Nitric oxide (NO) releasing poly ADP-ribose polymerase 1 (PARP-1) inhibitors targeted to glutathione S-transferase P1-overexpressing cancer cells

Article abstract of DOI:10.1021/jm401550d

We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1). Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that a Me₂N-N(O)=NO- group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of systemic toxicity.

Full text of DOI:10.1021/jm401550d

Article
pubs.acs.org/jmc
Nitric Oxide (NO) Releasing Poly ADP-ribose Polymerase 1 (PARP-1)
Inhibitors Targeted to Glutathione S‑Transferase P1-Overexpressing
Cancer Cells
Anna E. Maciag,*,† Ryan J. Holland,‡ Youseung Kim,‡ Vandana Kumari,§ Christina E. Luthers,‡
Waheed S. Sehareen,‡ Debanjan Biswas,‡ Nicole L. Morris,∥ Xinhua Ji,§ Lucy M. Anderson,‡
Joseph E. Saavedra,† and Larry K. Keefer‡
†Chemical Biology Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick,
Maryland 21702, United States
‡Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States
§Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States
∥Laboratory Animal Sciences Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research,
Frederick, Maryland 21702, United States
S
* Supporting Information
ABSTRACT: We report the antitumor effects of nitric oxide (NO) releasing derivatives of the PARP-1 inhibitor olaparib (1).
Compound 5b was prepared by coupling the carboxyl group of 3b and the free amino group of arylated diazeniumdiolated
piperazine 4. Analogue 5a has the same structure except that the F is replaced by H. Compound 13 is the same as 5b except that
a Me2N−N(O)NO− group was added para and ortho to the nitro groups of the dinitrophenyl ring. The resulting prodrugs are
activated by glutathione in a reaction accelerated by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed
in cancers. This metabolism generates NO plus a PARP-1 inhibitor simultaneously, consuming reducing equivalents, leading to
DNA damage concomitant with inhibition of DNA repair, and in the case of 13 inducing cross-linking glutathionylation of
proteins. Compounds 5b and 13 reduced the growth rates of A549 human lung adenocarcinoma xenografts with no evidence of
systemic toxicity.
maximum therapeutic benefit of these agents by blocking the
repair process. There are multiple ongoing clinical trials
evaluating the efficacy of PARP-1 inhibitors as chemo-
potentiators in several cancers, including non-small-cell lung
cancer (NSCLC). However, early phase clinical studies of
PARP-1 inhibitor, compound 1, in combination with top-
otecan,1 dacarbazine,2 or cisplatin plus gemcitabine3 showed
dose-limiting toxicity that was more pronounced than that seen
with the chemotherapeutic agents alone. Therefore, targeted
INTRODUCTION
■
Poly ADP-ribose polymerase 1 (PARP-1) is a critical enzyme in
the repair of DNA strand breaks. This 116 kDa nuclear protein
detects DNA single strand breaks and utilizes NAD+ as a
substrate to poly(ADP-ribosyl)ate nuclear proteins, resulting in
relaxation of chromatin and recruitment of other repair proteins
to the damaged site. PARP-1 is an attractive antitumor target
because of this vital role in DNA repair. The current clinical
approaches to the development of PARP-1 inhibitors include
either (1) the utilization as a single agent in BRCA1 or BRCA2-
deficient cancers where inhibition of PARP results in synthetic
lethality or (2) the utilization in combination with DNA
damaging therapeutics (radiation or chemotherapy) to increase
Received: October 4, 2013
Published: February 12, 2014
© 2014 American Chemical Society
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delivery of PARP inhibitors selectively to cancer cells could be a
solution to overcome these systemic toxicity problems.
Diazeniumdiolate-based nitric oxide (NO) releasing pro-
drugs developed in our laboratory have proven to be effective as
anticancer agents in a number of in vitro and in vivo models.4−9
The lead compound, O2-(2,4-dinitrophenyl) 1-[(4-
ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K,
2), exhibits a multifaceted mechanism of action initiated by
depletion of intracellular GSH and induction of oxidative stress,
followed by the activation of stress signaling, abrogated
mitochondria function, DNA damage, and apoptosis. DNA
single strand break damage (measured by Comet assay) was
observed in the H1703 NSCLC cell line after a 1 h treatment
with 2, and massive DNA damage was seen after 24 h. In
multiple myeloma cells, 2 caused DNA double strand break
damage.6 We hypothesized that inhibition of PARP may
enhance the potency of 2 and other NO-releasing prodrugs of
the arylated diazeniumdiolate class.
In this work, we have designed and synthesized NO-releasing
PARP-1-inhibitor prodrugs that are activated by reaction with
glutathione (GSH), a reaction that is accelerated by glutathione
S-transferase (GST), a family of phase II detoxification
enzymes. We show that these hybrid prodrugs are effective
against cancer cells in vitro and in vivo. Activation of the
compounds by GSTP1, an isoform of GST that is frequently
overexpressed in cancers, should result in the preferential
metabolism and thus release of cytotoxins in cancer cells,
potentially diminishing systemic toxicity associated with PARP
inhibition.
of two NOs plus known PARP inhibitors 6a and 6b, as
illustrated in Scheme 3.
Prodrug Activation and NO Release. The first step in the
activation of these prodrugs is the arylation of GSH, liberating a
diazeniumdiolate ion that spontaneously decomposes to NO,
freeing the PARP-inhibitory part of the molecule. Accordingly,
5a and 5b reacted smoothly with GSH to generate the expected
products, shown in Scheme 3. The rates of reaction of 5a and
5b with 4 mM GSH in aqueous pH 7.4 phosphate buffer (0.1
M) containing 50 μM diethylenetriaminepentaacetic acid at 37
°C were determined. The t1/2 for 5a was 11.2 min, and the t1/2
for 5b was 11.6 min. Liquid chromatography/mass spectrom-
etry (LC/MS) analysis of the reaction mixture after 2 half-lives
confirmed that 25% of the parent compound was still present
while 75% consisted of deacylated compound 1 (6b) and the
aryl-glutathione conjugate (7) (Scheme 3, Figure S1).
