DEGBELE
DESA
DEPBTOT
DEGBTOT
TDSNM
DGPBSA
DGGB
H1119
H1135
unstable
ꢀ49.3ꢁ0.2
–
–
–
–
–
–
–
–
1a
20.0ꢁ0.2
18.2ꢁ0.1
ꢀ5.6ꢁ0.0
ꢀ34.9ꢁ0.2
ꢀ36.7ꢁ0.2
ꢀ23.4ꢁ0.6
ꢀ11.5ꢁ0.6
ꢀ13.3ꢁ0.6
H1119
H1135
ꢀ54.2ꢁ0.2
ꢀ43.6ꢁ0.2
18.6ꢁ0.3
22.1ꢁ0.2
18.4ꢁ0.1
18.9ꢁ0.1
ꢀ6.9ꢁ0.0
ꢀ6.2ꢁ0.0
ꢀ42.5ꢁ0.2
ꢀ27.7ꢁ0.2
ꢀ42.7ꢁ0.2
ꢀ30.9ꢁ0.2
ꢀ22.4ꢁ0.6
ꢀ19.9ꢁ0.6
ꢀ20.1ꢁ0.6
ꢀ7.8ꢁ0.6
ꢀ20.3ꢁ0.6
ꢀ11.0ꢁ0.6
3a
4c
H1119
H1135
ꢀ55.7ꢁ0.1
ꢀ53.8ꢁ0.1
15.0ꢁ0.2
17.4ꢁ0.1
15.3ꢁ0.1
15.6ꢁ0.1
ꢀ6.6ꢁ0.0
ꢀ6.6ꢁ0.0
ꢀ47.4ꢁ0.2
ꢀ43.0ꢁ0.2
ꢀ47.0ꢁ0.2
ꢀ44.8ꢁ0.2
ꢀ22.0ꢁ0.6
ꢀ23.5ꢁ0.6
ꢀ25.4ꢁ0.6
ꢀ19.5ꢁ0.6
ꢀ25.0ꢁ0.6
ꢀ21.3ꢁ0.6
[a] DEVDW, van der Waals potential energy; [b] DEPBELE, electrostatic contributions to the binding energy, of which the polar solvent contributions were calcu-
lated with Poisson–Boltzmann equation; [c] DEGBELE, electrostatic contributions to the binding energy, of which the polar solvent contributions were calcu-
lated with Generalized Born equation; [d] DESA, nonpolar solvent contribution to solvation free energy; [e] DEPBTOT, the sum of DEVDW, DEPBELE and DESA
;
[f] DEGBTOT, the sum of DEVDW, DEGBELE and DESA; [g] TDSNM, entropy calculated with normal mode analysis; [h] DGPB, the calculated free energy of binding
using PB model; [i] DGGB, the calculated free energy of binding using GB model.
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Figure 2. Compound binding modes. Hypothesis HS1119 (a,c) and HS1135 (b,d) that emerged from pharmacophore and QSAR modeling. Features were ren-
dered as orange rings (aromatic ring), and green (hydrophobic), blue (donor), and pink (acceptor) spheres. All active compounds (shown in sticks) were
aligned to the pharmacophore hypothesis in panel a or b. The pharmacophore along with a reference compound is shown docked in the pocket of uPAR, in
ribbon representation (c,d). The residues making contact with the reference compound are rendered in thinner capped-sticks.
In comparison, 4b showed no cytotoxicity in the MDA-MB-231
cell line with 70% cell viability even at 100 mm (Figure 4).
To gain insight into the cell killing mechanism, a flow cytom-
etry analysis was performed with Annexin V-FITC and propidi-
um iodide (PI) staining for compounds 1a, 1 f, 1i, 3, and 4
(Table 6 and Figure 5). The level of apoptosis and necrosis in
MDA-MB-231 cells was assessed after exposure to increasing
concentrations of compound for 24 h as a percentage of An-
nexin V-positive/PI-negative cells (apoptotic) and Annexin V-
positive/PI-positive cells (necrotic), respectively (Table 6 and
Figure 5). The data reveal that only the pyrazole compounds
cause apoptosis, with 1 f and 1i exhibiting the most pro-
nounced effects. Control DMSO-treated cells showed about
3% apoptotic and 6% necrotic cells. At concentrations of 1,
10, 25 and 50 mm, compound 1 f resulted in 3, 45, 76 and 61%
apoptotic cells and 7, 15, 8 and 21% necrotic cells, respectively
(Figure 5). For compound 1i, 10, 25 and 50 mm resulted in 11,
6, 46, and 86% apoptotic cells, and 7, 9, 10, and 12% necrotic
cells, respectively. Finally, compound 1a exhibited less signifi-
cant effect with nearly 39 and 32% of cells undergoing apop-
tosis and necrosis at 50 mm, respectively.
eration and survival. Western blot analysis was carried out fol-
lowing plating of serum-starved cells onto fibronectin-coated
culture plates for 30 min in the presence of test compounds at
50 mm. Results show that pyrazole-based compounds 1 f, 1i
and 1a significantly impaired p44/42 MAPK phosphorylation.
Propylamine-based 2c exhibited moderate inhibition. In con-
trast, compounds 4, 4b and 3 showed no inhibition of p44/42
MAPK phosphorylation (Figure 6a), consistent with their com-
plete lack of cytotoxicity. Luciferase reporter assays performed
in MDA-MB-231 cells revealed that nuclear factor-kB (NF-kB),
Table 6. Mechanism of compound cell killing studied by flow cytometry.
Proportion of cells in apoptosis/necrosis [%]
Compd
DMSO
1 mm
10 mm
25 mm
50 mm
apoptosis
necrosis
apoptosis
necrosis
apoptosis
necrosis
apoptosis
necrosis
2
6
3
6
2
6
2
6
2
6
1
5
3
7
11
7
2
6
4
3
5
12
45
15
6
9
1
3
2
16
17
76
8
46
10
2
8
3
6
39
32
61
21
86
12
4
7
3
5
1a
1 f
1i
3
Western blot analysis was performed to evaluate the effects
of the compounds on mitogen-activated protein kinase
(MAPK) signaling, given that the pathway regulates cell prolif-
apoptosis
necrosis
4
14
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Figure 3. Compound binding mode stability. Root-mean-square deviation (RMSD) of compound versus time for 1b (a,b), 3a (e,f), and 4c (i,j), and correspond-
ing illustration of the motion of the compound for 1b (c,d), 3a (g,h), and 4c (k,l). RMSD values are determined for each compound relative to the initial
docked structure. Illustrations of superimposed snapshots collected over the course of the trajectories are shown in ribbon representation for uPAR and in
capped sticks for compounds (C, N, O, Cl and Br are shown in white, blue, red, pink and cyan, respectively). Carbon atoms are shown in yellow for the initial
docked structure.
hypoxia-inducible factor-1a (HIF1a) and extracellular signal-
regulated kinase (ERK) were active even at basal levels (Fig-
ure 6b). Compound 1 f impaired all three pathways. The effect
on HIF1a is consistent with inhibition of ERK1/2 phosphoryla-
tion, which is known to control activity of HIF1a. The effect on
NF-kB can also be attributed to inhibition of ERK phosphoryla-
tion. In both cases, however, it is possible that the ef-
fects are ERK-independent.
