Activation of the c-Jun N-terminal kinase/activating transcription factor 3 (ATF3) pathway characterizes effective arylated diazeniumdiolate-based nitric oxide-releasing anticancer prodrugs

Article abstract of DOI:10.1021/jm2004128

Improved therapies are needed for nonsmall cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Recently, we have shown that O ²-(2,4-dinitrophenyl) 1-[(4-ethoxyca

Full text of DOI:10.1021/jm2004128

Article
pubs.acs.org/jmc
Activation of the c-Jun N-terminal Kinase/Activating Transcription
Factor 3 (ATF3) Pathway Characterizes Effective Arylated
Diazeniumdiolate-Based Nitric Oxide-Releasing Anticancer Prodrugs
Anna E. Maciag,†,#,* Rahul S. Nandurdikar,‡,# Sam Y. Hong,‡ Harinath Chakrapani,§ Bhalchandra Diwan,†
Nicole L. Morris,∥ Paul J. Shami,⊥ Yih-Horng Shiao,‡ Lucy M. Anderson,‡ Larry K. Keefer,‡
and Joseph E. Saavedra†
†Basic Science Program, SAIC-Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702, United States
‡Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, United States
§Department of Chemistry, Indian Institute of Science Education and Research, Pune 411008, India
∥Laboratory Animal Sciences Program, SAIC-Frederick, Inc., National Cancer Institute, Frederick, Maryland 21702, United States
⊥Division of Hematology and Hematologic Malignancies, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112,
United States
S
* Supporting Information
ABSTRACT: Improved therapies are needed for nonsmall
cell lung cancer. Diazeniumdiolate-based nitric oxide (NO)-
releasing prodrugs are a growing class of promising NO-based
therapeutics. Recently, we have shown that O2-(2,4-
dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-
ium-1,2-diolate (JS-K, 1) is effective against nonsmall cell
lung cancer (NSCLC) cells in culture and in vivo. Here we
report mechanistic studies with compound 1 and its
homopiperazine analogue and structural modification of
these into more stable prodrugs. Compound 1 and its
homopiperazine analogue were potent cytotoxic agents against NSCLC cells in vitro and in vivo, concomitant with activation
of the SAPK/JNK stress pathway and upregulation of its downstream effector ATF3. Apoptosis followed these events. An aryl-
substituted analogue, despite extended half-life in the presence of glutathione, did not activate JNK or have antitumor activity.
The data suggest that rate of reactivity with glutathione and activation of JNK/ATF3 are determinants of cancer cell killing by
these prodrugs.
suppressor status. DNA strand-break damage measured by
comet assay was also observed.6 Similarly, in multiple myeloma
cells, 1 caused DNA double strand break damage.5
INTRODUCTION
■
Lung cancer is a leading cause of cancer deaths worldwide.
Nonsmall cell lung cancer (NSCLC) accounts for about 80% of
all lung cancers. Despite recent advances in therapy, the disease
remains largely incurable. For patients with advanced disease,
current platinum-based chemotherapy regimens result in
median survival of 7−10 months only.1
Compound 1 and its analogues release nitric oxide (NO) on
reaction with glutathione (GSH) (Scheme 1), and this reaction
is accelerated by glutathione S-transferases (GST).7 We have
previously shown that a homopiperazine analogue of 1,
compound 2 (Scheme 1), is less reactive than 1 with GST
and exhibits an extended half-life in the presence of GSH in
comparison to the parent drug in vitro.7 We hypothesized that
this property of the drug might translate to a prolonged lifetime
in the bloodstream and selective accumulation of the drug in
the tumor, thus leading to better antitumor efficacy.
O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-
yl]diazeniumdiolate (1, JS-K) has proven effective in several
in vivo cancer models.2−5 Recently, we have shown that 1 is
effective against NSCLC cells in vitro and in vivo.6 The
mechanism(s) of action of 1 is only partially understood. The
drug appears to have a multifaceted cell-type-dependent mode
of action with NO-mediated pathways and S-arylation of
cellular thiols being involved. In NSCLC cells, its effectiveness
depends on endogenous reactive oxygen species (ROS): the
compound was most effective in cell lines characterized by high
levels of endogenous ROS. The toxic effects involved
mitochondrially mediated apoptosis via caspase-dependent
mechanisms and were independent of the p53 tumor
In an attempt to create more stable analogues, we have
synthesized a small library of compounds by substituting the
C5-position of the aryl ring of the parent compounds with
methyl or methoxy groups to make the resulting compounds
Received: April 6, 2011
Published: October 17, 2011
© 2011 American Chemical Society
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with 2,4-dinitro-5-fluorotoluene (5) in DMSO at room
temperature furnished the tolyl analogues 6 and 7, respectively,
in good yields. Synthesis of the anisyl analogue 9 began with
the reported compound 8.3 Nucleophilic displacement of the
fluorine with methoxide ion in compound 8 was accomplished
using sodium methoxide in methanol to obtain the desired
anisyl compound 9 in 51% yield. An alternate strategy was used
for the synthesis of the homopiperazine anisyl analogue 11.