PARP-1 Enzyme Inhibition. The ability of the new
compounds to inhibit PARP-1 enzymatic activity was tested
using an assay that measures incorporation of biotinylated poly
ADP-ribose onto histone proteins. 5a and 5b were compared to
1 and authentic compounds 6a and 6b that are products of 5a
and 5b activation (Scheme 3). Compounds 6a and 6b had
PARP-1 inhibitory activities comparable to that of 1. PARP-1
enzyme IC50 values estimated for 6a, 6b, and 1 were 30.5, 6.9,
and 15.5 nM, respectively (Figure 1). Both prodrugs 5a and 5b,
without activation by GSH, were less potent, with IC50 of 123
nM for 5a and 58 nM for 5b.
In Vitro Antiproliferative Activity. Compounds 5a and
5b inhibited proliferation of an extensive panel of NSCLC cell
lines with IC50 concentrations ranging from 3 to 20 μM (Table
1). IC50 values for NO-releasing PARP-1 inhibitor prodrugs
were significantly lower than those of 1 for the most sensitive
cell lines.
The IC50 values correlated with endogenous ROS levels and
with levels of antioxidant enzyme peroxiredoxin 1 and DNA
repair enzyme 8-oxoguanine DNA glycosylase (OGG1, Figure
S2). Depleting the cell’s GSH during prodrug activation
combined with NO release led to induction of oxidative/
nitrosative stress (Figure S3) and cytotoxicity through cellular
stress overload, especially in cells with high endogenous ROS
levels. Since the proliferation screen and measurements of
PARP-1 inhibitory activities of both prodrugs showed fluoro-
substituted analogue 5b to be more potent than 5a, we decided
to focus on 5b for further development.
RESULTS AND DISCUSSION
■
Prodrug Design Considerations. Our specific strategy
here is to combine the structural features of the established
PARP-1 inhibitor 1 and arylated diazeniumdiolate 2, structures
shown in Scheme 1. Specifically, we wanted to produce
Scheme 1. Structures of the PARP-1 Inhibitor 1 and
a
Anticancer Drug Candidate 2
DNA Damage and Activation of Apoptosis. Activation
of PARP is an ATP-depleting process. Cellular levels of ATP
fall below a critical level, and cells with substantial DNA
damage cannot enter apoptosis, rendering them resistant to this
form of cell death or susceptible to necrosis. Therefore,
inhibition of PARP-1 in combination with substantial DNA
damage would conserve cellular energy and could allow tumor
cells to undergo apoptosis in response to DNA-damaging NO,
eliminating toxic effects or immune responses associated with
necrosis.
Twenty-four-hour treatment with NO-releasing PARP-1
inhibitor prodrug 5b resulted in significant DNA strand break
damage as observed by Comet assay. Treatment with 5b at 5
μM resulted in a stronger Comet signal, compared with 20 μM
1 (Figure 2A). A strong apoptotic signal (as evidenced by
cleaved caspase 7) was seen in cells treated with 5b, while the
same concentration of 1 did not trigger apoptosis (Figure 2B).
Cellular Uptake and GST-Catalyzed Metabolism. To
validate the release of the PARP-1 inhibitor in cells, we
a
Structural features to be merged in designing NO-releasing PARP
inhibitors for the present study are color coded: green for the arylating
fragment that is transferred to GSH in the activation step; red for the
PARP inhibitor moiety; and blue for the two molecules of cytolytic
nitric oxide released on activation.
molecules that link the PARP-inhibitory skeleton of 1 (the red
portion of structure 1 in Scheme 1; the black cyclopropane
carboxamide group is not necessary for PARP-1 inhibition)
with the electrophilic aryl ring shown in green and the caged
NO molecules shown in blue in Scheme 1. Accordingly, we
reacted known compounds 3a and 3b10 with 47 to obtain the
first two drug candidates, 5a and 5b, as shown in Scheme 2.
Our hypothesis was that attack by GSH on these molecules
would lead to consumption of a reducing equivalent through
irreversible arylation of the GSH, with simultaneous generation
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Scheme 2. Synthesis of NO-Releasing PARP-1 Inhibitors 5a and 5b
Scheme 3. Activation of 5b in the Presence of 4 mM GSH
Table 1. Antiproliferative Activities of NO-Releasing PARP-
1 Inhibitor Prodrugs Compared with 1 in NSCLC Cells
IC50 SD (μM)
cell line
5a
5b
1
H1568
H1703
H441
6.5 0.2
6.8 0.3
8.1 0.8
5.3 0.4
11.2 1.8
9.0 1.1
11.4 1.3
7.5 0.6
13.8 2.7
14.0 1.6
14.0 0.8
16.8 2.2
19.4 2.5
13.8 1.6
3.0 0.1
4.3 0.3
4.5 0.3
5.4 1.2
7.5 0.4
7.9 0.8
8.2 1.4
8.4 0.4
9.2 1.1
10.5 3.1
12.7 1.5
13.5 1.0
15.0 1.3
16.4 0.9
36 7.7
20 4.2
28 2.5
19 1.4
38 4.4
50 7.6
33 2.6
10 1.2
34 0.9
25 4.0
16 3.7
28 3.4
20 1.0
12 2.6
H1693
H2122
H322M
H1355
H23
H2030
H1944
H1792
A549
Figure 1. Inhibition of PARP-1 enzyme activity by 5a, 5b, 6a, and 6b,
compared with 1. Inhibitor concentrations were tested in the range
0.01 nM to 1 μM.