Effect of compounds on cell invasion, migration
and adhesion
To test the effects of compounds on MDA-MB-231 in-
vasion, a Matrigel-coated Boyden chamber was used
as previously described.[13] The compounds were ini-
tially tested at two concentrations (Figure 7a). First,
compounds 1b, 1 f, 2b, and 2c, 3 and 4b were
tested at 10 mm. Each compound inhibited invasion
with the pyrazole (1) and propylamine (2) derivatives
exhibiting highest activity, while the piperidinone (4)
and pyrolidinone (3) showing milder effects. At
25 mm, compounds 1 f, 2b and 2c completely inhibit-
ed invasion, while compounds 1b, 4b and 3 showed
moderate inhibition of invasion (Figure 7a). The ef-
fects of these compounds on migration were also
Figure 4. Cytotoxicity and cell viability studies. Effects of 1b, 1 f, 2c, 4b on MDA-MB-231,
AsPC-1 and PANC-1 cell viability as assessed by an MTT assay.
tested at 25 mm using a wound healing assay. Com-
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Figure 5. Flow cytometry analysis using Annexin V-FITC and propidium iodide (PI) staining. MDA-MB-231 cells were treated for 24 h with 1 f, 1a, 1i, 3 or 4
and analyzed for apoptosis and necrosis by flow cytometry.
Figure 6. Effect on signaling. a) Western immunoblotting showing the effects of compounds at 50 mm on phospho-p44/42 MAPK and total p44/42 MAPK in
MDA-MB-231 cell lysates prepared as described in the Experimental Section. Actin is shown as a loading control. Lane 1 (–) contains DMSO control. b) Lucifer-
ase reporter assay results evaluating the effect of 1 f on NF-kB, HIF1a, and MAPK/ERK reporter in MDA-MB-231 cells.
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Figure 7. Invasion and migration studies. a) Boyden chamber apparatus (with Matrigel) was used to assess the effects of compounds at 10 mm and 25 mm on
MDA-MB-231 invasion. b) Cell viability measured by MTT after 16 h within the time frame of the invasion studies. c) Effects of compounds at 25 mm on MDA-
MB-231 migration as assessed by a wound healing assay. d) Snapshots taken over the course of the wound healing experiment to illustrate the effect of the
compounds on migration.
pounds 1 f and 2c inhibited cell migration by 70 and 50%, re-
spectively (Figure 7c,d). In contrast, compounds 1b, 3, and 4b
had no effect on migration (Figure 7c,d). Cell viability was
studied using an MTT assay within a similar time frame (16 h)
used for invasion studies. At 10 mm, most compounds did not
exhibit any cytotoxicity except for 1 f, 2b and 2c, which inhibit
cell growth by 20, 60 and 80%, respectively (Figure 7c). Hence,
the effects of invasion observed at 10 mm for 1b, 1 f, 3, and 4b
are unlikely due to cell toxicity. At 25 mm, only pyrrolidinone 3
and piperidone 4b were non-cytotoxic, confirming that the
50% inhibition of cell invasion at this concentration is unlikely
due to effects on cell viability.
centration-dependent manner with an IC50 value of 28 mm (Fig-
ure 8b). Similarly at 25 mm, a complete inhibition of migration
is observed with 1 f as assessed by a wound healing assay (Fig-
ure 7b). We then tested the effects of piperidinone derivative
4b in cancer cell invasion and metastasis. Similar to pyrazole-
based compound 1 f, piperidinone-based 4b impaired invasion
in a concentration-dependent manner, with 50% inhibition ob-
served at 25 mm (Figure 7a). Importantly, 4b showed no cyto-
toxicity in cell viability studies conducted in parallel with inva-
sion studies. Furthermore, unlike 1 f, 4b showed no inhibition
of MMP-9 activity (Figure 8b) suggesting that the effects of 4b
on invasion are MMP-9-independent.
We used a Matrigel-coated Boyden chamber apparatus to
determine the effect of compounds 1 f and 4b on MDA-MB-
231 invasion in a concentration-dependent manner. Com-
pound 1 f inhibited invasion in a concentration-dependent
manner with an estimated IC50 value of less than 10 mm (Fig-
ure 8a). At 25 mm, a complete inhibition of invasion is ob-
served. To test whether the inhibition of invasion by 1 f is de-
pendent on the degradation of ECM by matrix metalloproteas-
es (MMPs), we performed gelatin zymography as previously
described.[13,32] Compound 1 f inhibits MMP-9 activity in a con-
Next, we tested the effect of 1 f on the integrin-mediated
process of cell adhesion.[18,33–35] MDA-MB-231 cells were added
to wells precoated with fibronectin. In the presence of 1 f,
a concentration-dependent impairment of cell adhesion is ob-
served with an estimated IC50 value of 25 mm (Figure 8c). In
contrast to pyrazole-based compound 1 f, piperidinone deriva-
tive 4b showed no impairment of cell adhesion (Figure 8c)
consistent on the lack of effect on migration (Figure 7b).
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direct the substituents bound to the central core into different
pockets, resulting in better shape complementarity with the
protein.
The difference in the structure of 1 and 4 is reflected in their
dramatically different cellular activity. In cell-based assays, pyra-
zole-containing compounds such as 1 f showed significantly
greater cytotoxicity. Flow cytometry analysis revealed that this
compound also causes significant apoptosis in MDA-MB-231
cells. In contrast, piperidinone compound 4b was non-cytotox-
ic even up to 100 mm. Furthermore, 1 f impaired MDA-MB-231
cell invasion, adhesion, and migration in a concentration-de-
pendent manner, whereas 4b inhibited invasion but had no
effect on adhesion or migration. The effects of 1 f on adhesion
and migration suggest that pyrazole-based compound 1 f
likely inhibits the interactions of uPAR with integrins. On the
other hand, the lack of effects of the piperidinone compound
on cell migration and adhesion suggest that it is unlikely to
perturb the uPAR–integrin association. Gelatin zymography
shows that 1 f impairs gelatinase (MMP-9) activity in a concen-
tration-dependent manner, whereas the piperidinone deriva-
tive did not, indicating that these compounds also differ in
their anti-invasion mechanism. It is well documented that bind-
ing of uPA to uPAR is critical for its activation, and this triggers
the proteolytic degradation of the ECM.[37,38] Activated uPA
converts the extracellular zymogen plasminogen into the
active serine protease plasmin, which degrades the ECM com-
ponents including fibrinogen, fibronectin and vitronectin, and
activates several matrix metalloproteases (MMPs).[39–42]
Finally, our signaling studies revealed that pyrazole-based
compounds such as 1 f, 1i and 1a significantly inhibit MAPK
signaling, whereas piperidinone (4 and 4b) and pyrrolidinone
(3) derivatives had no effect. The inhibition of MAPK phosphor-
ylation might explain the broad cellular activity exhibited by
1 f that includes inhibition of cell growth, migration, invasion
and adhesion, as well as apoptosis. The compound also im-
pairs HIF1a and NF-kB signaling. These effects could be due to
additional unknown off-targets. Future studies will utilize these
compounds as valuable leads to design derivatives with higher
affinities and to unravel the protein–protein interactions of
uPAR.
