Aryl bis(diazeniumdiolates): Potent inducers of S-glutathionylation of cellular proteins and their in vitro antiproliferative activities

Article abstract of DOI:10.1021/jm800831y

A number of bis(diazeniumdiolates) that we designed to release up to 4 mol of nitric oxide (NO) and that are structural analogues of the NO prodrug and anticancer lead compound O²-{2,4-dinitro-5-[4-(N-methylamino) benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2- diolate (PABA/NO) were synthesized and studied. A majority of these compounds yielded higher levels of NO, were better inhibitors of proliferation of a number of cancer cell lines, and more rapidly induced substantially increased levels of S-glutathionylation of cellular proteins in comparison with PABA/NO. In most cases, the antiproliferative activity and extents of S-glutathionylation correlated well with levels of intracellular NO release. We report bis(diazeniumdiolates) to be a class of S-glutathionylating agents with potent antiproliferative and S-glutathionylating activity.

Full text of DOI:10.1021/jm800831y

7944
J. Med. Chem. 2008, 51, 7944–7952
Aryl Bis(diazeniumdiolates): Potent Inducers of S-Glutathionylation of Cellular Proteins and
Their in Vitro Antiproliferative Activities
,§
Daniela Andrei,*,†,‡,§ Anna E. Maciag, Harinath Chakrapani,† Michael L. Citro, Larry K. Keefer,*,† and Joseph E. Saavedra
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702, and Basic
Research Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702
ReceiVed July 8, 2008
A number of bis(diazeniumdiolates) that we designed to release up to 4 mol of nitric oxide (NO) and that
are structural analogues of the NO prodrug and anticancer lead compound O2-{2,4-dinitro-5-[4-(N-
methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2- diolate (PABA/NO) were syn-
thesized and studied. A majority of these compounds yielded higher levels of NO, were better inhibitors of
proliferation of a number of cancer cell lines, and more rapidly induced substantially increased levels of
S-glutathionylation of cellular proteins in comparison with PABA/NO. In most cases, the antiproliferative
activity and extents of S-glutathionylation correlated well with levels of intracellular NO release. We report
bis(diazeniumdiolates) to be a class of S-glutathionylating agents with potent antiproliferative and
S-glutathionylating activity.
Introduction
is frequently overexpressed by cancer cells. Attack by glu-
tathione (GSH) on the 2,4-dinitroaryl ring results in the release
of a diazeniumdiolate anion, which dissociates to form NO
(Scheme 1).23 This process was shown to be catalyzed by GST
and thus presents one plausible mechanism for selective
generation of potentially cytotoxic nitric oxide in tumors.
Nitric oxide (NOa) is intimately involved in mediating a
plethora of biological processes.1-6 Numerous studies indicate
that nitric oxide is also a promising therapeutic agent for the
treatment of several physiological conditions and disease states,
including cancer, culminating in the pursuit of nitric oxide
prodrugs as potential drug candidates.7-11 The wide-ranging
physiological involvement and applicability of NO offer several
opportunities for drug discovery but also present enormous
challenges, most notably among these the delivery of nitric oxide
selectively to the desired physiological site in order to minimize
any potential side effects.