H2023
H460
amounts, suggesting very rapid, presumably catalyzed, cellular
metabolism (Figure 3).
evaluated cellular uptake and metabolism of 5b in A549
NSCLC cells. Cells were treated with 5b for varying time
periods, and cell lysates were analyzed for the presence of the
parent compound and its metabolites by LC/MS. The parent
compound was not detected at any measured time point
including a brief treatment of 5 min (asterisk (∗) in Figure 3).
At all time points measured up to 1 h, compound 6b and
arylated GSH metabolite 7 were detected in equally abundant
Lung cancer cells frequently express high levels of GSTP1,
the GST isoform that is often overexpressed in cancer cells. We
have screened a panel of 20 NSCLC cell lines for the level of
expression of GSTP1 protein using Western blot. This
experiment confirmed that the enzyme is highly expressed in
a majority of NSCLC cell lines, with little to none of the liver-
specific A1 isoform (GSTA1) detected (Figure S4). Therefore,
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Table 2. Selectivity of Prodrugs for GST-Catalyzed
Metabolism
t1/2 (min) in
4 mM GSH
k
GSH/kGSH
kGSTP/kGSH
kGSTA/kGSH
compd
(mean SD) (mean SD) (mean SD)
a
2
2.8 0.02
2.0 0.05
11.6 0.08
16.0 0.06
1
1
1
1
0.001
0.003
0.007
0.004
1.1 0.01
5.4 0.7
10.6 0.5
13.3 0.4
10.5 0.8
3.7 0.08
20.4 0.7
9.2 0.1
8
5b
13
a
Only two measurements made in the first half-life for the GSTA1
trials.
GSTA1; the A1 isoform was found to be a better catalyst for
glutathione attack than GSTP1 (Table 2, row 3). Further
synthetic approaches were taken to diminish GSTA1 catalysis.
Synthesis and Metabolism of a Bis-diazeniumdiolated
PARP-1 Inhibitor. In an effort to modify the structure of 5b to
make it less susceptible to GSTA1-induced metabolism and to
improve its suitability as a substrate for GSTP1, we employed a
strategy developed previously to do so with compound 2.11 In
that case, molecular modeling suggested that adding and
reducing steric bulk at different specific locations in the
structure of 2 would accomplish this goal. As predicted,
compound 8 (PABA/NO, structure in Scheme 4A) was
prepared and shown to be metabolized at similar rates by
GSTP1 and GSTA1, in contrast to 2, which was an order of
magnitude more efficiently metabolized by GSTA1 (Table 2,
rows 1 and 2).
Figure 2. Treatment with NO-releasing PARP-1 inhibitor prodrug 5b
resulted in DNA damage (A), as shown by the Comet assay in H1703
NSCLC cells. The DNA strand break damage caused by 5b was more
extensive than that resulting from 1 treatment. (B) Activation of
apoptosis as evidenced by cleavage of caspase 7. Actin served as a
loading control.
We chose a similar strategy in designing the bis-
diazeniumdiolated PARP-1 inhibitor hybrid 13 shown in
Scheme 4B as a synthetic target. Thus, Boc-protected
piperazine diazeniumdiolate 9 was reacted with aryl fluoride
substituted diazeniumdiolate 10 to give adduct 11, which upon
removal of the protecting group gave 12. This compound
contains nucleophilic nitrogen in the piperazine ring that was
reacted with 3b to produce the bis-diazeniumdiolated PARP-1
inhibitor 13.
Compound 13 was subjected to a similar reactivity and
metabolism analysis as 5b. Not only was compound 13 more
stable than 5b in reactions with GSH alone, but the rates of
catalysis suggest that compound 13 is better accommodated in
the active site of GSTP1 than that of GSTA1 (Table 2, row 4).
Molecular modeling experiments suggested that compound 13
fits nicely into the GSTP1 active site while a steric clash may
occur in the GSTA1 active site (Figure 4).
Compound 13 PARP-1 enzyme inhibitory activity in the
presence or absence of GSH and GSTP1 was also analyzed.
Mass spectrometric analysis of the cellular metabolism of
compound 13 in A549 lung adenocarcinoma cells and U937
human leukemia cells revealed that the compound is
metabolized as designed to release NO upon GSH/GST
attack. The dearylation of the first diazeniumdiolate moiety
results in spontaneous release of NO and frees the PARP-1-
inhibitory component. The resulting glutathione conjugate
reacts further with GSH, leading to attachment of two
glutathionyl substituents to the dinitrophenyl ring (Scheme
4C). Alternatively, the single glutathione conjugates could react
with protein thiols instead of GSH, resulting in a protein
adduct, in which GSH and the protein’s thiol group are
covalently cross-linked through the dinitrophenyl ring. This
thiol modification (cross-linking glutathionylation) appears to
be irreversible and likely plays an important role in mediating
the cytotoxic activity of bis-diazeniumdiolates.12 It is also
Figure 3. In vitro metabolism of compound 5b. The human A549
NCSLC cell line was treated with 10 μM 5b. Then the products were
extracted at various time points and analyzed by LC/MS/MS. Shown
are extracted ion chromatograms (EICs), which were extracted at
various m/z ratios including the [M + H]+ of 6b (retention time (tR)
of 11.9 min), arylated GSH (7, tR = 13.8 min), and compound 5b (tR =
18.2 min) (Scheme 3). The asterisk (∗) indicates where compound 5b
should be, based on the elution profile of a lysate spiked with authentic
compound 5b (bottom panel).
we studied the rate of reaction of 5b with GSH in the presence
of recombinant GSTP1 and found that the reaction was
enhanced over 10-fold when the enzyme was present (Table 2,
row 3). This strongly suggests that the prodrug, despite its
reactivity with GSH alone, will be preferentially activated when
GSTP1 is present. Importantly, the presence of GSTP1 did not
alter product distribution (Figure S5). Next, 5b was
independently reacted with glutathione in the presence of
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Scheme 4. (A) Design Considerations Leading to the Structure-Based Design of 8 as an Improvement over 2 in Terms of Their
Suitability as Substrates for GSTP1, (B) Synthesis of NO-Releasing PARP-1 Inhibitor 13, and (C) Metabolism of 13
predictable that this irreversible modification will affect proper
protein folding, which, if unresolved, leads to the unfolded
protein response (UPR) and endoplasmic reticulum (ER)
stress.