Figure 8. Invasion, MMP activity and adhesion studies. a) Boyden chamber
apparatus (with Matrigel) was used to assess the effect of 1 f (*) and 4b
(~) on MDA-MB-231 invasion in a concentration-dependent manner.
b) Effect of 1 f and 4b on MDA-MB-231 matrix metalloprotease-9 (MMP-9)
activity evaluated by using gelatin zymography. c) Effect of 1 f (*) and 4b
(~) on MDA-MB-231 cell adhesion to fibronectin.
Experimental Section
Biological methods
Discussion
Fluorescence polarization: Polarized fluorescence intensities were
measured using an EnVision multilabel plate readers (PerkinElmer)
with excitation and emission wavelengths of 485 and 530 nm,
respectively.[8,13] Samples were prepared in black BD Falcon 384-
well microplates with a final volume of 50 mL in duplicate. First, the
test compounds were serially diluted in DMSO and further diluted
in 1x phosphate-buffered saline (PBS) buffer with 0.01% Triton X-
100 to make final concentrations of 100 mm to 0.046 mm. Triton X-
100 was added to the buffer to avoid compound aggregation. Test
compound solution (35 mL) and PBS (10 mL) containing uPAR was
added to the wells, and plates were incubated for at least 15 min
to allow the compound to bind to the protein. Finally, fluorescently
labeled AE147 peptide AE147-FAM (K-S-d-Cha-F-s-k-Y-l-W-S-S-K,
where Cha is l-b-cyclohexyl-alanine) (5 mL) was added to make
Previously, we reported the discovery of anthraquinone-
based[13,27, 36] and pyrazole-based[8] compounds that bind to
uPAR and characterized their activity both in vitro and in vivo.
In this study, we report the design, synthesis and biological
characterization of a series of derivatives of the pyrazole (1), pi-
peridinone (4) and pyrrolidinone (3) cores that were shown to
bind to uPAR. Using fluorescence polarization (FP), we deter-
mined the inhibition constants (Ki) of these derivatives to be in
the range of 6 to 63 mm. Computational studies revealed that
the flat aromatic ring of the pyrazole-based compounds (1)
prefers a different binding mode to uPAR in comparison to de-
rivatives of 3 and 4. The chiral centers in the latter compounds
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a total volume of 50 mL in each well resulting in final uPAR and
peptide concentrations of 320 and 100 nm, respectively. The final
DMSO concentration was 2%, which had no effect on the binding
of the peptide. Controls included wells containing only the peptide
and wells containing both protein and peptide, each in quadrupli-
cates to ensure the validity of the reaction assay. A unit of millipo-
larization (mP) was used for calculating percentage inhibition of
the compounds. Using the Ki calculator available at http://
sw16.im.med.umich.edu/software/calc_ki/, inhibition constants
were measured.
gal filter units (Millipore, #UFC 501024), and electrophoresed on
7.5% sodium dodecyl sulfate (SDS) polyacrylamide gels containing
1 mgmLꢀ1 gelatin. After electrophoresis, the gels were washed
twice with 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 378C 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.
Western blot analysis: Six-well plates were coated with fibronectin
(30 ngmLꢀ1) at 48C overnight. Serum-starved MDA-MB-231 cells
(1.5ꢂ106) were plated onto each well in the presence of DMSO
(control) or 50 mm test compound for 30 min. Total cell lysates
were prepared in standard RIPA extraction buffer containing pro-
tease and phosphatase inhibitors (Sigma). An aliquot of protein
(30 mg) was separated by 10% SDS-PAGE and transferred to nitro-
cellulose membranes (Amersham, Arlington Heights, IL, USA). The
membranes were immunoprobed with phospho-p44/42 MAPK
(Thr202/Tyr204) rabbit monoclonal antibody (1:2000) or p44/42
MAPK mouse monoclonal antibody (1:2000) at 48C overnight.
Next, membranes were incubated with IRDye 800-conjugated goat
anti-mouse IgG (Rockland) or Alexa Fluor 680 goat anti-rabbit IgG
(Invitrogen) as secondary antibodies. Bands were detected using
a Li-Cor Odyssey Imaging System.
Reagents: Biotinylated anti-human uPAR antibody (BAF807) was
purchased from R&D Systems (Minneapolis, MN, USA). Phospho-
p44/42 MAPK (Thr202/Tyr204) rabbit monoclonal antibody (4370)
and p44/42 MAPK mouse monoclonal antibody (9107) were
obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Actin (C-2, sc-8432) antibody was purchased from Santa Cruz Bio-
technology Inc. (Santa Cruz, CA, USA).
Cell culture: MDA-MB-231 and PANC-1 cells were cultured in Dul-
becco’s Modified Eagle Medium (DMEM; Cellgro, Manassas, VA,
USA). AsPC-1 cells were cultured in RPMI-160 medium. Each
medium was supplemented with 10% fetal bovine serum (FBS),
1% penicillin/streptomycin, and cells were cultured in a 5% CO2
atmosphere at 378C.
Cell viability: MDA-MB-231, PANC-1 or AsPC-1 cells (104) were
plated at 100 mL/well in 96-well plates and incubated overnight.
Cells were treated with DMSO (control) or test compound at the
indicated concentration for 3 d. Viable cells were quantified by an
MTT assay at absorbances of 570 nm and 630 nm (reference back-
ground) as previously described.[8,32]
Wound healing assay: Confluent cell monolayers in 12-well plates
were wounded by scraping with a micropipette tip. The cells were
washed and then cultured in complete media containing test com-
pounds. The degree of wound closure was assessed using a Nikon
Diaphot 300 microscope in three randomly chosen regions by
measuring the distance between the wound edges just after
wounding and after 16 h and 40 h.[13]
Adhesion: 96-well plates were coated with 15 ngmlꢀ1 fibronectin
(Sigma–Aldrich, St. Louis, MO, USA) at 48C overnight, then blocked
with 2% bovine serum albumin (BSA) in PBS for 1 h, as described
previously.[8,13,43] After starving with serum-free medium for 4 h,
MDA-MB-231 cells (2.5ꢂ104 cellsmLꢀ1) were suspended in 100 mL
of DMEM containing 0.1% FBS with various concentrations of uPAR
compounds or DMSO control at 378C for 90 min. Medium was
then carefully suctioned out from each well. Each well was washed
three times with PBS, and the number of adherent cells was quan-
tified by MTT assay at 570 nm and 630 nm.
