Synthesis and characterization of a novel prostate cancer-targeted phosphatidylinositol-3-kinase inhibitor prodrug

Article abstract of DOI:10.1021/jm300881a

The phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway is constitutively activated in a substantial proportion of prostate tumors and is considered a key mechanism supporting progression toward an androgen-independent status, for which no effective therapy is available. Therefore, PI3K inhibitors, alone or in combination with other cytotoxic drugs, could potentially be used to treat cancer with a constitutive activated PI3K/Akt pathway. To selectively target advanced prostate tumors with a constitutive activated PI3K/Akt pathway, a prostate cancer-specific PI3K inhibitor was generated by coupling the chemically modified form of the quercetin analogue LY294002 (HO-CH₂-LY294002, compound 8) with the peptide Mu-LEHSSKLQL, in which the internal sequence HSSKLQ is a substrate for the prostate-specific antigen (PSA) protease. The result is a water-soluble and latent PI3K inhibitor prodrug (compound 11), its activation being dependent on PSA cleavage. Once activated, the L-O-CH₂-LY294002 (compound 10) can specifically inhibit PI3K in PSA-secreting prostate cancer cells and induce apoptosis with a potency comparable to that of the original LY294002 compound.

Full text of DOI:10.1021/jm300881a

Article
pubs.acs.org/jmc
Synthesis and Characterization of a Novel Prostate Cancer-Targeted
Phosphatidylinositol-3-kinase Inhibitor Prodrug
Daniele Baiz,† Tanya A. Pinder,‡ Sazzad Hassan,† Yelena Karpova,† Freddie Salsbury,# Mark E. Welker,*,‡
and George Kulik*,†
†Department of Cancer Biology and Comprehensive Cancer Center, Wake Forest School of Medicine, Medical Center Boulevard,
Winston-Salem, North Carolina 27157, United States
‡Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, United States
#Department of Physics, Wake Forest University, P.O. Box 7486, Winston-Salem, North Carolina 27109, United States
ABSTRACT: The phosphatidylinositol-3-kinase/Akt (PI3K/Akt)
pathway is constitutively activated in a substantial proportion of
prostate tumors and is considered a key mechanism supporting
progression toward an androgen-independent status, for which no
effective therapy is available. Therefore, PI3K inhibitors, alone or in
combination with other cytotoxic drugs, could potentially be used to
treat cancer with a constitutive activated PI3K/Akt pathway. To
selectively target advanced prostate tumors with a constitutive
activated PI3K/Akt pathway, a prostate cancer-specific PI3K inhibitor
was generated by coupling the chemically modified form of the quercetin analogue LY294002 (HO-CH2-LY294002, compound
8) with the peptide Mu-LEHSSKLQL, in which the internal sequence HSSKLQ is a substrate for the prostate-specific antigen
(PSA) protease. The result is a water-soluble and latent PI3K inhibitor prodrug (compound 11), its activation being dependent
on PSA cleavage. Once activated, the L-O-CH2-LY294002 (compound 10) can specifically inhibit PI3K in PSA-secreting prostate
cancer cells and induce apoptosis with a potency comparable to that of the original LY294002 compound.
cytoplasm and nucleus, where it phosphorylates downstream
substrates involved in angiogenesis, cell cycle progression,
growth, migration, proliferation, and survival.4
INTRODUCTION
■
Prostate cancer remains the second leading cause of cancer-
related death in men in the United States. Conventional
treatments such as surgery, radiation, and androgen suppression
are effective if prostate cancer is confined to the prostate.
Unfortunately, many patients with advanced metastatic cancer
treated with androgen ablation experience recurrence of
androgen-independent cancer, with limited or transient
response to other systemic chemotherapies.1,2 For this reason,
there is an urgent need for new specific and targeted agents to
treat androgen-independent prostate cancer. Several mecha-
nisms have been proposed to explain why prostate cancer cells
can grow in the absence or reduced presence of androgens.
Recent papers suggest that the phosphatidylinositol 3-kinase/
Akt (PI3K/Akt) pathway is one of the mechanisms that allow
prostate cancer cells to maintain continued proliferation in a
low-androgen environment.3
The PI3K pathway is a key signal transduction pathway
initiated by receptor tyrosine kinases that recruit and activate
the PI3K, resulting in an accumulation of phosphatidylinositol
3,4,5-triphosphate (PIP3) in plasma membrane. This lipid
second messenger recruits Akt and the phosphoinositide-
dependent protein kinase 1 (PDK1) to the cell membrane,
where Akt is phosphorylated by PDK1 at threonine 308.
Activated Akt recruits the mammalian target of rapamycin
(mTOR) that, acting with Rictor protein, forms the mTORC2
complex, which completes the activation of Akt by phosphor-
ylation at serine 473. Fully activated Akt translocates to the
Constitutive activation of the PI3K/Akt pathway in prostate
cancer is often led by functional loss of the tumor suppressor
PTEN (a phosphatase and tensin homologue deleted on
chromosome 10) that dephosphorylates PI3K substrates or by
activating mutations in the PI3 kinase itself that correlate with
increased Akt phosphorylation, higher Gleason grade, advanced
stage, and unfavorable prognosis.5,6 For these reasons, PI3K
inhibitors have been considered as an adjuvant therapy for
advanced prostate cancer; unfortunately, despite promising
effects in preclinical models, recent clinical trials did not show
benefits in prostate cancer-affected patients treated with PI3K
inhibitors (source www.ClinicalTrials.gov).