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Figure 4. Top panels: Transition state modeling of the 13−glutathione adduct in the GSTA1 and GSTP1 active sites. Bottom panel: Inhibition of
PARP-1 enzyme by compound 13 in the presence or absence of GSH/GSTP1. Activated compound is a much better PARP-1 inhibitor than the
prodrug.
Induction of Endoplasmic Reticulum Stress by
Compound 13. We exploited this hypothesis and found that
indeed treatment with 13 results in cellular protein
glutathionylation and ER stress (Figure 5A). Treated cells
were lysed and processed for immunoblotting under non-
reducing conditions, and protein/GSH cross-links were
detected with specific antibodies recognizing glutathione
conjugated to proteins. As shown in Figure 5A, glutathione
attached to protein was detected within 1 h of treatment, and
the signal became gradually stronger with time. The decrease in
protein/GSH conjugate signal observed at 8 h was only seen
upon the onset of apoptosis, as indicated by cleaved PARP
(Figure 5A). This essentially irreversible modification con-
sumes GSH and results in a shift in the cellular redox balance
toward a more oxidizing environment, generating oxidative/
nitrosative stress. ER stress induction was evidenced by
phosphorylation of eIF2α and up-regulation of CHOP, BiP,
and ATF4 proteins (Figure 5B). Activation of stress kinase p38
was seen within 15 min after treatment with 13 was initiated
(Figure 5C) and could be a result of ROS/RNS stress. We have
observed rapid and persistent phosphorylation of p38 in U937
leukemia cells upon treatment with compound 2.13
NO-Releasing PARP-1 Inhibitors Compare Favorably
with 1 in Vivo. Human lung adenocarcinoma cell line A549,
which expresses moderate levels of GSTP1, was chosen for
assessment of activity of the prodrugs in vivo against
xenografted cells in athymic mice. Compounds 5b and 13
were chosen for in vivo studies. Both compounds were
administered at 92 μmol/kg intravenously two times a week
for a 4-week period to assess anticancer activity. Figure 6 shows
significant reduction of the tumor growth in animals treated
with compound 13 (P < 0.02), compared to 1 or saline
controls. Compound 5b reduced growth of tumors to a lesser
extent. Importantly, treatment with either controls or
diazeniumdiolate-based drugs did not affect body weights
(see caption to Figure 6). Tumors from animals in each group
were resected and extracted for analysis of metabolites. Both
the parent drugs and the liberated PARP-1 inhibitor were
detected in some tumors from animals treated with compounds
5b and 13, indicating unambiguous delivery of compound to
tumors remote from the site of injection.
Combination with Bortezomib. The ubiquitin−protea-
some system is critical for the proliferation and survival of
cancer cells. Proteasome inhibition has become a very attractive
anticancer therapy. Bortezomib (PS-341, 14) is a boronic acid
dipeptide derivative that selectively and potently inhibits the
26S proteasome.14 It has clinically validated activity against
multiple myeloma and has undergone extensive evaluation in
NSCLC. Preliminary in vitro studies established that 14 alone
induces growth inhibition in several NSCLC cell lines.15−18 It
has been shown that, combined with cytotoxic agents in vitro,
14 enhanced the antitumor effect in NSCLC and other solid
To determine whether the induction of ROS/RNS and stress
signaling were associated with cell death, cells were treated with
compound 13 and then analyzed by Western blot for markers
of apoptosis. Apoptosis was activated through the extrinsic
pathway, as evidenced by caspase 8 activation within 6 h of
treatment with 1 μM 13 (Figure 5E). Cleaved effector caspases
3 and 7 and cleaved PARP signals were also observed (Figure
5E).
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Figure 5. (A) Compound 13 causes cross-linking of GSH to protein thiols (protein glutathionylation) that is irreversible and induces endoplasmic
reticulum (ER) stress in A549 lung adenocarcinoma cells (B). (C) Activation of the p38 stress signaling pathway in A549 cells after treatment with
13 occurred rapidly, within 15 min after treatment was initiated, and phosphorylation persisted for 8 h. (D) Tumor suppressor p53 and its effector,
Puma, are involved in cells bearing wild-type p53 (A549 NSCLC cell line). Phosphorylation of p53 at Ser15 is an indicator of DNA damage. (E)
Treatment with 13 induced the extrinsic apoptosis pathway in A549 cells. Cleavage of caspase 8 and the effector caspases 3 and 7, as well as PARP,
was observed: F.L., full length PARP; Cl, cleaved. Images are representative of at least three independent experiments.
tumors,19 and recently proteasome inhibition as a treatment for
NSCLC advanced to clinical trials.20
dramatically exacerbates DNA damage and cytotoxicity.
Second-generation molecules have been designed to be
preferentially metabolized by cells containing high levels of
GSTP1, e.g., cancer cells. We conclude that compound 13, a
bis-diazeniumdiolate, is capable of irreversibly cross-linking
GSH to protein thiols, causing the accumulation of misfolded
proteins, leading to ER stress (Figure 8). This additional
cytotoxic stress adds to the multifaceted proapoptotic
mechanisms of arylated diazeniumdiolate action.