Apoptosis assay: MDA-MB-231 cells were treated with the indicated
concentrations of compounds 1a, 1 f, 1i, 3 and 4 or 1% DMSO
control for 24 h, and flow cytometry analysis was performed as
described previously.[8]
Reporter assay: MDA-MB-231 cells were transfected with each re-
porter. 16 h after carrying out the transfection, the medium was
changed to complete medium (DMEM containing 10% FBS, 1%
nonessential amino acids, 100 UmLꢀ1 penicillin and 100 mgmLꢀ1
streptomycin). After 46 h of transfection, cells were treated with 1 f
at 25 mm. After 48 h of transfection, a dual-luciferase assay was
used to measure the ratio of firefly versus Renilla luciferase units.
The fold change was calculated by dividing the normalized lucifer-
ase ratio of each treated pathway-focused reporter by the normal-
ized luciferase ratio of the untreated pathway-focused reporter.
Experiments were performed in triplicate.
Invasion: Invasion assays were performed using BD Biocoat Matri-
gel invasion chambers (BD Biosciences, San Jose, CA, USA) as previ-
ously described.[8,13,44] The undersurface of the inserts was coated
with fibronectin (30 ngmLꢀ1) at 48C overnight. The inserts were
equilibrated with 0.5 mL of serum-free medium for 2 h at 378C.
After 4 h of serum starvation, cells were harvested and 5ꢂ104 cells
in 500 mL medium containing 0.1% FBS and the indicated com-
pounds or 1% DMSO control were plated onto the upper chamber.
500 mL of 10% FBS medium containing the same amount of com-
pound or DMSO control was added to the lower chamber. After
a 16 h incubation at 378C in 5% CO2, noninvaded cells were re-
moved from the upper chamber with a cotton swab, and the in-
vaded cells were fixed in methanol for 30 min at room temperature
and stained with hematoxylin stain Harris modified method (Fisher
Scientific, Waltham, MA, USA) for 1 h at room temperature. The fil-
ters were washed three time with water, and then air-dried, and
the number of invaded cells was counted in ten separate 200ꢂ
fields.
Computational methods
Pharmacophore hypotheses: Twelve of the highest affinity com-
pounds from pyrazole, pyrrolidinone and piperidinone derivatives
(four from each series) were selected to generate pharmacophore
hypotheses. Compound structures were prepared with LigPrep to
enumerate all possible enantiomers. Three-dimensional conforma-
tions of compounds were generated by docking compounds to
the uPA binding site on multiple uPAR crystal structures. uPAR crys-
tal structures were obtained from the RCSB Protein Data Bank
(PDB) both in its complex form (PDB codes: 1YWH,[46] 3BT2,[25]
3U73[35]) and in its apo form (PDB code: 3U74[35]). uPAR structures
were prepared with Protein Preparation Wizard in the Schrodinger
Gelatin zymography: MDA-MB-231 cells were treated with com-
pounds in serum-free medium for 24 h.[8,36,45] The conditioned
medium was collected and concentrated by Amicon Ultra centrifu-
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(Portland, OR, USA) modeling suite 2011 by removing solvent and
ligands, creating disulfide bonds, filling missing side chains, adding
hydrogen atoms, and optimizing hydrogen network to a pH neu-
tral environment. The docking of compounds to uPAR was carried
out using Autodock Vina (version 1.1.2).[47] All optional docking pa-
rameters were kept at default values, except that the num modes
was reset from 9 to 50 and exhaustiveness from 8 to 100. The
docked conformations of the compounds, whose predicted bind-
ing affinity (vina score) are stronger than ꢀ7.0 kcalmolꢀ1, were
kept for hypotheses generation.
The binding energy of compounds to uPAR protein was calculated
with the MM-PBSA approach.[52] The conformational stability of the
bound compound during simulations was analyzed using RMSD
from its initial binding mode. In total, 600 snapshots were extract-
ed evenly from the production trajectories that kept on the initial
binding mode and subject to MM-PBSA energy analysis. The MM-
PBSA Perl scripts in Amber9 were employed to determine the
binding energy.
Chemistry
The Phase program from Schrodinger was employed to generate
common pharmacophore hypotheses from docked conformations.
The standard features (acceptor, donor, hydrophobic, negative,
positive, and aromatic rings) implemented in the Phase program
were used. To find common pharmacophores, a restriction of
matching all 12 compounds to a five-site pharmacophore was ap-
plied. Further restrictions were applied to match at least two aro-
matic rings out of a maximum of three and one acceptor site out
of a maximum of two. After scoring the active compounds, 18 hy-
potheses survived with the default scoring function. Clustering on
the hypotheses decreased the number down to 16. The hypothe-
ses were then visualized in the presence of the uPAR receptor
structure. Six hypotheses were selected for further evaluation.
General: All chemicals were purchased from either Sigma–Aldrich
or Acros Organics and used as received. Column chromatography
was carried out using silica gel (25–63 m), also used as received. H
1
and 13C NMR were recorded in CDCl3 on a Bruker 500 MHz spec-
trometer. Chemical shifts (d) are reported in parts per million
(ppm) using residual CHCl3 as an internal reference. Coupling con-
stants (J), peak integrals and multiplicity are given in parentheses
using standard abbreviations. High-resolution mass spectra (HRMS)
were measured on an Agilent 6520 Accurate Mass Q-TOF instru-
ment.
1-(tert-Butoxycarbonyl)piperidine-4-carboxylic acid (6): To
a stirred solution of isonipecotic acid (77.4 mmol, 10.0 g) and
K2CO3 (154.8 mmol, 21.4 g) in H2O (150 mL) at 08C was added
dropwise a solution of Boc2O (77.4 mmol, 16.9 g) in THF (150 mL).