Diazeniumdiolate anions are prodrugs of nitric oxide that are
widely used as reliable sources of NO in a number of chemical
and biological studies.12-16 Our laboratory has developed a
number of promising diazeniumdiolate-based cancer drug
candidates17-22 such as O2-{2,4-dinitro-5-[4-(N-methylami-
no)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-
1,2-diolate, PABA/NO, which has shown high potency as an
antiproliferative agent.23-25 PABA/NO was shown to have
anticancer activity comparable to that of cisplatin in human
ovarian cancer xenograft studies in a rodent model.24 PABA/
NO and analogous members of the arylated diazeniumdiolate
class of prodrugs were conceived as substrates for glutathione
S-transferase (GST),26-28 a phase II detoxification enzyme that
Addition of glutathione to the cysteine residues in proteins
(S-glutathionylation) is a post-translational modification that
alters protein structure and function.29-34 A variety of chemical
pathways involving thiol oxidation or nitrosation have been
proposed to account mechanistically for protein S-glutathiony-
lation.35 Glutathionylation can serve as a protective mechanism
against cysteine oxidation in proteins and is recognized as an
important component of cellular redox signaling. Altered
S-glutathionylation levels are observed in several pathological
conditions such as melanoma, diabetes mellitus, HIV infection,
and ischemia.30 S-Glutathionylation of several proteins has been
reported; notable among these are PTP1B, caspase-3, HIV
protease, p38, ERK, and JNK. In addition to activation of stress
kinases such as p38 and JNK, PABA/NO (10 µM) also induced
rapid (<5 min) S-glutathionylation of ATP synthase, ꢀ-lactate
dehydrogenase, protein disulfide isomerase, actin, and
24,25
glucosidase-II.
Recently, we reported the synthesis and study of a number
of PABA/NO structural analogues.36 Abundant NO was released
from most of these prodrugs when treated with GSH. Further-
more, it was observed that analogues that released higher
amounts of NO had superior antiproliferative activity while
analogues that formed negligible amounts of NO under these
conditions were inactive against human leukemia HL-60 cells,
suggesting that nitric oxide is a component of the cytotoxicity
of such prodrugs. Hence, it was envisaged that any further
increase of the levels of nitric oxide may result in prodrugs
that have superior cytotoxic effects in comparison with PABA/
NO. Furthermore, nitrosative stress caused by NO and its redox
forms is also proposed to mediate protein S-glutathionylation
by PABA/NO (Scheme 1), suggesting that increased nitric oxide
levels in comparison with PABA/NO should also lead to higher
extents of S-glutathionylation.24,25
* To whom correspondence should be addressed. For D.A.: phone, 708-
524-6540; fax, 708-524-6055; e-mail, dandrei@dom.edu. For L.K.K.: phone,
301-846-1467; fax, 301-846-5946; e-mail, keefer@ncifcrf.gov.
†
Laboratory of Comparative Carcinogenesis, National Cancer Institute.
Present address: Chemistry Department, Dominican University, 7900
‡
W. Division Street, River Forest, IL 60305.
§
These authors contributed equally to this work.
SAIC-Frederick.
a Abbreviations: NO, nitric oxide; PABA/NO, O2-{2,4-dinitro-5-[4-(N-
methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-
diolate; GST, glutathione S-transferase; GSH, glutathione; DFM-FM
diacetate, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; DMSO,
dimethyl sulfoxide; DEA/NO, sodium 1-(N,N-diethylamino)diazen-1-ium-
1,2-diolate; DMA/NO, sodium 1-(N,N-dimethylamino)diazen-1-ium-1,2-
diolate; PROLI/NO, disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-
ium-1,2-diolate; HBSS, Hanks’s balanced salt solution; TBS-T, Tris-buffered
saline-Tween-20; PBS, phosphate buffered saline; MTT, 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
10.1021/jm800831y CCC: $40.75
2008 American Chemical Society
Published on Web 11/18/2008

Aryl Bis(diazeniumdiolates)
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7945
Scheme 1. Mechanism of Nitric Oxide Release from PABA/NO, an O2-Arylated Diazeniumdiolate That Is Designed To Be Activated
by Reaction with Glutathione23,a
a O-SH is a representative protein that can be S-glutathionylated, and O-S-SG is a representative S-glutathionylated protein.
Scheme 2. Design of Bis(diazeniumdiolate) Prodrugs That
Scheme 3. Synthesis of Bis(diazeniumdiolate) Prodrugs
Release Elevated Amounts of NO
One strategy to increase the amount of NO released is to
attach an additional diazeniumdiolate group to the aromatic ring;
attack by two GSHs would result in the formation of up to 4
mol of NO per mole of prodrug (Scheme 2). On the basis of
this design, we report the synthesis, glutathione-mediated nitric
oxide release, S-glutathionylating ability, and in vitro antipro-
liferative activity of a library of nitric oxide prodrugs possessing
two diazeniumdiolate groups.