Diazeniumdiolate sodium salt 4 was treated with 2,4-dinitro-5-
Scheme 1. Mechanism of Nitric Oxide Release from 1 and Its
Homopiperazine Analogue 2
9
t
fluoroanisole (10) in 5% NaHCO3 in BuOH:THF to afford
the desired compound 11 in 63% yield.
Decomposition Studies and Nitric Oxide Release. The
rates of reaction of 1 and its analogues with 4 mM glutathione
(GSH) in aqueous pH 7.4 phosphate buffered saline at 37 °C
were determined (Table 1). The results show that substituting
more resistant to glutathione/GST attack and thereby
extending their half-lives in the reaction with GSH. The
second-generation compounds were screened in vitro against a
panel of NSCLC cell lines, and the most promising prodrugs
were tested in vivo in an H1703 nonsmall cell lung cancer
xenograft model. We show that the homopiperazine analogue
of 1, compound 2, exhibits almost identical in vitro activity as
the parent drug. However, its in vivo efficacy appears to be
more pronounced. Substitution of the C5-position in the aryl
ring with a methyl group led to diminished reactivity of the
compound toward glutathione. However, this analogue did not
exhibit any antitumor efficacy in vivo. The reasons for this
difference were sought. Both 1 and 2 caused rapid and
sustained activation of the SAPK/JNK stress pathway in
NSCLC cells in culture and in vivo as well as significant cell
death in xenografted tumors. In contrast, O2-(2,4-dinitro-5-
anisyl) and O2-(2,4-dinitro-5-tolyl) analogues did not cause
SAPK/JNK pathway activation and histochemical analysis did
not reveal cell death in the tumors from animals treated with
O2-(2,4-dinitro-5-tolyl) analogue. We conclude that SAPK/
JNK pathway activation is important for this class of
compounds to induce tumor cell death in vivo.
Table 1. Stability of the Compounds in the Presence of 4
mM GSH, and in Vitro Antiproliferative Activities
IC50 (μM)
t
1/2(min) mean
compd
(SE)
H1703 H441 H1373 H2122 H1944
1
15.7 (1.4)
22.6 (0.4)
71.5 (4.3)
25.7 (1.0)
38.6 (2.8)
59.6 (4.2)
0.57
0.64
0.97
1.66
1.57
1.82
0.94
1.42
1.61
2.02
2.50
3.15
0.86
0.95
1.86
3.00
2.10
3.08
3.17
3.14
7.00
>10
>10
>10
>10
>10
>10
2
6
7
10.0
5.78
10.0
9
11
the phenyl ring with tolyl or anisyl rings significantly increased
the half-life of the reaction with 4 mM GSH (Table 1).
Replacing the piperazine moiety of 1 with homopiperazine also
resulted in a more stable compound (2). The decomposition
showed pseudofirst-order kinetics. To illustrate this, the plot for
the decomposition for compound 6 is shown in Supporting
Information Figure S1. We have previously reported this
phenomenon and shown that this analogue has diminished
reactivity with GST.7
In Vitro Efficacy. In a previous study,6 we screened a panel
of 18 NSCLC cell lines for sensitivity to 1. Compound 1
inhibited growth of all NSCLC cell lines with IC50
concentrations ranging from 0.33 to 17.64 μM. Six cell lines
were inhibited in the range of 0.33 to 1.01 μM and were thus as
sensitive as leukemia cells2 or multiple myeloma cells.5 In this
RESULTS
■
Synthesis. 1 and its homopiperazine analogue 2 (Scheme
1) were synthesized following the reported procedures.3,8
Syntheses of the tolyl and anisyl analogues are shown in
Scheme 2. Reactions of diazeniumdiolate sodium salts 3 and 4
Scheme 2. Synthesis of the Tolyl and Anisyl Analogues of 1 and 2
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work, we have chosen three 1-sensitive cell lines (H1703,
H441, and H1373) and two significantly less sensitive lines
(H2122 and H1944) for evaluation of growth-inhibitory
activities of the new analogues. The results are given in Table
1. Compounds 1 and 2 exhibited very comparable antiprolifer-
ative activities against the NSCLC cell lines (Table 1). This
finding is consistent with our previous comparison of
anticancer activities of 1 and 2 against a larger panel of cancer
cell lines from different sites.7 Surprisingly, increased stability of
6, 9, and 11 in the presence of glutathione did not result in
increased toxicity against cancer cells in culture.