The reaction mixture was gradually warmed to RT and stirred over-
night. The solvents were evaporated, and the residue was redis-
solved in CH2Cl2 (50 mL). The organic layer was washed with 1n
HCl (3ꢂ100 mL) and water (1ꢂ50 mL), dried over anhyd Na2SO4, fil-
tered and concentrated in vacuo to give pure 6 as a white powder
QSAR model development: A set of 30 compounds for training
and another 30 for testing, whose activity was uniformly distribut-
ed from most active to inactive across three different series, were
prepared for QSAR model development. With the QSAR Model De-
velopment Tools provided in the Phase program, we built atom-
based QSAR models using the partial least squared (PLS) regression
method applying a set of binary valued variables that encode
whether or not ligand atoms occupy various cube-shaped ele-
ments of space based on the hypotheses developed above. The
QSAR model performance was assessed using a number of metrics,
including R2 the coefficient of determination for regression, stabili-
ty (model stability to the change of training set composition), Q2
on the test set, and Pearson correlation (r) on the test set. Assum-
ing that good pharmacophore hypotheses will generate good
QSAR model performance, two pharmacophore models (HS1119
and HS1135) were selected from the hypothesis pool with the
aforementioned metrics, which were further assessed with exten-
sive molecular dynamics (MD) simulations and energy calculations.
1
(13.03 g, 75%): H NMR (500 MHz, CDCl3): d=4.02 (br s, 2H), 2.85
(t, J=11.5 Hz, 2H), 2.52–2.46 (m, 1H), 1.90 (d, J=11.5 Hz, 2H),
1.70–1.60 (m, 2H), 1.45 ppm (s, 9H); 13C NMR (126 MHz, CDCl3): d=
180.1, 154.7, 79.7, 40.7, 28.3, 27.6 ppm; HRMS: m/z [MH]ꢀ calcd for
C11H18NO4: 228.1241, found: 228.1240.
tert-Butyl 4-(2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-carbonyl)pi-
peridine-1-carboxylate (7): To a stirred solution of 6 (56.8 mmol,
13.03 g) and DMAP (5.68 mmol, 694 mg) in CH2Cl2 (10 mL) at 08C
were added DCC (62.5 mmol, 12.9 g) and 2,2-dimethyl-1,3-dioxane-
4,6-dione (62.5 mmol, 9.00 g) sequentially. The reaction mixture
was gradually warmed to RT and stirred overnight. The reaction
was filtered through filter paper, and the isolated precipitate was
washed with CH2Cl2 and then discarded. The resultant orange solu-
tion was concentrated in vacuo to give an orange oil. The impure
oil was not purified but used directly in next step. The product
was confirmed by MS: m/z 356 [M+H]+.
MD simulations and MM-PBSA calculations: One active com-
pound from each of three series of the compound was selected for
MD simulations. Here, we used compounds 1a, 3a and 4c to rep-
resent the three series. The compound binding pose that con-
formed to the corresponding hypothesis was used for setting up
the simulation. In total, six MD simulations were carried out on
three uPAR–compound complexes in two binding modes based on
hypotheses HS1119 and HS1135. The uPAR crystal structure with
PDB code 1YWH was used for all the simulations. The protocol for
setting up the MD simulations was described elsewhere.[48] Briefly,
the simulation of uPAR protein and compound complex solvated
by TIP3P[49] water molecules were carried out using AMBER9[50] MD
simulation package with ff99SB[51] protein force field. A deliberate
annealing process[48] was employed to equilibrate the solvated
structures before production runs were carried out. The pmemd in
AMBER was employed for production runs. By assigning different
initial velocities, four independent simulation trajectories 12 ns in
length were carried out for each uPAR–compound complex. The
first 2 ns on each trajectory was discarded for equilibration. MD
snapshots were saved every 2 ps, which results in 5000 structures
per trajectory.
tert-Butyl 4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate
(8): A solution of 7 in abs EtOH (200 mL) was heated at reflux for
48 h. The solution was concentrated in vacuo and purified by flash
chromatography (CH2Cl2, 100%) to give 8 as a reddish oil (14.36 g,
1
85%): Rf =0.2 (CH2Cl2, 100%); H NMR (500 MHz, CDCl3): d=12.09
(s, 0.14H, enol OH), 4.89 (s, 0.14H, enol C-H), 4.13 (q, J=7.0 Hz,
2H), 4.10–3.96 (m, 2H), 3.42 (s, 2H), 2.81–2.67 (m, 2H), 2.62–2.52
(m, 1H), 1.85–1.71 (m, 2H), 1.55–1.43 (m, 2H), 1.39 (s, 9H),
1.21 ppm (t, J=7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3): d=204.0,
180.2 (enol), 172.7 (enol), 167.0, 154.4, 87.52, 79.51, 61.3, 48.5, 47.1,
28.2, 27.1, 13.9 ppm; HRMS: m/z [M+H]+ calcd for C15H26NO5:
300.1805, found: 300.1808.
(E)-tert-Butyl 4-(3-ethoxy-2-(ethoxycarbonyl)acryloyl)piperidine-
1-carboxylate (9): Under argon, 8 (47.9 mmol, 14.36 g), triethylor-
thoformate (143.7 mmol, 24 mL), and Ac2O (95.8, 9 mL) were
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mixed, and the reaction was heated at reflux for 48 h. Low-boiling
impurities were evaporated off, and the crude product was purified
by flash chromatography (CH2Cl2, 100%) to give 9 as a yellowish
oil (14.92 g, 88%): Rf =0.2 (1% MeOH/CH2Cl2); 1H NMR (500 MHz,
CDCl3): d=7.59 (s, 0.54H, minor), 7.52 (s, 1H, major), 4.24 (q, J=
7.1 Hz, 2H), 4.21–4.09 (m, 7H), 4.08–3.92 (m, 4H), 3.09–3.01 (m,
0.58H, minor), 2.95–2.87 (m, 1H, major), 2.83–2.67 (m, 4H), 1.85–
1.67 (m, 4H), 1.58–1.47 (m, 4H), 1.41 (s, 19H), 1.37–1.26 (m, 9H),
1.23 ppm (t, J=7.1 Hz, 6H); 13C NMR (126 MHz, CDCl3): d=201.7,
165.7, 165.3, 162.3, 154.6, 112.6, 79.3. 72.2, 60.5, 48.0, 45.4, 28.3,
27.2, 15.2, 14.2 ppm (major isomer); d=199.6, 165.2, 154.6, 112.9,
72.7, 60.7, 28.0, 15.1, 14.1 ppm (minor isomer); HRMS: m/z [M+H]+
calcd for C18H30NO6: 356.2068, found: 356.2067.
vents were removed in vacuo, and the organic residue was redis-
solved in CH2Cl2 (10 mL). The organic phase was washed with satu-
rated aq NaHCO3 (2ꢂ10 mL) and brine (1ꢂ10 mL), dried over
MgSO4, and filtered. The solvent was removed in vacuo to yield de-
sired compound 1e–i. In some cases, purification by flash chroma-
tography was employed (5% (10% NH4OH+MeOH)/CH2Cl2).