One possible approach for improving the efficacy of PI3K
inhibitors to treat prostate cancer patients may be to convert
the PI3K inhibitor molecule into an inactive prodrug by
attaching a specific prostate-specific antigen (PSA) cleavable
peptide, increasing the delivery to tumor sites while minimizing
systemic toxicity. PSA is a protease with chymotrypsin-like
activity and is involved in the hydrolytic processing of
semenogelins, which are necessary for seminal fluid liquefac-
tion. In patients with prostate cancer, systemic PSA
concentration is high, but inactive, in blood serum as PSA is
Received: June 22, 2012
Published: August 27, 2012
© 2012 American Chemical Society
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complexed with the α1-antichymotrypsin (PSA-ACT) or α2-
macroglobulin.7,8 On the contrary, in the tumor environment,
PSA is free (fPSA) and enzymatically active, able to activate
cytotoxic prodrugs based on a PSA-cleavable peptidic
sequence.9
AutoDock’s estimator of binding affinity (Figure 1A, in blue).16
On the basis of this analysis, a phenyl ring in LY294002
In this work, we describe for the first time the synthesis and
characterization of a prostate cancer-specific PI3K inhibitor
prodrug based on the quercetin analogue LY294002 activated
by PSA cleavage. On the basis of previous reports of anticancer
toxins converted to PSA-activated prodrugs, we linked the PI3K
inhibitor LY294002 with the Mu-LEHSSKLQL peptide,
containing the HSSKLQ sequence specific for PSA cleav-
age.8,10−12 The generated PI3K inhibitor prodrug LY294002
(11) is water-soluble and is specifically activated in the media
conditioned by the prostate cancer cells C4-2 that secrete PSA.
Upon activation, the PI3K inhibitor prodrug 11 showed
consistent time-dependent and concentration-dependent in-
hibition of the PI3 kinase and induction of apoptosis. The
specificity of the PI3K inhibitor prodrug 11 for PSA-secreting
prostate cancer cells was confirmed using the BT-549 breast
cancer cell line and glioblastoma-astrocytoma U-87 MG cells,
which do not produce PSA: in these cells the PI3K pathway
activity is not affected by 11. Consistent with the requirement
of cleavage by PSA for drug activation, PI3K/Akt signaling in
BT-549 breast cancer cells is inhibited if 11 is previously
incubated in C4-2 conditioned medium or in the presence of
enzymatically active purified PSA.
Figure 1. Modeling of the interaction between LY294002 derivative
and PI3 kinase ATP-binding site. (A) Ribbon diagram of an X-ray
structure of the PI3K ATP-binding site (gray) with LY294002. An
active site Lys in bonds is shown for reference. Modeled structurally
similar docking poses for LY294002 (blue) and 10 (red) are shown for
comparison. (B) PI3K inhibitor prodrug (11) formula. Arrow points at
predicted prostate-specific antigen cleavage site.
Prostate tumor PSA-dependent inhibition of PI3K is
expected to produce dual benefits for patients: by completely
inhibiting the PI3K pathway at the tumor site and reducing side
effects due to inhibition of PI3K in normal tissues, systemic
exposure should be minimal, resulting only from the active drug
being redistributed from the tumor.13,14
structure was identified to attach a CH2OH moiety, which
subsequently was used to couple the Mu-LEHSSKLQL
sequence, containing the PSA substrate peptide (Figure 1B).
Because PSA is predicted to cleave Mu-LEHSSKLQL peptide
after glutamine (Q), it leaves leucine (L) attached to the phenyl
ring of the LY294002 via the CH2OH moiety. The AutoDock
modeling of interaction between this “activated prodrug” 10
and PI3K has shown that it can still fit into the ATP binding
site and, thus, is predicted to inhibit PI3K activity (Figure 1A,
in red).
Synthesis of Compound 11. We synthesized 8-(4-
(hydroxymethyl)phenyl)-2-morpholino-4H-chromen-4-one
(8), an analogue of LY294002 containing a CH2OH functional
group, which should not adversely affect the PI3K binding
ability but will permit the attachment of PSA substrate peptides
(Figure 2A; numbers hereafter refer to structures in Figure 2).