Results
Glutathione-Activated Nitric Oxide Release. The amount
of nitric oxide released from these compounds upon treatment
with glutathione was determined (Table 3). PABA/NO released
1.4 mol of nitric oxide per mole under these conditions. A
majority of these bis(diazeniumdiolate) prodrugs released at least
twice as much NO as PABA/NO. Excellent yields (g90%) of
nitric oxide from several compounds such as 4b, 4f, 4h, and 4j
were observed.
Antiproliferative Activity. Since PABA/NO has been shown
to exhibit cytostatic activity against the leukemia HL-60 cell
line,23 this line was used as an initial screen for the antiprolif-
erative activity of bis(diazeniumdiolates). All the PABA/NO
analogues prepared in this work, except 7, were found to possess
excellent antiproliferative activity, with IC50 values ranging from
0.9 to 4.0 µM (mean 1.7 µM) and in most cases better than
that of PABA/NO (Table 3).
Synthesis. Earlier, we reported the synthesis of PABA/NO
analogues from the sequential addition of a diazeniumdiolate
ion and then 4-aminobenzoic acid derivatives to 1,5-difluoro-
2,4-dinitrobenzene.23 Using a similar strategy, we prepared the
fluorides 3a, 3b, and 3c (Scheme 3). Treatment of 3a with a
number of diazeniumdiolate anions (2b-k, Table 1) afforded
4a-j in excellent yields (Table 2). By use of a similar procedure,
compounds 5a, 5c, and 5d were prepared from 3b, again in
excellent yields (Table 2). Compound 5b was synthesized from
3c in high yield (Table 2).
Finally, the diazeniumdiolate salt 2k was used to prepare
compound 6 and a prodrug form of 1-[2-(carboxylato)pyrrolidin-
1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), 7 (Scheme 4).
We assume that compound 4g is racemic, as the supplier did
not comment on its stereochemistry. Compounds 6, 5d, 4i, and
7 are assumed to be optically active and to have the configu-
ration of the amino acid proline from which they were all
ultimately prepared. The NMR spectra of 6 and 7 were those
of single compounds, with no evidence of a second diastereomer
that would be anticipated if significant racemization had
occurred.
Three analogues, 4a, 4i, and 5b, were then further tested for
their antiproliferative activity against a number of other human
cancer cell lines. Leukemia U-937, non-small-cell lung cancer
H441 and A549, colon cancer HCT-116, HCT-15, and HT-29,
ovarian cancer OVCAR-3 and OVCAR-5, and prostate cancer
PC-3 cell lines were used for this study. Again, the compounds

7946 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Andrei et al.
Table 1. Diazeniumdiolate Salts (R2N-N2O2-Na+) Used in the Synthesis of Aryl Bis(diazeniumdiolates)
Table 2. Bis(diazeniumdioalate) Prodrugs 4a-5d
4a, 4i, and 5b were found to be superior inhibitors of cell
proliferation and generally showed greater antiproliferative
activity in comparison with that of PABA/NO (Table 4), with
the leukemia cell line appearing to be the most sensitive.