1-(3,4-Dimethylphenyl)-N-(3-methoxy-5-(trifluoromethyl)phenyl)-
5-(piperidin-4-yl)-1H-pyrazole-4-carboxamide (1e): off-white solid
1
(42 mg, 36%): Rf =0.3 (5% (10% NH4OH+MeOH)/CH2Cl2); H NMR
(500 MHz, CDCl3): d=8.26 (s, 1H), 7.98 (s, 1H), 7.46 (s, 1H), 7.41 (s,
1H), 7.22 (d, J=8.0 Hz, 1H), 7.10–7.06 (m, 1H), 7.00 (d, J=8.0 Hz,
1H), 6.85 (s, 1H), 4.86 (br s, 2H), 3.81 (s, 2H), 3.25 (app d, J=
12.5 Hz, 2H), 3.12–3.05 (m, 1H), 2.64 (app t, J=11.0 Hz, 2H), 2.50–
2.39 (m, 2H), 2.32 (s, 3H), 2.29 (s, 3H), 2.30–2.25 (m, 2H), 1.67 ppm
(app d, J=12.5 Hz, 2H); 13C NMR (126 MHz, CDCl3): d=161.8, 160.3,
148.6, 139.8, 138.9, 138.6, 138.3, 136.6, 130.4, 127.3, 123.5, 115.1,
55.6, 45.1, 34.1, 28.2, 19.8, 19.5 ppm; HRMS: m/z [M+H]+ calcd for
C25H28F3N4O2: 473.2159, found: 473.2145.
tert-Butyl 4-(1-(3,4-dimethylphenyl)-4-(ethoxycarbonyl)-1H-pyra-
zol-5-yl)piperidine-1-carboxylate (10): 3,4-Dimethylphenyl hydra-
zine was prepared from its HCl salt by dissolving it in saturated aq
NaHCO3 and extracting the solution with CH2Cl2. The organic
phase was isolated, dried over anhyd Na2SO4, filtered and concen-
trated in vacuo to give the free hydrazine.
N-(2-(Benzyloxy)phenyl)-1-(3,4-dimethylphenyl)-5-(piperidin-4-
yl)-1H-pyrazole-4-carboxamide (1 f): white solid (51 mg, 41%):
Rf =0.3 (5% (10% NH4OH+MeOH)/CH2Cl2); 1H NMR (500 MHz,
CDCl3): d=8.50 (d, J=7.0 Hz, 1H), 8.31 (s, 1H), 7.69 (s, 1H), 7.45–
7.40 (m, 4H), 7.39–7.34 (m, 1H), 7.24 (d, J=8.0 Hz, 1H), 7.13 (s,
1H), 7.07–6.99 (m, 4H), 5.15 (s, 2H), 3.16–3.06 (m, 3H), 2.54 (app t,
J=11.0 Hz, 2H), 2.34 (s, 3H), 2.32 (s, 3H), 2.30–2.24 (m, 2H), 2.03
(br s, 1H), 1.60 ppm (app d, J=12.5 Hz, 2H); 13C NMR (126 MHz,
CDCl3): d=161.5, 149.6, 147.2, 138.6, 138.1, 137.9, 137.2, 136.4,
130.1, 128.8, 128.5, 128.4, 127.6, 127.5, 123.7, 123.5, 121.6, 119.8,
115.8, 111.7, 71.1, 46.7, 35.3, 31.6, 30.6, 29.0, 22.6, 19.8, 19.6,
14.1 ppm; HRMS: m/z [M+H]+ calcd for C30H33N4O2: 481.2598,
found: 481.2596.
To a stirred solution of the free hydrazine (2.56 g, 14.9 mmol) in
abs EtOH (0.3m, 140 mL) was added 9 (4.80 g, 13.5 mmol) in abs
EtOH (0.1m, 40 mL). The reaction was heated at reflux for 48 h.
EtOH was removed in vacuo, and the crude reddish brown residue
was purified by flash chromatography (MeOH/CH2Cl2, 1:99) to give
10 as a reddish brown oil (4.76 g, 82%): Rf =0.2 (1% MeOH/
CH2Cl2); 1H NMR (500 MHz, CDCl3): d=7.95 (s, 1H), 7.17 (d, J=
10.0 Hz, 1H), 7.08 (s, J=10.0 Hz, 1H), 6.96 (d, J=10.0 Hz, 1H), 4.25
(q, J=7.1 Hz, 2H), 4.13–4.01 (m, 2H), 3.09–3.02 (m, 1H), 2.61–2.45
(m, 1H), 2.28 (s, 3H), 2.26 (s, 3H), 2.25–2.15 (m, 2H), 1.84–1.71 (m,
1H), 1.51 (app d, 2H), 1.39 (s, 9H), 1.31 ppm (t, J=7.0 Hz, 3H).
5-(1-(tert-Butoxycarbonyl)piperidin-4-yl)-1-(3,4-dimethylphenyl)-
1H-pyrazole-4-carboxylic acid (11): To a stirred solution of 10
(4.76 g, 11.1 mmol) in 95% EtOH (0.1m) was added a 2.0m aq
NaOH solution (4.0 equiv, 22 mL). The reaction mixture was heated
at reflux for 20 h. EtOH was removed in vacuo, and the resulting
solid was acidified to pH 2 at 08C using 1m aq HCl. The reddish
brown solid was isolated by filtration and washed with cold water
1-(3,4-Dimethylphenyl)-N-(4-fluorophenethyl)-5-(piperidin-4-yl)-
1H-pyrazole-4-carboxamide (1g): white solid (61 mg, 58%): Rf =
0.3 (5% (10% NH4OH+MeOH)/CH2Cl2); 1H NMR (500 MHz, CDCl3):
d=7.59 (s, 1H), 7.26–7.16 (m, 3H), 7.09 (s, 1H), 7.02–6.97 (m, 3H),
6.01–5.98 (m, 1H), 3.63 (dd, J=12.5, 6.5 Hz, 2H), 3.13–3.05 (m, 3H),
2.89 (t, J=7.0 Hz, 2H), 2.56 (app t, J=11.0 Hz, 2H), 2.33 (s, 3H),
2.30 (s, 3H), 2.30–2.24 (m, 2H), 1.58 ppm (app d, J=12.5 Hz, 2H);
13C NMR (126 MHz, CDCl3): d=163.7, 162.6, 160.7, 148.5, 138.5,
138.1, 138.0, 137.0, 134.6, 130.2, 127.4, 123.6, 115.5, 115.3, 115.1,
46.1, 40.7, 35.0, 34.7, 30.0, 19.8, 19.5 ppm; HRMS: m/z [M+H]+
calcd for C25H30FN4O: 421.2398, found: 421.2386.