It has previously been shown that the proteasome inhibitor
14 has synergistic activity with compound 2.6 14 induced ROS
generation in H460 NSCLC cells.21 Having shown that
cytotoxicity of diazeniumdiolate-based NO-releasing prodrugs
correlated with intracellular ROS levels, we hypothesized that
co-treatment of lung adenocarcinoma cells with 14 and our
prodrugs would result in enhanced cytotoxicity through ROS/
RNS stress. Also, proteasome inhibitors are known to induce
the unfolded protein response and ER stress. Therefore, a
combination of 14 with compound 13, which causes
irreversible, aryl-cross-linking glutathionylation of cellular
proteins and induces ER stress, might result in enhanced
cytotoxicity. Indeed, combination treatment of 14 and 13
exhibited enhanced toxicity as evaluated by colony forming
assay (Figure 7). This promising result suggests that further in
vitro and in vivo trials of compound 13 combined with 14 are
indicated.
EXPERIMENTAL SECTION
■
General. Starting materials were purchased from Aldrich Chemical
Co. (Milwaukee, WI) unless otherwise indicated. NMR spectra were
recorded on a Varian UNITY INOVA spectrometer. Chemical shifts
(δ) are reported in parts per million (ppm) downfield from
tetramethylsilane. Ultraviolet (UV) spectra were recorded on an
Agilent model 8453 or a Hewlett-Packard model 8451A diode array
spectrophotometer. Elemental analyses were performed by Midwest
Microlab (Indianapolis, IN). Chromatography was performed on a
Biotage SP1 flash purification system. Prepacked silica gel flash
chromatography columns were purchased from Silicycle (Quebec City,
Canada) or from Yanazen Science Inc. (San Bruno, CA). Compounds
1,10 2,22 6a,10 6b,10 and 811 were prepared as previously described. In
addition to NMR, UV, and HPLC with HRMS, CHN combustion
analysis was performed to determine purity of compounds. All
compounds were ≥95% pure.
CONCLUSIONS
■
We have designed prodrugs combining an arylated diazenium-
diolate with a PARP-1 inhibitor to achieve simultaneous
delivery of a DNA damaging agent and a DNA repair inhibitor
to the cell. Previous work has shown that arylated
diazeniumdiolates cause significant DNA strand break damage.
Concurrent treatment with a DNA strand break repair inhibitor
Synthesis of 5a. 3-[(4-Oxo-3H-phthalazin-1-yl)methyl]benzoic
acid, 3a, was prepared as described by Menear et al.10 To a slurry of
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Figure 6. (A) Compound 13 significantly reduced growth of NSCLC
cells in vivo. Compounds were administered intravenously at 92 μmol/
kg, 2 times a week for 4 weeks, and tumors were measured with a
caliper. Values are medians, and the relevant 95% confidence interval
bars are shown (Mann−Whitney test). Stars indicate the significance
of the differences between 13-treated and 1-treated and control mice
at each time point. The treatment did not affect body weights. The
average body weight for all mice was 22.9 0.31 g (mean SE) at the
beginning of the experiment. At the termination, the average weights
of the control groups were 25.6 0.76 g (n = 12), 25.0 0.78 g (n =
10), and 25.0 0.77 g (n = 11) for 1, vehicle, and saline control,
respectively. The weights of animals treated with 13 were 25.3 0.49
g (n = 10), and those treated with 5b were 27.7 0.82 g (n = 11).
Figure 8. Mechanism of action of bis-diazeniumdiolate/PARP-1
inhibitor, compound 13.
Synthesis of 5b. A solution of 685 mg (2.3 mmol) of 3b10 in 30
mL of N,N-dimethylformamide was cooled to 4 °C. HATU (950 mg,
2.5 mmol) was added, and the resulting slurry was stirred under
nitrogen. A solution of 765 mg (2.19 mmol) of 45 and 753 μL (5
mmol) of diisopropylethylamine in 60 mL of dimethylformamide was
added, and the resulting solution was stirred at room temperature
overnight. The solution was cooled to 4 °C and ice−water (200 mL)
was added, giving a solid precipitate. The product was collected by
filtration, washed with water, and allowed to dry to give 1.3 g of crude
material. Recrystallization from ethanol afforded 853 mg of pure 5b:
mp 150−153 °C; UV (acetonitrile) λmax 295 nm (ε = 20.5 mM−1
1
cm−1); H NMR (DMSO-d6) δ 3.41 (b, 1H), 3.58 (b, 1H), 3.72 (b,
1H), 3.84 (b, 1H), 4.33 (s, 2H), 7.25 (t, 1H) J = 9.0 Hz, 7.37−7.46
(m, 2H), 7.82 (t, 2H) J = 9.0 Hz, 7.87−7.97 (m, 3H), 8.24 (d, 1H) J =
7.8 Hz, 8.54−8.57 (dd, 1H) J = 2.4, 7.8 Hz, 8.86 (d, 1H) J = 2.4 Hz,
12.58 (s, 1H); 13C NMR (DMSO-d6) δ 36.9, 45.2, 50.5, 116.9, 118.52,
119.5, 122.6, 124.0, 125.8, 126.5, 127.9, 129.3, 130.2, 131.9, 132.6,
133.9, 135.2, 137.3, 142.6, 145.2, 154.1 (d) JC−F = 246 Hz, 159.8,
164.4; HRMS (ESI) m/z calculated for C26H22FN8O8 [M + H]+ =
593.1545, found 593.1538, Δ ppm = 1.1. Anal. Calcd for
C26H21FN8O8·H2O: C, 51.15; H, 3.80; N, 18.35; F, 3.11. Found: C,
51.45; H, 3.70, N, 18.41; F, 3.42.