The synthesis of 8 was modeled after the synthetic strategy
presented by Abbott and Thompson.17 Commercially available
aromatic ester 2,3-dihydroxybenzoic acid (1) was converted to
methyl 2,3-dihydroxybenzoate (2) by acid-catalyzed addition of
methanol. The least sterically hindered hydroxyl was then
converted to a triflate by treatment with trifluoromethylsulfonic
anhydride to produce methyl-2-hydroxy-3-{[(trifluoromethyl)-
sulfonyl]oxy}benzoate (3). The enolate of N-acetylmorpholine
(4) was then generated with lithium diisopropyl amide and
condensed with the ester in 3 to produce 2-hydroxy-3-(3-
morpholin-4-yl-3-oxopropanoyl)phenyl trifluoromethanesulfo-
nate (5). 5 underwent cyclodehydration after being treated
with triflic anhydride to give 2-morpholin-4-yl-4-oxo-4H-
chromen-8-yl trifluoromethanesulfonate (6). 6 was then
DESIGN AND SYNTHESIS
■
PI3K Inhibitor Prodrug (Compound 11) Design. To
generate a PI3K inhibitor prodrug activated by the prostate-
specific antigen, we chose the quercetin analogue LY294002
(developed by Eli Lilly Co.). The stability of this agent in
aqueous solutions made it a reagent of choice for numerous
tissue culture studies of PI3K signaling. To determine the
position in the LY294002 structure where an inhibitory peptide
could be attached, we conducted modeling studies of
interactions between the ATP binding pocket of the PI3
kinase and LY294002. These modeling studies have been
performed using an existing crystal structure of the PI3 kinase
to which LY294002 has been bound as a template.15
AutoDock3 software with AutoDock Tools was used to
generate the appropriate charge, solvent, and van der Waals
parameters for LY294002 and the PI3 kinase.16 Electrostatic
and van der Waals potential grids for docking were generated
on the basis of these parameters and were centered at the
center of geometry for LY294002 in the crystal structure,
although LY294002 was removed before grid creation.
To ensure both high resolution and sampling of a large
region of protein for possible binding sites, the grids were
generated at 0.375 Å resolution in a cube of 30 × 30 × 30 Å.
LY294002 was redocked into the active site with 256 runs of
AutoDock’s Lamarckian genetic algorithm. Default parameters
were used except in the number of runs (256) and the size of
the initial pool (30000 initial dockings). Subsequent analysis
showed that the LY294002 was docked with a similar poise as
in the crystal structure with 4.0 μM predicted affinity, using
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Figure 2. Synthesis of LY294002 analogues. Once 8 was prepared (panel A), it was used to attach leucine, generating 10 (panel B), or the Mu-
LEHSSKLQ-peptide (panel C), generating 11, the PI3K inhibitor prodrug.
subjected to Suzuki coupling with commercially available 4-
(hydroxymethyl)phenylboronic acid (7) to produce 8. Once
the LY294002 analogue with the primary alcohol functional
group (8) was prepared, it could be used to attach the leucine
or the Mu-LEHSSKLQL-peptide (Figure 2B,C).
First, we synthesized 4-(2-morpholino-4-oxo-4H-chromen-8-
yl)benzyl-2-(tert-butoxycarbonylamino)-4-methylpentanoate
(9), where 8 is directly attached to BOC-protected leucine. The
BOC protecting group can be removed by p-TsOH when
desired to produce 4-(2-morpholino-4-oxo-4H-chromen-8-yl)
benzyl-2-amino-4-methylpentanoate (10). 10 represents what
would remain after PSA has cleaved off the substrate peptide
from the prodrug. The peptide Mu-LEHSSKLQL was attached
to 8 by AnaSpec, Inc. (Fremont, CA), to yield Mu-
LEHSSKLQL-8-(4-(hydroxymethyl)phenyl)-2-morpholino-
4H-chromen-4-one (11) as a white powder. Mass calculated as
1487.7 was found to be 1488.0 by LC-MS analysis, conducted
at AnaSpec, Inc. (provided with the certificate of analysis).
BIOLOGICAL RESULTS
■
Compound 10 Inhibits the PI3 Kinase. The androgen-
independent prostate cancer C4-2 cells that secrete PSA were
chosen for this study as a model of advanced and PSA-secreting
prostate cancer and used for a preliminary experiment. In
parallel, as a negative control, we used the breast cancer BT-549
cell line, which does not secrete PSA. Both cell lines are PTEN-
deficient and show constitutive activation of the PI3K/Akt
signaling pathway.
As reported, PSA is predicted to cleave 11 after the
glutamine, leaving a leucine attached to the phenyl ring of
the LY294002. We started by testing whether the “activated
prodrug” 10 retained inhibitory properties toward PI3K
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inhibition, similarly to unmodified LY294002, as predicted by
AutoDock modeling.
The activated prodrug 10 inhibited the PI3 kinase with a
potency comparable to that of LY294002, resulting in a dose-
dependent reduction of p-Akt T308 levels in both cell lines 30
min after administration (Figure 3). Dose−response analysis
shows that 30 μM 10 completely inhibited Akt phosphorylation
in both cell lines.
Figure 3. Compound 10 exerts PI3K inhibitory properties similar to
those of LY294002. At 30 min after the administration of the
LY294002 or compound 10, the “activated prodrug”, dose-dependent
decreases of p-Akt T308 were detected both in prostate cancer C4-2
and in breast cancer BT-549 cells versus control (cells treated with
DMSO). Similar results were obtained in three independent
experiments.
Compound 11 Inhibits the PI3 Kinase in PSA-
Secreting Prostate Cancer Cells. Effects of 11 were
monitored in prostate cancer C4-2 and breast cancer BT-549
cells from 30 min to 6 h. A time-dependent reduction of p-Akt
T308 levels was observed in prostate cancer C4-2 cells versus
control (cells treated with DMSO). Notably, no significant
differences in p-Akt T308 levels were detected in breast cancer
BT-549 cells treated with 11 during this time period (Figure
4A). To determine whether PI3 kinase inhibition is due to the
cleavage of Mu-LEHSSKLQL peptide by PSA and conversion
of 11 into 10, we monitored concentrations of 10 in the media
conditioned by either prostate cancer C4-2 or breast cancer
BT-549 cells. LC-ESI-MS analysis showed a significant increase
of 10 in the media conditioned by prostate cancer C4-2 cells
(that secrete PSA) at 30 min and 3 and 6 h after administration,
whereas no significant increase was found in the media
conditioned by breast cancer BT-549 cells compared to control
(cells treated with PBS) (Figure 4B). In addition, the tPSA
assay confirmed that during these time points, PSA
concentrations were significantly increased in prostate cancer
C4-2 conditioned media, whereas no PSA was detected in
breast cancer BT-549 conditioned media (Figure 4C). These
results suggest that PSA−peptide cleavage is required for
activation of 11.