In Vivo Studies. NSCLC cell line H1703, with established
in vivo sensitivity to 1,6 was chosen for assessment of activity of
the prodrugs in vivo against xenografted cells in athymic mice.
To compare the effects of prolonging the half-life of the
compound on the in vivo efficacy, compounds 2 and 6 were
chosen for further studies. The compounds were formulated in
Pluronic P123. Compounds 1 and 2 were administered at 6
μmol/kg. Compound 6, which is significantly more stable to
nucleophilic attack by GSH, was delivered at 8 μmol/kg. The
drugs were administered intravenously, three times a week for a
3-week period, to assess anticancer activity. Treatment with
either 1 or 2 significantly reduced growth of H1703 human
NSCLC cells when compared with cells in control animals
treated with vehicle only. Despite the higher dosage of 6,
treatment did not result in reduction in H1703 tumor growth
in vivo. Figure 1 shows a comparison of the tumor weights from
Activation of a SAPK/JNK Stress Pathway. We have
shown previously that treatment with 1 resulted in oxidative/
nitrosative stress and DNA damage in H1703 cells.6 Both
oxidative stress and DNA damage are widely observed to
activate a SAPK/JNK pathway, leading to activation of
transcription factors and expression of stress-response genes.
It has been documented that SAPK/JNK activation is necessary
for apoptosis induced by various stress stimuli in several cell
types.10,11
Treatment of H1703 cells with 1 μM compound 1 resulted
in rapid phosphorylation of SAPK/JNK (through its upstream
kinase SEK1/MKK4) and its downstream effectors, tran-
scription factors ATF2 and c-jun, and in increased activating
transcription factor 3 (ATF3) protein (Figure 2A). Phosphor-
ylation of SAPK/JNK was detected within 30 min of treatment.
Activation of ATF2 and c-jun was inhibited by pretreatment
with JNK inhibitor 1,9-pyrazoloanthrone (Figure 2B). Only 1
or 2 caused SAPK/JNK activation; treatment with the other
four compounds did not lead to SAPK/JNK phosphorylation
(Figure 2C).
Preincubation of the H1703 cells with ROS scavenger Tiron
attenuated SAPK/JNK phosphorylation resulting from 1 or 2
(Figure 2D), which suggests activation of the pathway by
oxidative stress. As a loading control, membranes were
reprobed with a SAPK/JNK antibody. No variation in the
amount of SAPK/JNK protein was detected.
ATF3 may be strongly induced in a variety of tissues by
different stress signals.12 Several pathways, including JNK,
could be responsible for ATF3 induction in a p53-dependent or
-independent manner.13,14 Accumulating evidence suggests that
ATF3 plays a significant role in apoptosis. When overexpressed,
ATF3 induced apoptosis in ovarian cancer cells15 and sensitized
HeLa cells to chemotherapy.16 ATF3 was also shown to play a
role in β-cell apoptosis.14 In the present study, ATF3 protein
expression was induced by 1 μM 1 or 2 within 2 h of the
treatment (Figure 2A,E). Other analogues did not upregulate
ATF3. ATF3 upregulation was partially blocked by pretreat-
ment with 1,9-pyrazoloanthrone, which confirms that its
activation was in part JNK-dependent (Figure 2B). Figure 2E
shows a time course of ATF3 activation by 1 or 2 but not 6.
ATF3 was activated through an increase in mRNA. Real-time
PCR (RT-PCR) analysis of ATF3 mRNA level revealed its
rapid upregulation after treatment with 1. We observed 8-fold
increase in ATF3 mRNA level after 1.5 h treatment with 1 μM
compound 1 and 10-fold increase with 5 μM 1 (Figure 2F).
Treatment with 1 or 2 Resulted in Arrest of the Cell
Cycle in G2/M Phase and Commitment to Apoptosis.
Treatment of H1703 cells with 1 or 2, but not 6, for 1, 4, or 6 h
resulted in cleavage of PARP, a reliable indicator of apoptosis
(Figure 3A). This was confirmed in a further experiment in
which cleavage of caspases 3 and 7 were also measured (Figure
3B). A preliminary study utilizing fluorescence-activated cell
sorting (FACS) further confirmed apoptosis and indicated G2/
M growth arrest. After 6 h treatment with 1 μM 1, 37% of
H1703 cells were in the G2 phase and 14.4% were apoptotic.
Control (DMSO-treated) H1703 cells processed in parallel
exhibited 8% in G2 phase and 0.05% cells found to be
apoptotic.