1
to give 11 as a tan solid (3.57 g, 80%): H NMR (500 MHz, CDCl3):
d=8.06 (s, 1H), 7.29–7.22 (m, 1H), 7.12 (s, 1H), 7.03 (d, J=8.0 Hz,
1H), 4.27–4.01 (m, 2H), 3.12 (app t, J=12.0 Hz, 1H), 2.69–2.55 (m,
2H), 2.34 (s, 3H), 2.32 (s, 3H), 2.29–2.20 (m, 2H), 1.57–1.54 (m, 2H),
1.46 ppm (s, 9H); 13C NMR (126 MHz, CDCl3): d=168.1, 154.9, 150.6,
143.5, 138.3, 138.1, 136.8, 130.2, 127.5, 123.6, 111.1, 79.6, 35.1, 28.5,
28.4, 19.8, 19.6 ppm; HRMS: m/z [M+H]+ calcd for C22H30N3O4:
400.2231, found: 400.2246.
1-(3,4-Dimethylphenyl)-N-(4-isopropylphenyl)-5-(piperidin-4-yl)-
1H-pyrazole-4-carboxamide (1h): white solid (60 mg, 58%): Rf =
0.3 (5% (10% NH4OH+MeOH)/CH2Cl2); 1H NMR (500 MHz, CDCl3):
d=7.87 (s, 1H), 7.82 (s, 1H), 7.23–7.17 (m, 4H), 7.11 (s, 1H), 7.04–
7.02 (m, 1H), 3.12–3.05 (m, 4H), 2.89–2.84 (m, 1H), 2.53 (app t, J=
11.0 Hz, 2H), 2.33 (s, 3H), 2.30 (s, 3H), 2.30–2.24 (m, 2H), 1.60 (app
d, J=12.5 Hz, 2H), 1.22 ppm (d, J=7.0 Hz, 6H); 13C NMR (126 MHz,
CDCl3): d=161.9, 149.2, 145.0, 138.7, 138.1, 138.0, 137.0, 135.6,
130.1, 127.5, 126.8, 123.6, 120.6, 115.5, 46.3, 34.9, 33.5, 30.2, 24.0,
19.8, 19.5 ppm; HRMS: m/z [M+H]+ calcd for C26H33N4O: 417.2649,
found: 417.2637.
tert-Butyl 4-(4-((3,5-dimethylphenyl)carbamoyl)-1-(4-isopropyl-
phenyl)-1H-pyrazol-5-yl)piperidine-1-carboxylates
12e–i:
To
a stirred solution of 11 (1.0 equiv) and DMAP (0.1 equiv) in CH2Cl2
(0.1m) at 08C was added DCC (1.1 equiv) and the appropriate ani-
line (1.0 equiv) sequentially. The reaction was gradually warmed to
RT and stirred for 48 h. CH2Cl2 was removed in vacuo, and the
crude product was subjected to flash column chromatography
(EtOAc/hexane, 30:70) to remove unreacted starting material. The
isolated semi-pure material was used immediately in the next step
without further purification. The product was confirmed by
1H NMR but not characterized further due to the impure nature of
the material.
1-(3,4-Dimethylphenyl)-5-(piperidin-4-yl)-N-(4-propylphenyl)-1H-
pyrazole-4-carboxamide (1i): off-white solid (68 mg, 65%): Rf =0.3
1
(5% (10% NH4OH+MeOH)/CH2Cl2); H NMR (500 MHz, CDCl3): d=
7.85 (s, 1H), 7.81 (s, 1H), 7.46–7.42 (m, 2H), 7.22 (d, J=8.0 Hz, 1H),
7.16–7.10 (m, 3H), 7.05–7.03 (m, 1H), 3.13–3.02 (m, 3H), 2.57–2.47
(m, 4H), 2.33 (s, 3H), 2.30 (s, 3H), 2.30–2.24 (m, 2H), 1.66 (br s, 1H),
1.61–1.55 (m, 4H), 0.93 ppm (t, J=7.0 Hz, 3H); 13C NMR (126 MHz,
CDCl3): d=161.9, 149.5, 138.8, 138.7, 138.0, 137.9, 137.1, 135.5,
1-(3,4-Dimethylphenyl)-N-(3,5-dimethylphenyl)-5-(piperidin-4-
yl)-1H-pyrazole-4-carboxamides 1e–i: To a stirred solution of
12e–i (1.0 equiv) in CH2Cl2 (0.5m) at 08C was added TFA (0.5m).
The reaction mixture was warmed to RT and stirred for 1 h. The sol-
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130.1, 128.9, 127.5, 123.6, 120.4, 115.5, 46.7, 37.4, 35.2, 30.8, 24.5,
19.8, 19.5, 13.7 ppm; HRMS: m/z [M+H]+ calcd for C26H33N4O:
417.2649, found: 417.2646.
2H), 2.40 (t, J=7.0 Hz, 1H), 2.36 (t, J=7.0 Hz, 1H), 2.23–2.16 (m,
1H), 2.10–2.00 ppm (m, 1H).
anti-N-(2,4-Dimethoxyphenyl)-2-(4-fluorophenyl)-1-(4-methoxy-
phenyl)-6-oxopiperidine-3-carboxamide (4): To a stirred solution
of 16 (457 mg, 1.33 mmol), EDC (255 mg, 1.33 mmol), and HOBt
(54 mg, 0.40 mmol) in CH2Cl2 (7 mL) at 08C was added dropwise 2,
4-dimethoxyaniline (0.189 mL, 1.33 mmol). The reaction was
warmed to RT and stirred for 2 d. The mixture was concentrated
and partitioned between 1m aq HCl and EtOAc (1:1, 20 mL?) The
organic layer was separated, washed with saturated aq NaHCO3
(3ꢂ15 mL) and brine (1ꢂ20 mL), dried over anhyd Na2SO4, filtered
and evaporated in vacuo. The crude solid was first purified by flash
chromatography (EtOAc, 100%) and then triturated with hexanes
to give 4 as a pinkish solid (444 mg, 70%): 1H NMR (500 MHz,
CDCl3): d=8.10 (d, J=9.0 Hz, 1H), 7.30 (s, 1H), 7.21–7.15 (m, 2H),
7.02 (d, J=9.0 Hz, 2H), 6.93 (t, J=9.0 Hz, 2H), 6.73 (d, J=9.0 Hz,
2H), 6.45 (d, J=9.0 Hz, 1H), 6.40 (d, J=2.5 Hz, 1H), 5.26 (d, J=
7.5 Hz, 1H), 3.77 (s, 3H), 3.73 (s, 3H), 3.70 (s, 3H), 2.90–2.71 (m,
3H), 2.40–2.04 ppm (m, 2H); 13C NMR (126 MHz, CDCl3): d=169.9,
168.9, 161.1, 158.0, 156.8, 149.3, 136.0, 133.9, 129.1, 129.0, 128.9,
121.02, 120.3, 115.7, 115.5, 114.1, 103.7, 98.5, 66.1, 55.6, 55.5, 55.2,
51.1, 31.5, 31.2, 23.2, 22.6, 14.1 ppm; HRMS: m/z [M+H]+ calcd for
C27H28FN2O5: 479.1977, found: 479.1987.