Figure 4. Compound 11 selectively inhibits PI3 kinase in PSA-
secreting prostate cancer cells. (A) Prostate cancer C4-2 and breast
cancer BT-549 cells were treated with 11 for indicated periods of time.
Time-dependent reduction of p-Akt T308 was seen in prostate cancer
C4-2 cells (top panel). No significant differences in p-Akt T308 levels
were detected in breast cancer BT-549 cells treated with 11 in the
same time frame (bottom panel). A representative Western blot, of
three independent experiments, is shown. (Control = cells treated with
To further demonstrate that inhibition of the PI3K/Akt
pathway by 11 depends on the presence of PSA in the media,
we incubated 11 in the medium conditioned by prostate cancer
C4-2 cells and then, after an incubation of 6 h, we transferred
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glioblastoma-astrocytoma U-87 MG cells that do not secrete
PSA (Figure 6A,B). Consistent with earlier results, p-Akt T308
levels were reduced in LNCaP cells incubated with 11 and in
C4-2 cells in a dose-dependent fashion (Figure 6A). This effect
was not seen in U-87 MG cells or in BT-549 cells (Figure 6B),
supporting the notion that secretion of PSA in the media
(confirmed by tPSA assay) (Figure 6C) is needed to cleave the
Mu-LEHSSKLQL peptide.
Compound 11 Promotes Apoptosis in C4-2LucBAD
Prostate Cancer Cells. As we have previously shown,
LY294002 induces apoptosis in C42LucBAD cells that
ectopically express BAD, a BH3-only pro-apoptotic protein of
the Bcl-2 family.18−21
Figure 4. continued
PBS.) (B) LC-ESI-MS analysis of cell media treated with 11 showed
significantly increased concentrations of 10 in the media conditioned
by C4-2 cells, but no increase in BT-549 conditioned media. Results
are expressed as the mean SEM, n = 3; (∗) p < 0.039 and (∗∗) p <
0.012 versus control, represented by BT-549 or C4-2 conditioned
media. (C) Total PSA assay (tPSA) showing significant increase of
secreted PSA in prostate cancer C4-2 cells in conditioned media. No
PSA was detected in breast cancer BT-549 cells conditioned media.
Results are expressed as the mean SEM, n = 3; (∗) p < 1.1 × 10−5,
(∗∗) p < 4.1 × 10−6, and (∗∗∗) p < 3.26 × 10−5 versus control,
represented by BT-549 or C4-2 media treated with 30 μM 11.
Administration of 11 inhibited the PI3K/Akt pathway and
induced apoptosis in C42LucBAD cells, as evident from
increased caspase 3 activity and cleavage of caspase substrate
PARP (Figure 7). Somewhat lower levels of caspase activity
induced by 11 are likely due to extra time needed for
conversion of 11 into an active PI3K inhibitor. These
experiments unequivocally demonstrate that 11 is converted
by PSA cleavage into an active PI3K inhibitor that selectively
blocks the PI3K/Akt pathway and induces apoptosis in PSA-
secreting prostate cancer cells.
the C4-2 conditioned media (containing also 11 activated by
PSA cleavage) into Petri dishes containing breast cancer BT-
549 cells. As shown in Figure 5A, addition of 11 previously
DISCUSSION AND CONCLUSIONS
■
In this paper, we have demonstrated for the first time the
conversion of a non-tissue-specific PI3K inhibitor (LY294002)
into an inactive and latent prodrug (11), which can be
specifically activated by the PSA protease. Earlier publications
have shown that various nonspecific cytotoxic drugs, including
thapsigargin, 5-fluorodeoxyuridine, and doxorubicin, could be
converted into inactive prodrugs by attaching a PSA-cleavable
substrate peptide; however, no publications have reported an
attempt to convert a PI3 kinase inhibitor (or any kinase
inhibitor) into latent prodrugs activated by PSA cleavage.22−24
Converting a PI3K inhibitor into a latent and prostate cancer-
specific prodrug may allow an increased drug dosage,
accomplishing complete inhibition of PI3K activity in prostate
cancer cells while at the same time reducing systemic cytotoxic
effects exerted by non-tissue-specific PI3K inhibitors. Com-
pared to nontargeted PI3K inhibitors, a PI3K inhibitor prodrug
is expected to be more effective against prostate tumors with a
constitutively activated PI3K pathway, which become depend-
ent on PI3K signaling for their growth and survival.25,26
Figure 5. PSA converts compound 11 into an active PI3K inhibitor.