Figure 1. H1703 cells growth in vivo was significantly inhibited by 1 or
2 but not by 6. Compounds 1 and 2 were administered iv at 6 μmol/
kg and compound 6 at 8 μmol/kg. The injections were performed
three times a week for three weeks. Growth of tumors treated with 1
or 2 was inhibited, in contrast to 6, for which tumor weights did not
differ significantly from the controls. Values are means ± SE (Mann−
Whitney test); numbers of mice for the control and treated groups,
respectively, were 13 and 14 for 1, 14 and 15 for 2, and 13 and 14 for
6. The treatment did not affect body weights. The average body
weights at the beginning of the experiment were 22.5 ± 0.4 g, 22.5 ±
0.4 g, and 22.2 ± 0.4 g for groups 1, 2 and 6, respectively. At the end
of the treatment, the weights were: in group 1, 24.6 ± 0.9 g and 24.9 ±
0.4 g; in group 2, 24.6 ± 0.6 g and 24.9 ± 0.4 g; and in group 6, 24.6 ±
0.6 g and 24.5 ± 0.6 g, vehicle control and compound-treated,
respectively.
drug-treated animals relative to vehicle-treated controls at the
termination of the experiment. Treatment with 1 led to 49%
reduction of the tumor weights (P < 0.05). Treatment with 2
resulted in 63% reduction of the tumor weights (P < 0.01).
Importantly, treatment with either vehicle or diazeniumdiolate-
based drugs did not affect body weights (see legend to Figure
1).
Silencing of ATF3 Prevents 1-Induced Apoptosis. The
H1703 cells were treated with siRNA to ATF3 for 48 h,
followed by treatment with compound 1 or DMSO (control)
for 8 h. In the cells pretreated with siRNA to ATF3, treatment
with 1 did not result in upregulation of ATF3 protein
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Figure 2. Only 1 and 2 activated the SAPK/JNK stress pathway in H1703 cells. Representative blots are shown. (A) Activation of SAPK/JNK and its
downstream effectors upon 1 treatment. (B) Pretreatment of the H1703 cells with 1,9-pyrazoloanthrone (50 μM, 30 min) inhibited 1-induced
phosphorylation of JNK and its downstream effectors ATF2 and c-jun. Blocked upregulation of ATF3 protein confirms that its activation was in part
JNK-dependent. (C) Only 1 and 2 induced JNK phosphorylation. (D) Pretreatment with ROS scavenger Tiron partially blocked JNK activation by
1 or 2. Cells were pretreated with 10 mM Tiron for 1 h, followed by 30 min incubation with 1 or 2. (E) ATF3 protein expression was induced by 1
or 2 but not by 6. H1703 cells were treated with 1 μM compound or DMSO for 1, 4, or 6 h, then lysed and processed by Western blotting for ATF3
protein. (F) ATF3 mRNA levels in H1703 cells treated with 1 were evaluated by quantitative RT-PCR. β-actin was used as an internal control. Data
are reported as mean ± SE.
Figure 3. Activation of apoptosis as evidenced by PARP cleavage and
Figure 4. Silencing of ATF3 protects from 1-induced apoptosis.
H1703 cells were treated with siRNA to ATF3 or controls for 48 h,
followed by treatment with 1 μM compound 1 or DMSO (control) for
8 h, then lysed and processed for Western blotting for cleaved PARP
p85 and cleaved caspase 3 proteins. β-actin was used as a loading
control. M, mock (reagent control); si, siRNA to ATF3; NS,
nonsilencing siRNA control.
activation of effector caspases 3 and 7. (A) H1703 cells were treated
with 1 μM compound or DMSO for 1, 4, or 6 h, then lysed and
processed by Western blotting for PARP protein. PARP cleavage was
induced by 1 and 2 but not by 6. (B) Cells were treated with 1 μM 2
for the time periods indicated or DMSO for 6 h. Stars indicate full-
length PARP (116 kDa), and arrows indicate cleaved fragments (89
and 24 kDa).
that ATF3 is involved in induction of apoptosis in H1703 cells
after treatment with 1.
expression (Figure 4). Activation of apoptosis was also
inhibited: in the cells pretreated with siRNA to ATF3,
induction of apoptosis resulting from treatment with 1 was
inhibited, as evidenced by lack of cleaved PARP signal and
cleaved caspase-3 signal (Figure 4). Control cells (reagent-
control or nonsilencing control treated) responded to the
treatment with strong apoptotic signals. This strongly suggests
SAPK/JNK Activation and ATF3 Upregulation is Also
Observed in Xenograft Tumors. Next, we investigated if
these differences in activating JNK pathway could be observed
in vivo, in xenograft tumors. H&E staining analysis revealed
significant tissue death in 1- or 2-treated tumors, in contrast to
the tumors from animals treated with compound 6 or vehicle
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control (Figure 5). Immunohistochemical analysis of the
tumors revealed phosphorylation of SAPK/JNK, as well as
DISCUSSION
■
Lung cancer is one of the most common and deadly
malignancies, and novel treatment strategies are urgently
needed. In our previous report,6 we correlated the anticancer
activity of compound 1 against NSCLC cells with the
endogenous levels of ROS/RNS. We reported multiple
mechanisms playing a role in the drug’s cytotoxicity, including
depletion of glutathione, DNA damage, and MnSOD protein
tyrosine nitration leading to mitochondrially mediated
apoptosis.