4-Methoxy-N-(4-methoxybenzylidene)aniline (13): In
a round
bottom flask with p-anisidine (1.0 g, 8 mmol) was added CH2Cl2
(40 mL), anhyd MgSO4 (6 g), followed by p-anisaldehyde (0.97 mL,
8 mmol). The reaction was stirred for 72 h at RT, then filtered to
remove MgSO4 and concentrated in vacuo to give 13 as an off-
white solid (1.92 g, 99%): 1H NMR (500 MHz, CDCl3): d=8.40 (s,
1H), 7.83 (d, J=8.5 Hz, 2H), 7.21 (d, J=8.5 Hz, 2H), 6.97 (d, J=
9.0 Hz, 2H), 6.92 (d, J=9.0 Hz, 2H), 3.86 (s, 3H), 3.83 ppm (s, 3H).
1,2-bis(4-Methoxyphenyl)-5-oxopyrrolidine-3-carboxylic
acid
(14): A solution of imine 13 (200 mg, 0.83 mmol) and succinic an-
hydride (200 mg, 0.83 mmol) in xylenes (1 mL) was heated at reflux
for 24 h. The reaction was slowly cooled to RT and extracted with
saturated aq NaHCO3 (3ꢂ10 mL). The aqueous extracts were
washed with hexane and then acidified to pH 2 by addition of
concd HCl to give a white precipitate, which was subsequently ex-
tracted into Et2O (3ꢂ15 mL). The combined Et2O extracts were
dried over anhyd MgSO4, filtered and concentrated in vacuo to
give 14 as
a
yellow–white powder (181 mg, 64%): 1H NMR
(500 MHz, CDCl3): d=7.21 (d, J=9.0 Hz, 2H), 7.16 (d, J=9.0 Hz,
2H), 6.83 (d, J=9.0 Hz, 2H), 6.77 (d, J=9.0 Hz, 2H), 5.38 (d, J=
5.0 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 3H), 3.20–3.14 (m, 1H), 3.05 (dd,
J=17.5, 9.5 Hz, 1H), 2.96 ppm (dd, J=17.5, 7.0 Hz, 1H).
Abbreviations
Acetic anhydride (Ac2O); bovine serum albumin (BSA); N,N’-dicyclo-
hexylcarbodiimide (DCC); 4-dimethylaminopyridine (DMAP); dime-
thylsulfoxide (DMSO); di-tert-butyl dicarbonate (BOC2O); Dulbecco’s
modified Eagle medium (DMEM); 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide (EDC); fetal bovine serum (FBS); hydroxybenzo-
triazole (HOBt); matrix metalloprotease (MMP); molecular dynamics
(MD); nonessential amino acids (NEAA); phosphate-buffered saline
(PBS); sodium dodecyl sulfate (SDS); tetrahydrofuran (THF); tri-
fluoroacetic acid (TFA).
N-(2-Ethoxyphenyl)-1,2-bis(4-methoxyphenyl)-5-oxopyrrolidine-
3-carboxamide (3): To a stirred solution of 14 (181 mg, 0.53 mmol)
and DMAP (6 mg, 0.053 mmol) in CH2Cl2 (5 mL) at 08C was added
DCC (120 mg, 0.58 mmol) followed by o-phenetidine (0.069 mL,
0.53 mmol). The reaction was warmed to RT and stirred for 3 d. The
reaction was filtered through Celite, and the filtrate was concen-
trated in vacuo. The crude solid was purified by flash chromatogra-
phy (EtOAc/hexane, 60:40) to give 3 as a white solid (146 mg,
1
70%): H NMR (500 MHz, CDCl3): d=8.38 (d, J=8.0 Hz, 1H), 7.58 (s,
1H), 7.21–7.15 (m, 4H), 7.06–7.01 (m, 1H), 7.00–6.93 (m, 1H), 6.84–
6.79 (m, 3H), 6.79–6.74 (m, 2H), 5.34 (d, J=7.0 Hz, 1H), 4.04–3.94
(m, 2H), 3.75 (s, 3H), 3.71 (s, 3H), 3.24–3.11 (m, 2H), 3.00–2.93 (m,
1H), 1.26 ppm (t, J=7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3): d=
172.3, 168.7, 159.4, 157.3, 147.0, 131.4, 130.1, 128.1, 127.1, 125.4,
124.2, 120.9, 119.9, 114.4, 114.0, 110.9, 66.6, 64.0, 55.2, 55.1, 50.3,
34.8, 14.5 ppm; HRMS: m/z [M+H]+ calcd for C27H29N2O5:
461.2071, found: 461.2080.
Acknowledgments
This research was supported by the US National Institutes of
Health (NIH) (CA135380 to S.O.M.), an Indiana Genomics Initiative
(INGEN) Grant from Lilly Endowment, Inc. (S.O.M.), the Indiana
University Melvin and Bren Simon Cancer Center Translational Re-
search Acceleration Collaboration (ITRAC) (S.O.M.), the Showalter
Trust (S.O.M.), the Indiana University Biomedical Research Fund
(S.O.M.), and a Research Scholar Grant from the American
Cancer Society (RSG-12-092-01 to S.O.M.).
N-(4-Fluorobenzylidene)-4-methoxyaniline (15): In
a
round
bottom flask with p-anisidine (1.0 g, 8 mmol) was added CH2Cl2
(40 mL), anhyd MgSO4 (6 g), followed by 4-fluorobenzaldehyde
(0.86 mL, 8 mmol). The reaction was stirred for 72 h at RT, then fil-
tered to remove MgSO4 and concentrated in vacuo to give 15 as
1
an off-white solid (1.91 g, 99%): H NMR (500 MHz, CDCl3): d=8.44
(s, 1H), 7.88 (dd, J=8.5, 5.5 Hz, 2H), 7.23 (d, J=8.5 Hz, 2H), 7.15 (t,
J=9.0 Hz, 2H), 6.94 (d, J=9.0 Hz, 2H), 3.84 ppm (s, 3H).
Keywords: cancer · inhibitors · invasion · metastasis · protein–
protein interaction · small molecules · uPAR · urokinase ·
urokinase receptors
anti-2-(4-Fluorophenyl)-1-(4-methoxyphenyl)-6-oxopiperidine-3-
carboxylic acid (16): A solution of imine 15 (910 mg, 3.97 mmol)
and glutaric anhydride (453 mg, 3.97 mmol) in xylenes (5 mL) was
heated at reflux for 72 h. The reaction was slowly cooled to RT and
extracted with saturated aq NaHCO3 (3ꢂ30 mL). The aqueous ex-
tracts were washed with hexane and then acidified to pH 2 by ad-
dition of concd HCl to give 16 as a white precipitate (457 mg,
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Received: August 21, 2013
Published online on October 2, 2013
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