(A) Addition of 11 (incubated for 6 h in media conditioned by
prostate cancer C4-2 cells) resulted in significant reduction of p-Akt
T308 in breast cancer BT-549 cells. (Control = cells treated with
PBS.) (B) Compound 11 incubated for 6 h with a range of
concentrations (0−104 ng/mL) of active PSA and added to breast
cancer BT-549 cell media for 30 min resulted in significant decreases
of p-Akt T308, if compared to control. (Control = cells treated with
DMSO.) Similar results were obtained in three independent
experiments.
Although this narrows the spectrum of potentially targeted
tumors compared to “nonspecific” cytotoxic drugs, there are
several advantages of targeting the PI3K signaling pathway in
prostate cancer. First, normal tissues do not require constitutive
PI3K activity; thus, the presence of the PI3K inhibitor outside
tumor sites is not expected to produce substantial toxic side
effects, as evident from phase I clinical trials with several
nontargeted PI3K inhibitors.27,28 Second, as constitutively
activated PI3K activity is connected with increased resistance
to apoptosis in prostate cancer, a prostate cancer-selective PI3K
inhibitor is expected to sensitize prostate tumors to other
anticancer therapeutics.29,30 Third, as the PI3K inhibitor
prodrug cannot enter into cells (and be metabolized) until
“released” from inhibiting peptide, these agents should have an
improved pharmacokinetic profile compared to unmodified
PI3K inhibitor, as reported for SF1126, a LY294002 derivative
coupled with a RGDS peptide.14 Also, unlike the original
LY294002, which has poor water solubility and must be
dissolved in dimethyl sulfoxide, the peptide-coupled prodrug-
incubated in the media conditioned by prostate cancer C4-2
cells significantly reduced p-Akt T308 levels in breast cancer
BT-549 cells (which do not secrete PSA). In contrast, 11
incubated in the media conditioned by breast cancer BT-549
cells had no effects on p-Akt T308 levels. Finally, to confirm
that inhibition of PI3K activity by 11 was due to peptide
cleavage by PSA (and not by unrelated proteases secreted by
prostate cancer C4-2 cells), 11 was incubated for 6 h at 37 °C
with increasing concentrations of enzymatically active PSA
purified from human semen. Addition of 11 (preincubated with
PSA) to BT-549 cells for 30 min reduced p-Akt T308 levels in a
PSA dose-dependent fashion (Figure 5B). Notably, 11 without
enzymatically active PSA had no effect on p-Akt T308 levels in
BT-549 cells.
To further demonstrate that activation of 11 is PSA-
dependent, we extended the screening to hormone-sensitive
prostate cancer LNCaP cells (that secrete PSA) and to
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Figure 6. Selective inhibition of PI3K in PSA-secreting prostate cancer cells by compound 11. (A) Incubation of prostate cancer LNCaP cells and
C4-2 cells with 11 at 3 h resulted in a dose-dependent reduction of p-Akt T308 levels. (Control = cells treated with PBS.) (B) No significant
reduction of p-Akt T308 levels was observed in glioblastoma-astrocytoma U-87 MG cells or breast cancer BT-549 cells. (Control = cells treated with
PBS.) Similar results were obtained in three independent experiments. (C) Total PSA (tPSA) assay confirms secretion of PSA in prostate cancer
LNCaP and C4-2 cell media at 3 h. (Control = cell media.) As expected, breast cancer BT-549 and glioblastoma-astrocytoma U-87 MG cells did not
secrete PSA. Results are expressed as the mean SEM, n = 3; (#) p < 4.2 × 10−9 and (∗) p < 6.3 × 10−10 versus control.
condensed by rotary evaporation and purified on alumina using 95:5
ethyl acetate/ethanol as an eluent to give 8-(4-(hydroxymethyl)-
phenyl)-2-morpholino-4H-chromen-4-one as a pale yellow powder
LY294002 here presented is water-soluble and, thus, could be
more easily formulated as an orally or intravenously delivered
therapeutic.
1
(0.062 g, 0.184 mmol, 67%): Rf 0.5 (95:5 ethyl acetate/ethanol); H
Future experiments in mouse models of prostate cancer will
examine pharmacokinetics, pharmacodynamics, and antitumor
effects of 11. Should these experiments demonstrate superior
properties of 11 compared to unmodified LY294002, it will
justify modification of other PI3K inhibitors into PSA-activated
prodrugs and clinical testing of these new prodrug-PI3K
inhibitors in patients with advanced prostate cancer. Activating
mutations of PI3K pathway are observed in almost all advanced
prostate tumors; therefore, introducing improved prostate-
selective PI3K inhibitors into the clinic may benefit a
substantial number of prostate cancer patients.
NMR (300 MHz, CDCl3) δ 8.18 (dd, J = 1.7, 7.9 Hz, 1H), 7.50 (m,
6H), 5.52 (s, 1H), 4.80 (s, 2H), 3.74 (t, J = 4.8 Hz, 4H), 3.36 (t, J =
5.1 Hz, 4H); 13C NMR (75.47 MHz, CDCl3) δ 177.23, 162.57,
150.57, 140.93, 135.49, 133.56, 130.03, 129.46, 126.82, 125.05, 124.76,
123.35, 87.03, 65.88, 64.75, 44.74. HRMS [M + H]+ calculated for
C20H19NO4, 338.1392; found, 338.1393.