Here we presented evidence that both 1 and its
homopiperazine analogue 2 activated the SAPK/JNK stress
signaling pathway, and this induction could be partially
suppressed by a ROS scavenger. Involvement of the SAPK/
JNK pathway in activation of apoptotic cell death has been
reported,10 as well as its involvement in ATF3 induction.17
SAPK/JNK activation in response to 1 has been shown in
hepatoma4 and multiple myeloma cells.5
ATF3 can be greatly induced by cellular stress, including UV
irradiation, ROS, or NO. Its induction often associates with
cellular damage, suggesting an important role during stress
response.12,18,19 ATF3 upregulation can be mediated by ATF2,
another member of the ATF/CREB family. Although ATF2
expression has been associated with maintaining a malignant
phenotype, possible tumor suppressor functions also have been
reported.20 An increase in ATF3 expression in the treated cells
as well as xenograft tumors is interesting for two reasons. First,
ATF3 has been implicated in apoptosis,14−16 which raises the
possibility that this transcription factor is a novel mediator of
the antitumor effects of 1 or 2. Second, ATF3 has been
reported to be induced by NO;21 strong immunostaining of
ATF3 in the tumors from animals treated with 1 or 2 suggests
that the prodrugs are stable enough in the circulation to be able
to deliver cytotoxic NO to the tumor.
Figure 5. H&E staining shows significant areas of cell death (stars) in
tumors from animals treated with 1 or 2 in contrast to 6 or vehicle-
treated controls. Tumors treated with 1 or 2 show strong staining for
phosphorylated SAPK/JNK and ATF3. Controls or tumors treated
with 6 show very weak staining. Representative tumors are shown.
upregulation in ATF3 protein level in the tumors treated with 1
or 2. Tumors treated with vehicle control or 6 did not stain for
P-SAPK/JNK, and the ATF3 signal was comparable to that of
the control, much weaker than that resulting from the
treatment with 1 or 2 (Figure 5, Supporting Information
Table S2).
Scheme 3. Proposed Cellular Mechanisms of NO Prodrug Toxicity
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pressure. The crude material was purified by flash column
chromatography (1:1 hexane/ethyl acetate) to afford product 7 (190
mg, 46%) as a yellow solid. UV (ethanol) λmax (ε) 304 nm (17.7
mM−1 cm−1). 1H NMR (DMSO-d6, 70 °C) δ 1.09 (t, J = 7.0 Hz, 3H),
1.83−1.89 (m, 2H), 2.62 (s, 3H), 3.40 (t, J = 5.8 Hz, 2H), 3.64 (t, J =
5.9 Hz, 2H), 3.89 (t, J = 5.7 Hz, 2H), 3.95−4.00 (m, 4H), 7.60 (s,
1H), 8.66 (s, 1H). 13C NMR (DMSO-d6, 70 °C) δ 14.81, 20.88, 25.71,
44.17, 45.66, 50.25, 50.62, 61.25, 120.54, 123.68, 135.03, 142.39,
142.85, 151.90, 155.48. Anal. (C15H20N6O8) C, H, N.
The methyl and methoxy substituents in compounds 6, 7, 9,
and 11 were introduced in order to increase the electron
density at the carbon bearing the diazeniumdiolate moiety.
Increased electron density would render these analogues less
susceptible to nucleophilic attack and thus hamper rate-limiting
formation of the Meisenheimer complex along with the
subsequent elimination of the diazeniumdiolate anion. Indeed,
this modification to the structure led to an increased half-life of
the compounds, which did not, however, translate to increased
antiproliferative activity. The substituted analogue lacked the
ability to induce SAPK/JNK phosphorylation and ATF3
induction both in vitro and in vivo.
Our results suggest that rate of reactivity with GSH is a
critical factor for the effectiveness of this class of compounds,
perhaps due to increased ROS consequent to GSH depletion.