Protein S-Glutathionylation. All but one of the bis(diaz-
eniumdiolate) prodrugs induced rapid protein S-glutathionylation
in human leukemia HL-60 cells (Figure 1). Treatment with
compound 7 did not result in protein S-glutathionylation. The

Aryl Bis(diazeniumdiolates)
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7947
Scheme 4. Synthesis of Bis(diazeniumdiolate) Prodrug Forms 6
and 7
Figure 1. (A) Equal numbers of HL-60 cells were treated with 5
µM PABA/NO, with the bis(diazeniumdiolate) prodrugs, or with
the vehicle DMSO. Ten micrograms of cellular protein lysates was
separated by SDS-PAGE under nonreducing conditions and im-
munoblotted using antiglutathione antibodies. (B) The immunoblots
shown in (A) were stripped and reprobed with ꢀ-actin antibodies
as a loading control. The band visible in the DMSO control in (A)
is ꢀ-actin, which has been shown to be spontaneously glutathiony-
lated at physiological conditions.37,38
Table 3. Glutathione-Activated Nitric Oxide Release from
Bis(diazeniumdiolate) Prodrugs and PABA/NO and Their in Vitro
Antiproliferative Activity in the HL-60 Human Leukemia Cell Line
compd
moles of NO released
IC50 (µM)a
TGI (µM)b
PABA/NO
1.4
2.7
3.7
3.1
3.5
3.5
3.7
3.0
3.6
3.0
3.8
3.1
2.9
3.0
3.0
2.5
3.5
3.9
1.2
1.6
0.9
1.9
1.2
1.7
1.5
2.3
1.0
1.0
2.1
0.9
2.9
1.8
4.0
9.6
>10
2.0
3.9
2.7
4.9
5.0
4.9
4.9
4.9
2.0
2.3
5.0
3.0
>10
4.0
>10
>10
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
5a
5b
5c
5d
6
7
Figure 2. Intracellular levels of NO measured as DAF-NO derivative
fluorescence intensities in arbitrary units (AU). Error bars represent
standard deviations of three independent experiments carried out with
the final concentration of the compound being 5 µM.
a IC50 ) concentration required to inhibit cell proliferation by 50%. b TGI
) concentration providing total growth inhibition.
levels of S-glutathionylation for two compounds (4i and 6) were
comparable to those of PABA/NO. The 13 remaining com-
pounds caused massive glutathionylation of intracellular pro-
teins, much more intense than the levels resulting from PABA/
NO.
PABA/NO were found to produce significantly higher levels
of fluorescence, indicating greater amounts of intracellular nitric
oxide than that produced by PABA/NO (Figure 2). However,
analogues 4i, 6, and 7 formed intracellular levels of NO
comparable to or lower than those of PABA/NO. The amounts
of NO released intracellularly did not correlate significantly with
those obtained upon reaction with glutathione in simple
phosphate buffer (P ) 0.08). Lack of consistency between the
buffered GSH solution and intracellular NO release measure-
ments may reflect differences in cell permeability of individual
compounds.
Intracellular Nitric Oxide Release. The intracellular level
of nitric oxide after bis(diazeniumdiolate) treatment was quanti-
fied by measuring the extent of reaction with the nitric oxide-
sensitive fluorophore, 4-amino-5-methylamino-2′,7′-difluorof-
luorescein diacetate (DAF-FM diacetate). HL-60 cells were
preloaded with DAF-FM diacetate, followed by treatment with
the bis(diazeniumdiolate) prodrugs or the solvent (DMSO)
control or PABA/NO. Most bis(diazeniumdiolate) analogues of
Table 4. Antiproliferative Activity of 4a, 4i, 5b, and PABA/NO against Selected Cancer Cell Lines
4a 4i
5b
PABA/NO
cell line
IC50 (µM)
TGI (µM)
IC50 (µM)
TGI (µM)
IC50 (µM)
TGI (µM)
IC50 (µM)
TGI (µM)
U-937
H441
A549
HCT-116
HCT-15
HT-29
OVCAR-3
OVCAR-5
PC-3
1.3
5.0
9.1
2.4
2.5
4.5
1.3
3.3
5.0
3.0
7.0
>30
4.0
4.0
10
2.3
0.8
2.1
5.1
1.6
1.7
2.4
1.7
2.5
3.3
1.2
4.5
6.0
3.6
3.3
4.5
3.8
4.0
7.0
0.9
1.7
3.9
1.5
1.6
2.5
1.4
1.9
3.3
3.0
4.0
10
4.0
4.5
6.5
4.0
5.5
8.0
2.7
4.9
>10
5.5
>10
>10
>10
>10
>10
9.5
5.5
5.7
7.0
5.0
7.8
9.0
7.0
>10
>10
>10

7948 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Andrei et al.
amounts of nitric oxide released upon treatment with GSH was
derived. For example, the IC50 value of 4j, which released 3.8
mol of NO, was 1.0 µM while 4i showed a similar inhibitory
effect but released only 3.0 mol of NO under similar conditions.