4-(2-Morpholino-4-oxo-4H-chromen-8-yl)benzyl-2-(tert-butoxy-
carbonylamino)-4-methylpentanoate (9). 8-(4-(Hydroxymethyl)-
phenyl)-2-morpholino-4H-chromen-4-one, 8 (0.259 g, 0.766 mmol),
was dissolved in DCM (20 mL). N-α-tert-Butoxycarbonyl-L-leucine (2
equiv, 0.354 g, 1.532 mmol), DCC (2 equiv, 0.316 g, 1.532 mmol),
and DMAP (1 equiv, 0.094 g, 0.766 mmol) were added, and the
solution was stirred at room temperature overnight. DCM (90 mL)
was added, and the solution was washed with cold 1 M HCl (80 mL),
NaHCO3 (60 mL), and brine (60 mL). Organic fractions were dried
with Na2SO4, and the reaction mixture was condensed by rotary
evaporation and purified on alumina using ethyl acetate as an eluent to
give 4-(2-morpholino-4-oxo-4H-chromen-8-yl)benzyl-2-(tert-butoxy-
carbonylamino)-4-methylpentanoate as a clear oil (0.161 g, 0.293
mmol, 38%): Rf 0.6 (100% ethyl acetate); 1H NMR (300 MHz,
CDCl3) δ 8.16 (dd, J = 1.8, 7.8 Hz, 1H), 7.56−7.37 (m, 6H), 5.52 (s,
1H), 5.22 (s, 2H), 4.96 (d, J = 8.6 Hz, 1H), 4.37 (m, 1H), 3.73 (t, J =
4.4 Hz, 4H), 3.34 (t, J = 4.9 Hz, 4H), 2.03 (m, 2H), 1.62 (m, 1H),
1.43 (s, 9H), 0.92 (dd, J = 1.1, 6.5 Hz, 6H); 13C NMR (75.47 MHz,
EXPERIMENTAL SECTION
■
Chemical Synthesis. 8-(4-(Hydroxymethyl)phenyl)-2-morpholi-
no-4H-chromen-4-one (8). 2-Morpholin-4-yl-4-oxo-4H-chromen-8-yl
trifluoromethanesulfonate, 7 (100 mg, 0.263 mmol), was dissolved in
5:1 MeCN/EtOH (10 mL). K2CO3 (2.5 equiv, 0.091 g, 0.658 mmol)
was added, and the solution was degassed with N2. 4-
(Hydroxymethyl)phenylboronic acid (1.21 equiv, 0.048 g, 0.318
mmol) and Pd(OAc)2 (0.1 equiv, 0.006 g, 0.0263 mmol) were
added, and the solution was refluxed overnight. The solution was
cooled to room temperature, filtered through Celite, washed with
MeCN (10 mL), and dried with Na2SO4. The reaction mixture was
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The purity of compounds was measured by LC-ESI-MS analysis
using an Accela Open 1200 UHPLC coupled to an LTQ XL Orbitrap
mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA),
resulting to be ≥95%. The purity of 11 was >95% after LC-ESI-MS
analysis, conducted at AnaSpec, Inc., as verified by the certificate of
analysis.
Cell Cultures. The hormone-responsive prostate cancer LNCaP
and breast cancer BT-549 cell lines were purchased from ATCC
(Manassas, VA, USA). The androgen-independent prostate cancer
LNCaP subline C4-2 and the glioblastoma-astrocytoma U-87 MG cells
were generous gifts from Dr. Leland Chung (Cedars-Sinai Medical
Center, Los Angeles, CA, USA) and Dr. Frank Furnari (University of
California, San Diego, CA, USA), respectively.
All cells were maintained in a humidified atmosphere (5% CO2) at
37 °C, using RPMI 1640 (C4-2, C4-2LucBAD, and BT-549), T-
Medium (LNCaP), and DMEM (U-87 MG) supplemented with 10%
FBS (5% for LNCaP). BT-549 medium was also supplemented with
0.023 U/mL of insulin. All tissue culture reagents were purchased from
Invitrogen (Carlsbad, CA, USA).
Experimental Setup. If not otherwise indicated, cells were seeded
at 4 × 105/6 cm Petri dish and kept in culture for 48 h to reach about
80% confluence before the start of any experiments. Cells were kept in
a serum-free media condition (LNCaP cells were kept in medium
supplemented with 2.5% FBS). C4-2LucBAD cells were kept in serum-
free medium for 18 h to increase PSA concentration.
Total PSA Electrochemiluminescence Immunoassay. Total
PSA was assayed with a Cobas Elecsys Total PSA according to the
manufacturer’s protocol, using a Roche Elecsys 2010 Chemistry
Analyzer (Roche, Basel, Switzerland). Each sample was assayed in
triplicate.