Figure 7. Colony forming assay (A549 cells) indicated enhanced
cytotoxicity of the combination of compound 13 with proteasome
inhibitor 14.
557 mg (1.6 mmol) of the hydrochloride salt of 4,5 448 mg (1.6
mmol) of 3a, and 608 mg (1.6 mmol) of HATU in 5 mL of
dimethylacetamide was added 1.02 mL (6 mmol) of diisopropylethyl-
amine. The resulting solution was stirred at room temperature for 1 h,
followed by addition of 40 mL of water. The resulting precipitate was
collected by filtration and recrystallized from ethanol, giving 827 mg of
an off-white solid: mp 114−117 °C; UV (acetonitrile) λmax 294 nm (ε
= 18.9 mM−1 cm−1); 1H NMR (DMSO-d6) δ 3.63−3.68 (m, 8H), 4.38
(s, 2H), 7.28−7.30 (m, 1H), 7.39−7.46 (m, 3H), 7.81−7.98 (m, 4H),
8.26 (d, 1H) J = 7.4 Hz, 8.56−8.59 (dd, 1H) J = 2.7, 7.4 Hz, 8.88 (d,
1H) J = 2.7 Hz, 12.06 (s, 1H); 11C NMR (DMSO-d6) δ 37.7, 50.2,
116.5, 122.5, 125/6, 125.9, 126.5, 127.8, 128.3, 129.2, 129.6, 130.2,
130.7, 132.0, 133.9, 135.6, 137.3, 138.93, 142.6, 145.3, 153.2, 159.8,
Synthesis of Compound 11. A partial solution of 2.52 g (0.0087
mol) of O2-(2,4-dinitro-5-fluorophenyl) 1-(N,N-dimethylamino)-
diazen-1-ium-1,2-diolate (10)5 in 120 mL of tert-butanol was stirred
at room temperature. A solution of 2.34 g (0.0087 mol) of sodium 1-
[4-(tert-butoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (9)23
in 120 mL of 5% aqueous sodium bicarbonate was added gradually
through a pressure equalizing addition funnel, and the resulting
mixture was stirred at room temperature overnight. The reaction
mixture was treated with 150 mL of water and the resulting yellow
solid was collected by filtration, washed with water, and dried to give
1.33 g of product. The material was placed on a silica gel column and
eluted with hexane/ethyl acetate to give 3.6 g (80%) of 11 as a yellow
powder: mp 123−125 °C; UV (acetonitrile) λmax 292 nm (ε = 23
1
169.4; HRMS (ESI) m/z calculated for C26H23N8O8 [M + H]+
=
mM−1 cm−1); H NMR (CDCl3) δ 1.49 (s, 9H), 3.28 (s, 6H), 3.60−
575.1639, found 575.1548, Δ ppm = 1.5. Anal. Calcd for C26H22N8O8·
H2O: C, 52.70; H, 4.08; N, 18.91. Found: C, 52.66; H, 3.73, N, 18.50.
3.61 (m, 4H), 3.65−3.67 (m, 4H), 7.57 (s, 1H), 8.86 (s, 1H); 13C
NMR (CDCl3) δ 28.3, 41.8, 50.5, 81.0, 105.3, 125.5, 154.2, 154.3.
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Anal. Calcd for C17H25N9O10: C, 39.61; H, 4.89; N, 24.46. Found: C,
39.44; H, 4.79; N, 24.09.
PARP Inhibition Assay. PARP enzyme inhibition was measured
using an HT Universal Colorimetric 96-well PARP assay kit (Trevigen,
Gaithersburg, MD), according to the manufacturer’s protocol with the
following small modifications: When inhibitory activities of activated
prodrugs were studied, GSH (4 mM) was added in the presence and
absence of GSTP1 in PBS, pH 7.4. Reactions were initiated by the
addition of prodrug after a 10 min incubation at 37 °C and carried out
for another 10 min. Substrate concentrations were 10 μM with a
GSTP1 concentration of 40 nM. The absorbance at 450 nm was
measured.
Cell Culture and Proliferation Assay. Cell lines were obtained
from the American Type Culture Collection (Manassas, VA) and
cultured according to the supplier’s protocol. For proliferation assays,
cells were seeded at 1 × 104 per well (H1693, H322M, H1703, H1944,
H1355, H2122, H441, H1568) or 5 × 103 per well (H460, H1792,
A549, H2023, H2030, H23) in 96-well plates and allowed to adhere
for 24 h. Compounds were prepared as 10 mM stock solutions in
DMSO. Increasing drug concentrations in 10 μL of PBS were added to
100 μL of the culture medium and incubated for 72 h. The MTT assay
(Promega, Madison, WI) was performed according to the
manufacturer’s protocol. Each concentration was represented in six
repeats, and the screening was performed as at least two independent
experiments. IC50 values were calculated by using Sigma Plot software
(Systat Software, Inc., San Jose, CA).
Catalysis of NO-Releasing PARP Inhibitor Activation by
Glutathione S-Transferase. Kinetic experiments were performed at
37 °C using a standard UV−visible spectrophotometer. GSH (4 mM)
was added in the presence and absence of GSTP1 or GSTA1 in 0.1 M
phosphate buffer solution, pH 7.4, containing 50 μM diethylene-
triaminepentaacetic acid (DTPA). Reactions were initiated by the
addition of substrate after the GSH-containing buffer and enzyme
reached thermal equilibrium. Typical substrate concentrations were 10
μM with a GST concentration of 40 nM. In each experiment the data
were analyzed at 302 nm and the rate was derived by fitting the data to
an exponential curve typical for first order processes.