While the analogue 6 showed good antiproliferative activity
against human leukemia HL-60 cells, the inhibitory activity of
the PROLI/NO analogue 7 was diminished in comparison with
all other prodrugs, although it formed 3.5 mol of NO when
treated with GSH. This observation is consistent with our earlier
finding that the 2,4-dinitrophenyl prodrug form of PROLI/NO
did not significantly inhibit proliferation of HL-60 cells even
at 50 µM.39
The total growth inhibition (TGI) index for several of these
analogues was found to be less than 5 µM. Also, when one of
the diazeniumdiolate groups was diazeniumdiolated dimethyl-
amine 2a, the size and nature of the substituent at the 5-position
of the aromatic ring did not appear to play any significant role
in the antiproliferative activity (IC50 values of 4a-j and 5a
ranged from 0.9 to 2.3 µM; TGIs were 2-5 µM).
Figure 3. Intracellular levels of NO measured as DAF-NO derivative
fluorescence intensities in arbitrary units (AU). Error bars represent
standard deviations of three independent experiments.
Intracellular NO release correlated well with the antiprolif-
erative activities and S-glutathionylating abilities, however. For
example, from the intracellular NO release study with DAF-
FM diacetate, compound 7, which had the lowest levels of
fluorescence among the analogues, showed both diminished
antileukemic activity and lowered S-glutathionylation levels. All
compounds with superior TGIs (e5 µM), with the exception
of 4i, also formed greater intracellular levels of NO in
comparison with PABA/NO (TGI > 10 µM). The intracellular
NO release from 4i was comparable with that of the prolinol
analogue 6, but both these analogues had diminished intracellular
NO-releasing capacities in comparison with 5d. This observation
was consistent over the concentration range 0.5-5 µM (Figure
3). At this time, we are unable to provide a rationale to account
for these observations. However, the levels of intracellular nitric
oxide formed from the prolinol analogues 4i, 5d, and 6
correlated well with their S-glutathionylating abilities, suggesting
that cell permeability is critical to the observed biological effects
of this series of compounds.
Proliferation of the lung cancer cell line A549, which was
resistant to PABA/NO, was significantly inhibited by 4i and
5b, whose IC50 values were 5.1 and 3.9 µM, respectively, and
moderately inhibited by 4a (IC50 ) 9.1 µM). Compound 4i was
the most consistent inhibitor of proliferation in the range of
cancer cell lines tested, and in all cases, the TGI was less than
10 µM. Intracellular NO release and S-glutathionylation by 4i
were at levels comparable to that of PABA/NO, showing that
factors other than nitric oxide generation contribute to antipro-
liferative activity.
Scheme 5. Proposed Mechanism of Nitric Oxide Release from
Bis(diazeniumdiolate) Prodrugs
We were curious about the structure-property relationships
among 4i, 5d, and 6. All three bear a prolinol diazeniumdiolate
substitutent at one position of the dinitrophenyl ring, the
difference being that the other substituent is the diazeniumdiolate
of dimethylamine (4i), diethylamine (5d), or a second prolinol
(6). All three had low IC50 values in the HL-60 cell screen (1.0,
1.8, and 4.0 µM, respectively, Table 3), but the diethylamine
derivative was far more active than the other two as an inducer
of S-glutathionylation (Figure 1) and intracellular release of
active NO (Figure 2). While we were confident that these were
reproducible observations, a referee’s question led us to run an
additional experiment. In this case, the three compounds were
compared with PABA/NO at four different concentrations
ranging from 0.5 to 5 µM. The results, summarized in Figure
3, once again showed that 5d was substantially more active than
the other three. The mechanistic origins of this difference remain
to be determined.