Western Blotting. Media were removed and cells washed gently
two times using cold phosphate-buffered saline (PBS) on ice. Lysates
were generated using cold 20 mM HEPES, 150 mM NaCl, 1 mM
EDTA, 0.5% Na+deoxycholate, 1% Nonidet P-40, and 1 mM DTT, pH
7.4, buffer containing protease inhibitors (10 μg/mL aprotinin, 10 μg/
mL leupeptin, 10 μg/mL pepstatin, 1 mM benzamidine, and 1 mM
PMSF) and phosphatase inhibitors (1 mM NaVO4, 50 mM β-
glycerophosphate, 40 mM p-nitrophenylphosphate, 40 mM NaF, and
1 μg/mL microcystin). All reagents were purchased from Sigma-
Aldrich (St. Louis, MO, USA). Cell lysates were clarified by
centrifugation at 15000 rpm for 15 min at 4 °C, and the supernatant
was collected and protein content measured by a Bradford assay (Bio-
Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s
directions. Proteins were separated on SDS-PAGE on 12% gels and
transferred onto a 0.45 μm nitrocellulose membrane (PerkinElmer,
Waltham, MA, USA). Western blotting was performed with the
Odyssey CLx Infrared Imaging System (Li-Cor Biosciences, Lincoln,
NE, USA), according to the manufacturer’s instructions. Rabbit
polyclonal anti-p-Akt Thr308, anti-Akt, and anti-cleaved PARP
(Asp214) were purchased from Cell Signaling Technology (Beverly,
MA, USA). Mouse monoclonal anti-β-actin was purchased from
Sigma-Aldrich. Secondary goat anti-mouse IRDye 680 and goat-anti-
rabbit IRDye 800 were both purchased from Li-Cor Biosciences.
Protein bands were quantified using ImageJ software (National
Institutes of Health, Bethesda, MD, USA).
Caspase 3 Assay. Apoptosis in whole cell populations was
assessed by monitoring caspase 3 activity with the specific fluorogenic
substrate Ac-DEVD-7-amido-4-trifluoromethylcoumarin (Bachem,
Torrance, CA, USA), as previously reported.30 On ice, adherent and
floating cells were collected and lysed using a cold lysis buffer (1%
Nonidet P-40, 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1 mM
DTT, and 5 μg/mL aprotinin, leupeptin, and pepstatin, respectively).
Fluorescence was recorded each 15 min for 1 h using a VersaFluor
(Bio-Rad). Caspase 3 activity was expressed in arbitrary units and
calculated using Excel 2010 (Microsoft Corp., Redmond, WA, USA).
LC-ESI-MS Analysis. LC-ESI-MS analysis was performed using an
Accela Open 1200 UHPLC coupled to an LTQ XL Orbitrap mass
spectrometer (Thermo Fisher Scientific). Separation was accom-
plished using a Zorbax Eclipse XDB-C18 (1.8 μm, 2.1 × 50 mm)
analytical column with a Zorbax extend C18 narrow-bore guard
Figure 7. Compound 11 induces apoptosis in C4-2LucBAD cells. (A)
Compound 11 induced a dose-dependent reduction of p-Akt T308 in
C4-2LucBAD cells in 3 h. (Control = cells treated with PBS.) (B)
Caspase 3 assay performed in C4-2LucBAD cells 6 h after the
administration of 10 and 11. Results are expressed as the mean
SEM, n = 2; (#) p < 0.0062, (##) p < 0.012; (∧) p < 0.0015; (∧∧) p <
8.3 × 10−5, (∗) p < 0.0055, (∗∗) p < 0.0022, (§) p < 0.003, and (§§) p
< 0.001 versus control. (Control = cells treated with DMSO.) (C)
Samples from (B) were analyzed for the cleaved PARP (89 kDa),
confirming activation of caspases effector after the administration of 10
or 11. (Control = cells treated with DMSO.) Similar results were
obtained in three independent experiments.
CDCl3) δ 177.10, 173.37, 162.48, 155.39, 150.47, 136.25, 135.38,
133.47, 129.50, 127.94, 125.18, 124.75, 123.31, 87.02, 79.90, 66.34,
65.84, 52.13, 44.66, 41.58, 28.25, 24.75, 22.81, 21.82. HRMS [M +
Na]+ calculated for C31H38N2O7, 573.2577; found, 573.2643.
4-(2-Morpholino-4-oxo-4H-chromen-8-yl) benzyl-2-amino-4-
methylpentanoate (10). 4-(2-Morpholino-4-oxo-4H-chromen-8-yl)
benzyl-2-(tert-butoxycarbonylamino)-4-methylpentanoate, 9 (0.070 g,
0.127 mmol), was dissolved in MeCN (15 mL). p-TsOH·H2O (2
equiv, 0.044 g, 0.254 mmol) was added and the solution stirred at
room temperature for 42 h. p-TsOH·H2O (1 equiv, 0.022 g, 0.127
mmol) was added and the solution stirred for an additional 23 h. The
reaction mixture was condensed by rotary evaporation. The solid
residue was dissolved in cold H2O (6 mL), and ethyl acetate (10 mL)
was added. The aqueous layer was separated, and saturated NaHCO3
(5 mL) was added. An aqueous layer was extracted with ethyl acetate
(3 × 10 mL). Organic fractions were dried with Na2SO4, and solvent
was removed by rotary evaporation to give 4-(2-morpholino-4-oxo-
4H-chromen-8-yl)benzyl 2-amino-4-methylpentanoate as a white solid
(0.033 g, 0.073 mmol, 58%): 1H NMR (300 MHz, CDCl3) δ 8.16 (dd,
J = 1.6, 7.8 Hz, 1H), 7.46 (m, 6H), 5.52 (s, 1H), 5.22 (s, 2H), 3.71 (t, J
= 4.8 Hz, 4H), 3.57 (m, 1H), 3.34 (t, J = 5.0 Hz, 4H), 1.91 (m, 2H),
1.62 (m, 1H), 0.91 (t, J = 6.5 Hz, 6H); 13C NMR (75.47 MHz,
CDCl3) δ 177.53, 176.60, 162.93, 150.93, 136.78, 136.04, 133.91,
130.12, 130.00, 128.52, 125.66, 125.21, 123.77, 87.50, 66.55, 66.29,
45.13, 44.48, 29.17, 25.19. HRMS [M + H]+ calculated for
C26H30N2O5, 451.2233; found, 451.2302.