An alternative explanation may be the fact that by being less
reactive with GSH, 6 is less efficient in delivering NO (the
primary toxin in these prodrugs) to the malignant cells
(Scheme 3). Stereoelectronic perturbations associated with
methyl and methoxy substitutions of the aromatic ring may also
contribute to the observed differences in in vivo activity. Most
likely, our observations are due to a combination of these
factors.
O2-(2,4-Dinitro-5-methoxyphenyl) 1-[(4-Ethoxycarbonyl)-
piperazin-1-yl]diazen-1-ium-1,2-diolate (9) (JS-56−32). To a
solution of 8 (246 mg, 0.612 mmol) in 10 mL of dichloromethane was
added 5.4 M sodium methoxide in methanol (0.113 mL, 0.612 mmol).
The resulting orange solution faded to a light yellow−orange solution.
TLC analysis on silica gel using 10:1 dichloromethane:ethyl acetate
indicated that all the starting material had reacted within the first 5
min of reaction. The solution was diluted with 25 mL of
dichloromethane, washed with water, dried over sodium sulfate,
filtered through a layer of anhydrous magnesium sulfate, and
evaporated under vacuum to give 208 mg of a resin. The resin
solidified upon trituration with ether, and the resulting yellow powder
was collected by filtration giving product 10 (128 mg, 51%): mp 142−
144 °C; UV (ethanol) λmax (ε) 276 nm (10.2 mM−1 cm−1). 1H NMR
(CDCl3) δ 1.29 (t, J = 7.0 Hz, 3H) 3.57−3.62 (m, 4H), 3.72−3.75 (m,
4H), 4.09 (2, 3H), 4.19 (q, J = 7.0 Hz, 2H) 7.11 (s, 1H), 8.79 (s, 1H).
13C NMR, (CDCl3) δ 14.61, 42.20, 50.71, 57.54, 62.10, 101.52,
125.57, 154.21, 157.84, 165.87, 175.96. Anal. (C14H18N6O9) C, H, N.
O2-(2,4-Dinitro-5-methoxyphenyl) 1-[(4-Ethoxycarbonyl)-
homopiperazin-1-yl]diazen-1-ium-1,2-diolate (11) (RN-2−
50). To a solution of 4 (254 mg, 1.0 mmol) in 5% sodium
bicarbonate (8 mL) was added a solution of 10 (216 mg, 1.0 mmol) in
1:1 THF/tBuOH (8 mL) at room temperature. After 12 h, the
reaction mixture was diluted with 15 mL of ether, and the organic layer
was separated. The aqueous layer was extracted with ether (2 × 10
mL). The combined organic layer was dried over anhydrous sodium
sulfate, and solvent was evaporated under reduced pressure. The crude
material was purified by flash column chromatography (1:1 hexane/
ethyl acetate) to afford product 11 (270 mg, 63%) as a yellow solid:
UV (ethanol) λmax (ε) 305 nm (14.1 mM−1 cm−1). 1H NMR (DMSO-
d6, 70 °C) δ 1.09 (t, J = 7.00 Hz, 3H), 1.88 (broad, 2H), 3.40 (t, J =
6.0 Hz, 2H), 3.65 (t, J = 5.4 Hz, 2H), 3.90 (t, J = 5.5 Hz, 2H), 3.94−
4.01 (m, 4H), 4.06 (s, 3H), 7.29 (s, 1H), 8.68 (s, 1H). 13C NMR
(DMSO-d6, 70 °C) δ 14.92, 25.54, 44.20, 45.59, 50.13, 50.56, 58.49,
61.28, 120.00, 125.28, 129.30, 133.03, 154.48, 157.90. Anal.
(C15H20N6O9) C, H, N.
Formulated Prodrug Stability Studies. First, 490 μL of 2.25%
Pluronic P123 in phosphate-buffered saline (PBS) was aliquoted into
glass HPLC vials and maintained at 50 °C. To this solution, 10 μL of
50 mM prodrug in DMSO was added and maintained at 50 °C for 10
min. Then a glutathione stock solution (40 mM) was prepared in 0.1
M phosphate buffer, pH 7.4. To 800 μL of 0.1 M phosphate buffer in a
glass HPLC vial, 100 μL of glutathione stock solution and 100 μL of
formulated prodrug were added. The disappearance of the prodrug
was monitored using an Agilent 1100 series HPLC fitted with a C-18
reverse phase column (Phenomenex Luna 250 mm × 4.60 mm)
operating at 300 nm and run isocratically with acetonitrile:water
(75:25).