Conclusions
A number of bis(diazeniumdiolated) dinitrobenzene deriva-
tives that generate up to 4 mol of NO per mole of prodrug were
synthesized and found to have excellent protein S-glutathiony-
lating ability in human leukemia HL-60 cells. The antiprolif-
erative activities of a majority of these prodrugs were superior
to those of PABA/NO, suggesting them as an improved class
of antitumor agents with potent ability to induce S-glutathio-
nylation of proteins in cells treated with them.
Discussion
The results show that adding a second diazeniumdiolate group
to the aromatic ring of previously reported arylated diazenium-
diolate prodrugs generally increased the amount of NO released
(Scheme 5) on reaction with glutathione in neutral buffer
solution relative to the mono(diazeniumdiolated) analogue
PABA/NO, as expected, providing significantly higher antipro-
liferative activity than PABA/NO across a diverse panel of
tumor cell lines.
Experimental Section
NO was purchased from Matheson Gas Products (Montgomer-
yville, PA). Other starting materials were purchased from Aldrich
Chemical Co. (Milwaukee, WI) unless otherwise indicated. NMR
spectra were recorded on a Varian UNITY INOVA spectrometer;
Within the group of bis(diazeniumdiolates) reported here,
however, no significant correlation between IC50 values and

Aryl Bis(diazeniumdiolates)
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7949
chemical shifts (δ) are reported in parts per million (ppm) downfield
from tetramethylsilane. Unless otherwise specified, the solvent used
in all NMR experiments was CDCl3. Quantification of NO after
treatment of compounds with glutathione (GSH) was carried out
by chemiluminescence with a thermal energy analyzer model 502A
instrument (Thermedics, Inc., Woburn, MA) or a Sievers 280i nitric
oxide analyzer (Boulder, CO). 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). Compounds
2a,40 2b,41 2c,42 2d-e,20 2g,42 2h,41 2i,43 2j,44 2k,45 3a,20 and
3c20 were prepared using reported methods.
Sodium 1-(4-N,N-Diethylcarbamoylpiperazin-1-yl)diazen-1-
ium-1,2-diolate (2f). Compound 2f was prepared according to ref
18, but the procedure reported for it there was incorrectly labeled
as that of “JS-K” in the Materials and Methods section of that paper.
The correct procedure for 2f is as follows (summarized from ref
18). A solution of 5.3 g (0.029 mol) of N,N-diethylcarbamoylpip-
erazine in 15 mL of methanol was placed in a Parr bottle and treated
with 6.7 mL (0.031 mol) of methanolic sodium methoxide. The
solution was purged with 40 psi of NO and stirred at 25 °C for
72 h. The solid precipitate was filtered, washed with ether, and
dried to give 4.54 g (60%) of diazeniumdiolate salt: UV (0.1 M
NaOH) λmax (ε) 250 nm (9.8 mM-1 cm-1); 1H NMR (0.1 M NaOD
in D2O) δ 1.13 (t, J ) 7.1 Hz, 6H), 3.13-3.16 (m, 4H), 3.27 (q,
J ) 7.1 Hz, 4H), 3.43-3.47 (m, 4H); 13C NMR (0.1 M NaOD in
D2O) δ 29.7, 33.2, 36.5, 39.0 152.7.
0.97 (t, J ) 7.2 Hz, 3H), 1.64-1.73 (m, 2H), 3.28 (s, 6H),
3.62-3.65 (m, 4H), 3.72-3.75 (m, 4H), 4.08 (t, J ) 6.8 Hz, 2H),
7.54 (s, 1H), 8.82 (s, 1H); 13C NMR δ 10.2, 22.1, 41.6, 42.0, 50.3,
67.5, 104.7, 125.4, 131.2, 131.4, 153.8, 154.2, 155.0. Anal.