In Vitro Metabolism of Compounds 5b and 13. The
metabolism of each compound was studied in the human A549
NSCLC cell line and the human U937 leukemia cell line. In each case
cells were plated in 75 cm2 flasks and incubated overnight at 37 °C.
The A549 cell line was treated with 10 μM of each compound and
incubated for varying time points. At each time point the cells were
lysed via scraping in 400 μL of 10 mM HCl and 400 μL of HPLC
grade acetonitrile. The U937 cell line was treated with 5 μM of each
compound and by two cycles of freeze (−80 °C) and thaw (37 °C) in
400 μL of 10 mM HCl and 400 μL of HPLC grade acetonitrile. To
each lysate was added 200 μL of a 5% 5-sulfosalicylic acid solution.
The precipitate was removed by centrifugation at 12000g for 15 min,
followed by syringe filtration of the supernatant and analysis by LC/
MS.
Synthesis of Compound 12 as the Hydrochloride Salt. To a
solution of 3.6 g (0.007 mol) of 11 in 300 mL of ethyl acetate was
added 70 mL of 2 M HCl in ether. The resulting solution was stirred at
room temperature. The hydrochloride salt precipitated from the
solution gradually over a 72 h period. The product was collected by
filtration, washed with ethyl acetate, and allowed to dry, giving 3.1 g
(98%) of 12: mp 135−137 °C; UV (ethanol) λmax 288 nm (ε = 28
mM−1 cm−1); 1H NMR (DMSO-d6) δ 3.26 (s, 6H), 3.33−3.35 (b, 9H;
4H for piperazine, 5H for water), 3.89−3.91 (m, 4H), 7.82 (s, 1H),
8.90 (s, 1H), 8.41 (b, 2H); 13C NMR (DMSO-d6) δ 41.6, 47.2, 105.6,
125.7, 131.5, 131.9, 153.6, 154.1. This salt was used for the next step
without further purification.
Synthesis of Compound 13. To a solution of 2.05 g (0.006 87
mol) of 3b10 and 2.85 g (0.0075 mol) of HATU in 150 mL of N,N-
dimethylformamide was added 2.6 mL (0.015 mol) of DIPEA. To the
solution was gradually added 3.1 g (0.006 87 mol) of 12 in 150 mL of
N,N-dimethylformamide, and the resulting solution was stirred at
room temperature overnight. The solution was treated with 250 mL of
cold aqueous ammonium chloride solution, and the resulting
precipitate was collected by filtration and washed with water. The
solid containing water and dimethylformamide was taken up in
dichloromethane, dried over sodium sulfate, filtered through a layer of
anhydrous magnesium sulfate, and evaporated in vacuo. The yellow
moist solid was triturated with ether to give, after filtration, 2.16 g of
13. Recrystallization from ethanol gave a product of 88% purity.
Purification was carried out with preparative HPLC: mp 122−125 °C;
1
UV (0.2% DMSO/ethanol) λmax 287 nm (ε = 32 mM−1 cm−1); H
NMR (acetone-d6) δ 3.30 (s, 6H), 3.58−3.97 (m, 8H), 7.14−7.22 (m,
1H), 7.44−7.54 (m, 2H), 7.79 (s, 1H), 7.80−7.88 (m, 1H), 7.94−7.96
(m, 2H), 8.32−8.34 (m, 1H), 8.86 (s, 1H), 11.75 (s, 1H); 13C NMR
(acetone-d6) δ 37.9, 41.7, 51.0, 51.2, 105.5, 110.75, 116.5, 116.9, 124.8,
125.9, 126.4, 127.2, 129.5, 132.1, 134.1, 136.0, 145.6, 153.4, 155.1,
155.6 (d) JC−F = 201.0 Hz, 160.3, 165.1. For further decomposition
and biological screening, a portion of the compound was purified on a
Phenomenex Luna C18 column, 3 μm, 150 mm × 2.0 mm, with a
gradient consisting of water and acetonitrile containing 0.1% formic
acid. HRMS (ESI) m/z calculated for C28H27FN11O10 [M + H]+ =
696.1921, found 696.1928, Δ ppm = 0.96. Anal. Calcd for
C28H26N11FO10·H2O: C, 47.13; H, 3.95; F, 2.66; N, 21.59. Found:
C, 47.14; H, 4.08; F, 2.73; N, 21.45.
Determination of Intracellular Reactive Oxygen/Nitrogen
Species and Nitric Oxide. Intracellular levels of reactive oxygen/
nitrogen species were quantified by oxidation of the ROS/RNS-
sensitive fluorophore 5-(and -6)-chloromethyl-2′,7′-dichlorodihydro-
fluorescein diacetate (DCF-DA, Invitrogen, Carlsbad, CA). Cells
growing on six-well plates (6 × 105/well) were loaded with 5 μM
DCF-DA in Hanks’ balanced salt solution (HBSS) at 37 °C and 5%
CO2. After 30 min of incubation, HBSS containing the probe was
removed, cells were rinsed with HBSS, and 3 mL of fresh HBSS was
added to each well followed by addition of compounds (10 μM) or
DMSO as a control. After 60 min the cells were collected by scraping
in HBSS, and DCF fluorescence was measured by using a PerkinElmer
Life and Analytical Sciences (Waltham, MA) LS50B luminescence
spectrometer with the excitation source at 488 nm and emission at 530
nm.
The intracellular level of nitric oxide and its oxidation products after
treatment with compounds was estimated by using the fluorophore 4-
amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) diacetate
(Invitrogen). Cells growing in six-well plates were loaded with 2.5
μM DAF-FM diacetate in HBSS at 37 °C and 5% CO2. After 30 min
of incubation the cells were rinsed with HBSS to remove excess probe.