EXPERIMENTAL SECTION
■
General. Starting materials were purchased from Aldrich Chemical
Co. (Milwaukee, WI) unless otherwise indicated. 2,4-Dinitro-5-
fluorotoluene was purchased from Oakwood Products, Inc., West
Columbia, SC. NMR spectra were recorded on a Varian UNITY
INOVA spectrometer; chemical shifts (δ) are reported in parts per
million (ppm) downfield from tetramethylsilane. The NMR spectra of
compounds 7 and 11 were recorded at 70 °C in DMSO-d6. 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). Compounds 1−43,
83, and 109 were prepared using reported methods. All the new
compounds were found to be >95% pure as determined by NMR,
elemental analyses, and HPLC.
O2-(2,4-Dinitro-5-methylphenyl) 1-[(4-Ethoxycarbonyl)-
piperazin-1-yl]diazen-1-ium-1,2-diolate (6) (JS-55−111). A sol-
ution of 3 (543 mg, 2.5 mmol) in 10 mL of DMSO was stirred at
room temperature. A solution of 5 (500 mg, 2.5 mmol) in 5 mL of
DMSO was added through a syringe. The solution turned green upon
addition and faded to yellow gradually. The solution was stirred at
room temperature for 72 h, flooded with 25 mL of ice−water, and
extracted with ether. The organic layer was dried over sodium sulfate
and filtered through a layer of anhydrous magnesium sulfate.
Evaporation of the solvent gave a yellow solid that was recrystallized
from ether−petroleum ether to give compound 6 (409 mg). The
aqueous/DMSO layer was allowed to stand overnight, producing an
additional 181 mg of product 6 (total yield: 64%). mp 81−83 °C; UV
(ethanol) λmax (ε) 252 nm (12.2 mM−1 cm−1), λmax (ε) 290 nm (11.6
Cell Culture and Proliferation Assay. Cell lines derived from
human nonsmall cell lung cancers were obtained from American Type
Culture Collection (ATCC, Manassas, VA) and are designated by their
NCI numbers. Cells were maintained in RPMI 1640 medium (Gibco,
Invitrogen, Carlsbad, CA), supplemented with 10% fetal calf serum
(Gemini Bio-Products, Sacramento, CA), 100 U/mL penicillin/
streptomycin, and 2 mM glutamine at 37 °C and 5% CO2.
1
mM−1 cm−1). H NMR (CDCl3) δ 1.29 (t, J = 7.0 Hz, 3H), 2.75 (s,
3H), 3.60−3.63 (m, 4H), 3.72−3.75 (m, 4H), 4.19 (q, J = 7.0 Hz, 2H)
7.44 (s, 1H), 8.78 (s, 1H). 13C NMR (CDCl3) δ 14.62, 21.61, 42.24,
50.68, 62.08, 120.70, 123.05, 123.85, 141.94, 151.89, 155.01. Anal.
(C14H18N6O8) C, H, N.
O2-(2,4-Dinitro-5-methylphenyl) 1-[(4-Ethoxycarbonyl)-
homopiperazin-1-yl]diazen-1-ium-1,2-diolate (7) (RN-2−45).
To a solution of 4 (254 mg, 1.0 mmol) in 5 mL of DMSO was added a
solution of 5 (200 mg, 1.0 mmol) in 1 mL of DMSO at room
temperature. After 12 h, reaction was quenched with ice−water and
extracted with ether. The combined organic layer was dried over
anhydrous sodium sulfate, and solvent was evaporated under reduced
For proliferation assays, cells were seeded at 2 × 104 /well in 96-
well plates, allowed to adhere for 24 h, and then were treated with the
drug or DMSO as a control for 48 h. Final concentration of DMSO
did not exceed 0.1%. Compounds were prepared as 10 mM stock
solution in DMSO (Sigma, St. Louis, MO). Increasing drug
concentrations in 10 μL of phosphate-buffered saline (PBS) were
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dx.doi.org/10.1021/jm2004128|J. Med. Chem. 2011, 54, 7751−7758

Journal of Medicinal Chemistry
Article
added to 100 μL of the culture medium and incubated at 37 °C for 72
h. The CellTiter 96 nonradioactive cell proliferation assay (MTT
assay, Promega, Madison, WI) was performed according to the
manufacturer’s protocol. Each compound concentration was repre-
sented in six repeats, and the screening was performed as at least two
independent experiments. IC50 values were calculated using Sigma Plot
software (Systat Software).
Statistical Analysis. All experiments were performed at least
three times, each time at least in triplicate. Results are presented as
averages ± SE. Statistical tests were carried out using GraphPad Instat
version 3.00 (GraphPad Software). Pairwise comparisons included the
t test, with the Welch correction or application of the Mann−Whitney
test as appropriate. Significance of correlations was assessed by the
Pearson linear correlation or the Spearman test as appropriate.