(C16H23N9O10) C, H. N: calcd 25.14; found 24.62.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-{[(4-n-butoxycarbonyl)piperazin-1-yl]diazen-1-ium-
1,2-diol-2-ato}benzene (4c). A solution of 2e (270 mg, 1.00 mmol)
in 10 mL of acetone was added to a solution of 3a (250 mg, 0.86
mmol) in 10 mL of acetone. Purification of the crude material was
carried out by recrystallization from ethanol to give 4c (480 mg,
93%): mp 113-114 °C; UV (ethanol) λmax (ε) 290 nm (24 mM-1
1
cm-1); H NMR δ 0.95 (t, J ) 7.3 Hz, 3H), 1.35-1.44 (m, 2H),
1.61-1.68 (m, 2H), 3.28 (s, 6H), 3.61-3.63 (m, 4H), 3.71-3.73
(m, 4H), 4.12 (t, J ) 6.7 Hz, 2H), 7.55 (s, 1H), 8.83 (s, 1H); 13C
NMR δ 13.7, 19.1, 30.9, 41.6, 42.1, 50.3, 65.9, 104.9, 125.4, 131.4,
131.6, 153.8, 154.2, 155.0. Anal. (C17H25N9O10) C, H. N: calcd
24.46; found 24.01.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-{[4-(N,N-diethylaminocarbonyl)piperazin-1-yl]diazen-
1-ium-1,2-diol-2-ato}benzene (4d). A solution of 2f (255 mg, 1.00
mmol) in 10 mL of acetone was added to a solution of 3a (250
mg, 0.86 mmol) in 10 mL of acetone. Purification of the crude
material was carried out on silica gel with 7:3 ethyl acetate/hexane
as the eluant. The product 4d was obtained as a pale-yellow solid
(487 mg, 94%): mp 133-134 °C; UV (ethanol) λmax (ε) 289 nm
(25 mM-1 cm-1); 1H NMR δ 1.15 (t, J ) 7.1 Hz, 6H), 3.23-3.30
(m, 10H), 3.42 (t, J ) 5.1 Hz, 4H), 3.63-3.68 (m, 4H), 7.57 (s,
1H), 8.85 (s, 1H); 13C NMR δ 13.2, 41.7, 41.8, 45.6, 50.2, 105.1,
125.5, 131.5, 131.7, 153.8, 154.0, 154.3, 163.6. Anal. (C17H26N10O9)
C, H, N.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-{[4-(pyrimidin-2-yl)piperazin-1-yl]diazen-1-ium-1,2-
diol-2-ato}benzene (4e). A solution of 2g (200 mg, 0.81 mmol) in
8 mL of acetone was added to a solution of 3a (184 mg, 0.63 mmol)
in 8 mL of acetone. Purification of the crude material was carried
out by recrystallization from ethanol to give 4e (420 mg, 85%):
mp 139-140 °C; UV (ethanol) λmax (ε) 290 nm (23 mM-1 cm-1);
1H NMR δ 3.28 (s, 6H), 3.69-3.72 (m, 4H), 4.07-4.09 (m, 4H),
6.61 (t, J ) 4.8 Hz, 1H), 7.59 (s, 1H), 8.35 (d, J ) 4.8 Hz, 2H),
8.86 (s, 1H); 13C NMR δ 41.8, 42.3, 50.5, 105.0, 111.1, 125.6,
131.6, 131.7, 154.0, 154.3, 157.8, 160.8. Anal. (C16H19N11O8) C,
H, N.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-[(piperidin-1-yl)diazen-1-ium-1,2-diol-2-ato]benzene (4f).