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Journal of Medicinal Chemistry
Article
column (5 μm, 2.1 × 12.5 mm) (Agilent Technologies, Santa Clara,
CA, USA). The following mobile phases were used for the separation:
solvent A, water/0.1% formic acid; and solvent B, methanol/0.1%
formic acid. The gradient used for separation was 95 to 5% solvent A
over 10 min at a flow rate of 250 μL/min. Using the mass of 496.2569
0.005, optimized positive mode ESI conditions were found as
follows: sheath and auxiliary gas, 60 and 4 arbitrary units, respectively;
spray voltage, 4 kV; capillary temperature and voltage, 325 °C and 49
V, respectively; tube lens voltage, 115 V. Full scan high-resolution
mass spectra were collect at 60000 Hz from m/z 150 to 2000. The full
scan data for standard curve samples and unknowns were processed
with Xcalibur software to determine concentrations (Thermo Fisher
Scientific).
C4-2 Conditioned Medium Assay. C4-2 and BT-549 cells were
cultured as described above. 11 was added to the medium with C4-2
cells and incubated for 6 h at 37 °C in a humidified atmosphere (5%
CO2) in serum-free medium. Medium was then collected and added to
BT-549 cells for 3 h. Inhibition of the PI3 kinase pathway was followed
by monitoring the p-Akt T308 levels by Western blotting.
Cleavage of Compound 11 with Purified PSA. Compound 11
was incubated in 50 mM Tris-HCl−0.1 M NaCl, pH 7.8, buffer for 6 h
at 37 °C with purified active human PSA (Merck, Darmstadt,
Germany) and then added to BT-549 cells for 30 min, cultured as
previously described. Inhibition of PI3 kinase was followed by
monitoring p-Akt T308 levels by Western blotting.
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AUTHOR INFORMATION
■
Corresponding Author
(11) LeBeau, A. M.; Banerjee, S. R.; Pomper, M. G.; Mease, R. C.;
Denmeade, S. R. Optimization of peptide-based inhibitors of prostate-
specific antigen (PSA) as targeted imaging agents for prostate cancer.
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*(M.E.W.) Phone: +1-(336)-758-3898. E-mail: welker@wfu.
edu (G.K.) Phone: +1-(336)-713-7650. E-mail: gkulik@
wakehealth.edu.
(12) Vlahos, C. J.; Matter, W. F.; Brown, R. F.; Traynor-Kaplan, A.
E.; Heyworth, P. G.; Prossnitz, E. R.; Ye, R. D.; Marder, P.; Schelm, J.
A.; Rothfuss, K. J.; Serlin, B. S.; Simpson, P. J. Investigation of
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Rev. Drug Discov. 2011, 10, 563−564.
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Murali, R.; Lu, Y.; Mills, G. B.; Kundra, V.; Shu, H. K.; Peng, Q.;
Durden, D. L. A vascular targeted pan phosphoinositide 3-kinase
inhibitor prodrug, SF1126, with antitumor and antiangiogenic activity.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Marcus W. Wright (Wake Forest University,
Department of Chemistry) for LC-ESI-MS analysis, Tina
Snider (Department of Urology, Wake Forest Baptist Health)
for performing ECLIA tPSA assays, and Karen Kline for editing.
This work was supported by WFU Cross-Campus Collabo-
rative Research Program and R01CA118329 to G.K.
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ABBREVIATIONS USED
■
BAD, Bcl-2-associated death promoter; Bcl-2, B-cell lymphoma
2; Bcl-XL, B-cell lymphoma extra-large; DCC, N,N′-dicyclohex-
ylcarbodiimide; DCM, dichloromethane; DMAP, 4-dimethyla-
minopyridine; DMSO, dimethyl sulfoxide; DTT, dithiothritol;
PARP, poly(ADP-ribose) polymerase; PBS, phosphate buffer
saline; PI3K, phosphatidylinositol-3-kinase; PSA, prostate-
specific antigen; PTEN, phosphatase and tensin homologue
deleted on chromosome 10
(18) Zha, J.; Harada, H.; Yang, E.; Jockel, J.; Korsmeyer, S. J. Serine
phosphorylation of death agonist BAD in response to survival factor
results in binding to 14-3-3 not BCL-X(L). Cell 1996, 87, 619−628.
(19) Datta, S. R.; Dudek, H.; Tao, X.; Masters, S.; Fu, H.; Gotoh, Y.;
Greenberg, M. E. Akt phosphorylation of BAD couples survival signals
to the cell-intrinsic death machinery. Cell 1997, 91, 231−241.
(20) del Peso, L.; Gonzalez-Garcia, M.; Page, C.; Herrera, R.; Nunez,
G. Interleukin-3-induced phosphorylation of BAD through the protein
kinase Akt. Science 1997, 278, 687−689.
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