In Vivo Treatments. All animals used in this research project
were cared for and used humanely according to the following policies:
The U.S. Public Health Service Policy on Humane Care and Use of
Animals, 1996; the Guide for the Care and Use of Laboratory Animals,
1996; and the U.S. Government Principles for Utilization and Care of
Vertebrate Animals Used in Testing, Research, and Training, 1985. All
NCI-Frederick animal facilities and the animal program are accredited
by the Association for Assessment and Accreditation of Laboratory
Animal Care International.
ASSOCIATED CONTENT
■
S
* Supporting Information
Results from elemental analysis; decomposition of compound 6
with GSH; immunohistochemical analysis of xenograft tumors.
This material is available free of charge via the Internet at
http://pubs.acs.org.
H1703 cells were harvested at 80% confluence, washed with PBS,
and suspended in PBS. The cells were injected at 5 × 106 sc into a
flank of 7-week-old female athymic NCr-nu/nu mice (Charles River).
The drug injections were initiated when the tumors reached at least 2
mm × 2 mm × 2 mm (typically 4 weeks). Animals were treated three
times a week for three weeks with iv tail vein injections of either
vehicle (2.25% P-123 in PBS) or 1 or 2 (6 μmol/kg in the vehicle) or
6 (8 μmol/kg). Body weights were measured before each drug
injection. Animals were sacrificed two hours after the last drug
injection, and the tumors were excised and weighed. There were 13−
15 mice/group at termination. The nonparametric Mann−Whitney
test was utilized for statistical comparisons of tumor weights.
Treatment with siRNA. H1703 cells growing on 6-well plates at
60% confluence were transfected with siRNA to ATF3 (Santa Cruz
Biotechnology, Santa Cruz, CA) or AllStars nonsilencing siRNA
control (Qiagen, Valencia, CA), using HiPerfect transfection reagent
(Qiagen). After 48 h, cells were treated with 1 μM compound 1 or
equal volume of DMSO for 8 h, and then lysed and processed for
immunoblotting.
Immunoblotting and Immunohistochemistry. Western blot
analysis was performed as previously described.22 Primary antibodies
for caspases 3, 7, PARP, P-SEK1/MMK4, P-SAPK/JNK and SAPK/
JNK, P-ATF2, P-c-jun (Cell Signaling Technology, Danvers, MA),
cleaved PARP p85 (Promega, Madison, WI), and ATF3 (Santa Cruz
Biotechnology, Santa Cruz, CA) were used. SAPK/JNK inhibitor 1,9-
pyrazoloanthrone was obtained from Calbiochem, EMD Chemicals,
San Diego, CA.
Paraffin-embedded xenograft tumor sections were analyzed for P-
SAPK/JNK and ATF3 by immunohistochemistry using the antibody
against P-SAPK/JNK (Cell Signaling, no. 4668) or ATF3 (Novus
Biologicals, NBP1−02935). The staining intensity of the cells was
categorized as negative (−), very weak (∓), weak (+), moderate (++),
or strong (+++).
RT-PCR Analysis of ATF3 mRNA Expression. Total RNA from
H1703 cells treated with 1 or DMSO control was isolated using
RNeasy Mini kit (Qiagen, Valencia, CA). Residual genomic DNA was
removed using DNA-free kit (Ambion/Applied Biosystems, Austin,
TX). For ATF3 mRNA analysis, cDNA was synthesized using the
SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA).
TaqMan probes (Applied Biosystems, Foster City, CA) were used for
real-time PCR amplification. The relative abundance of ATF3
transcript was normalized with β-actin internal control.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 301-846-1246. Fax: 301-846-5946. E-mail: maciaga@
mail.nih.gov.
Author Contributions
#A.E.M. and R.S.N. contributed equally to this work.
ACKNOWLEDGMENTS
■
We thank Ken Kosak (University of Utah) for providing the
Pluronics P123 formulation, Donna Butcher (PHL, SAIC-
Frederick, Inc., NCI-Frederick) for immunohistochemical
analysis, Kathleen Noer and Guity Mohammadi (Frederick-
CCR Flow Cytometry Core, SAIC-Frederick, Inc., NCI-
Frederick) for FACS analysis, and Dr. Debanjan Biswas for
critical comments on the manuscript. This research was
supported by the Intramural Research Program of National
Institutes of Health, National Cancer Institute, and with federal
funds from the National Cancer Institute under contract
HHSN261200800001E.
ABBREVIATIONS USED
■
NO, nitric oxide; JS-K, O2-(2,4-dinitrophenyl) 1-[(4-
ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate;
NSCLC, nonsmall cell lung cancer; GST, glutathione S-
transferase; GSH, glutathione; DFM-FM diacetate, 4-amino-5-
methylamino-2′,7′-difluorofluorescein diacetate; DMSO, di-
methyl 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-diphenyltetrazo-
lium bromide
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