Test compounds in fresh HBSS were added to the cells at 10 μM final
concentration. After 30 min of incubation, the fluorescence of the
benzotriazole derivative formed on DAF-FM’s reaction with aerobic
NO was analyzed by using a PerkinElmer Life and Analytical Sciences
LS50B luminescence spectrometer with the excitation source at 495
nm and emission at 515 nm. All experiments were performed at least
three times, each time at least in triplicate.
The system used for analysis is an Agilent 1200 HPLC instrument
coupled with an Agilent 6520 accurate-mass quadrupole time-of-flight
(Q-TOF) LC/MS/MS instrument. Positive ions were generated with
the Agilent multimode source in mixed mode. Separations were
performed on a Phenomenex Luna column, C18 5 μm, 2.1 mm × 150
mm, at a flow rate of 0.2 mL/min under H2O/acetonitrile/0.1% formic
acid gradient conditions.
Comet Assay. The alkaline comet assay was performed as
described.24
Molecular Modeling. Transition state modeling of the 13−
glutathione adduct in the GSTA1 and GSTP1 active sites was carried
out as described.25 Briefly, the initial models of the Meisenheimer
complex of compound 13 (GS13−) bound to GSTP1 or GSTA1 was
built on the basis of the GSTCD− in the GSTP1·GSTCD− structure
(PDB entry 1AQX)26 or in the GSTA1·GSTCD− model complex.25
The initial GSTA1·GSTCD− model complex was built on the basis of
the crystal structures of the GSTCD− found in the active sites of
GSTM1 (PDB entry 4GST) and GSTP1 (PDB entry 1AQX), and
then it was docked into the active site of GSTA1 in complex with the
GSH adduct of ethacrynic acid (PDB entry 1GSE). The GSTP1·
GS13− and GSTA1·GS13− complexes built in dimeric forms because
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GSH interacts with the side chains from both subunits of GST. The
dimeric model complexes were subject to geometry optimization using
Notes
The authors declare no competing financial interest.
the MacroModel module of Schrodinger, LLC (MacroModel, version
̈
9.6).27 OPLS_2005 force field with efficient continuum solvation
models was used during energy minimization.
ACKNOWLEDGMENTS
■
We thank Dr. Sergey Tarasov and Marzena A. Dyba of the
Biophysics Resource in the Structural Biophysics Laboratory,
NCI-Frederick, for assistance with the high resolution mass
spectrometry studies. This project has been funded in part with
federal funds from the National Cancer Institute, National
Institutes of Health, under Contract HHSN26120080001E.
The content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S.
Government. This research was supported [in part] by the
Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research.
Immunoblot Analysis. Western blot analysis was performed as
described.7 Most of the primary antibodies were from Cell Signaling
Technology (Danvers, MA), with the exception of ATF3 which was
from Santa Cruz Biotechnology (Santa Cruz, CA).
To analyze for glutathionylation of cellular proteins, lysates were
separated by gel electrophoresis (4−12% NuPAGE gel) under
nonreducing conditions, transferred to PVDF membranes, and probed
with anti-glutathione conjugated to protein monoclonal antibody
(Virogen).
Establishment of Xenograft Tumors and Drug Injections.
Human lung adenocarcinoma cell line A549 was obtained from the
American Type Culture Collection and cultured in RPMI 1640
medium with 10% fetal bovine serum, according to the cell supplier’s
protocol, for a maximum of four passages before use. Cells were
harvested at 70−80% confluence, washed with phosphate buffered
saline, suspended in phosphate buffered saline, and implanted
subcutaneously at 5 × 106 cells/0.2 mL into NCr nu/nu athymic
mice, obtained from Charles River. Frederick National Laboratory for
Cancer Research is accredited by AAALAC International and follows
the Public Health Service Policy for the Care and Use of Laboratory
Animals. Animal care was provided in accordance with the procedures
outlined in the “Guide for Care and Use of Laboratory Animals”
(National Research Council, 1996; National Academy Press,
Washington, DC). When the tumors reached approximately 3 mm
× 3 mm, the mice were distributed randomly into groups of 12 for
treatment.
Compounds were injected into the tail veins of the mice, at 92
μmol/kg body weight (in 20 μL of DMSO solution), 2 times per week
for 4 weeks. Control groups were treated with saline or DMSO. Mice
were weighed, and tumors were measured 2 times per week. Tumor
volumes in mm3 were estimated by the formula (π/2 × length ×
width2). Mice were euthanized almost immediately after the last
treatment. Blood was collected under isoflurane anesthesia into
Eppendorf tubes containing 50% acetonitrile/10 mM HCl for drug
metabolite analysis. Tumors were also removed and frozen
immediately for drug metabolite analysis.
ABBREVIATIONS USED
■
NO, nitric oxide; JS-K, O2-(2,4-dinitrophenyl) 1-[(4-
ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate;
NSCLC, non-small-cell lung cancer; GST, glutathione S-
transferase; GSH, glutathione; DMSO, dimethyl sulfoxide;
HBSS, Hanks’s balanced salt solution; TBS-T, Tris-buffered
saline−Tween-20; PBS, phosphate buffered saline; MTT, 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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ASSOCIATED CONTENT
■
S
* Supporting Information
Activation of 5b in the presence of 4 mM GSH, correlations of
IC50 of 5b with ROS, PRX1, and OGG1, data showing ROS/
RNS stress in cells treated with 5a and 5b, GSTP1 and GSTA1
protein expression levels in NSCLC cell lines, product
distribution in the reaction of 5b with GSH in the presence
or absence of GSTP1, and NMR spectra of new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 301-846-1246. Fax: 301-846-5946. E-mail: maciaga@
mail.nih.gov.
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