The product 4f (375 mg, 90%) was obtained from 2h (250 mg,
0.86 mmol) and 3a (190 mg, 1.13 mmol). The crude compound 4f
was purified by recrystallization from ethanol: mp 134-135 °C;
UV (ethanol) λmax(ε) 292 nm (26 mM-1 cm-1); 1H NMR δ
1.57-1.63 (m, 2H), 1.80 (m, 4H), 3.28 (s, 6H), 3.61-6.64 (m,
4H), 7.59 (s, 1H), 8.86 (s, 1H); 13C NMR δ 23.2, 24.3, 29.7, 41.7,
51.6, 110.0, 125. 5, 131.5, 154.3. Anal. (C13H18N8O8) C, H, N.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-[(2-methylpiperidin-1-yl)diazen-1-ium-1,2-diol-2-
ato]benzene (4g). This reaction was carried out as described in
the general procedure using 2i (260 mg, 1.45 mmol) and 3a (300
mg, 1 mmol). Purification of the crude material was carried out on
silica gel with 7:3 ethyl acetate/hexane as the eluant to give 4g
(350 mg, 81%): mp 114-115 °C; UV (ethanol) λmax (ε) 286 nm
(22 mM-1 cm-1); 1H NMR δ 1.13 (d, J ) 6.0 Hz, 3H), 1.44-1.62
(m, 3H), 1.66-1.74 (m, 1H), 1.80 (quintet, J ) 6.0 Hz, 2H),
1.87-1.93 (m, 1H), 3.28 (s, 6H), 3.51-3.56 (m, 1H), 3.77-3.85
(m, 1H), 7.68 (s, 1H), 8.85 (s, 1H); 13C NMR δ 16.2, 21.1, 24.9,
31.7, 41.8, 51.2, 55.7, 105.6, 125.4, 131.8, 153.9, 154.3. Anal.
(C14H20N8O8) C, H, N.
O2-(2,4-Dinitro-5-fluorophenyl) 1-(N,N-Diethylamino)diazen-
1-ium-1,2-diolate (3b). A partial solution of 2 g (9.8 mmol) of
1,5-difluoro-2,4-dinitrobenzene in 25 mL of tert-butyl alcohol was
placed in a round-bottom flask and stirred at room temperature. A
solution of 0.70 g (4.4 mmol) of 2b in 15 mL of 5% sodium
bicarbonate was added dropwise at a rapid rate. The reaction mixture
was stirred for 30 min, and a thick yellow solid was formed. Water
was added, and the product was collected by filtration and
recrystallized from methanol to give 3b (2.2 g, 70%): mp 59-60
1
°C; UV (ethanol) λmax (ε) 254 nm (12 mM-1 cm-1); H NMR δ
1.26 (t, J ) 7.1 Hz, 6H), 3.65 (q, J ) 7.1 Hz, 4H), 7.44 (d, J )
11.7 Hz, 1H), 8.90 (d, J ) 7.5 Hz, 1H); 13C NMR δ 11.40, 46.52,
2
106.31 (d, JC-F ) 27.6 Hz), 130.71, 132.56, 155.19, 157.29,
160.01. Anal. (C10H12FN5O6) C, H, F, N.
General Procedure for the Synthesis of Bis(diazeniumdi-
olates). A slurry of diazeniumdiolate salt (1.3 mmol) in 10 mL of
acetone is added slowly to a solution of 1-diazeniumdiolated
5-fluoro-2,4-dinitrobenzene (1 mmol) in 10 mL of acetone that was
previously cooled to 0 °C under nitrogen. The reaction mixture is
allowed to warm gradually to room temperature. The progress of
reaction is monitored by TLC. The product is extracted with
dichloromethane and washed with 5% sodium bicarbonate solution.
The organic layer is dried over anhydrous sodium sulfate, filtered,
and evaporated under vacuum to give the crude product. Purification
is carried out by recrystallization or flash chromatography.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-{[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-
diol-2-ato}benzene (4a). A solution of 2c (282 mg, 1.3 mmol) in
10 mL of acetone was added to a solution of 3a (289 mg, 1 mmol)
in 10 mL of acetone. Purification of the crude material was carried
out on silica gel with 7:3 ethyl acetate/hexane as the eluant. The
product 4a was obtained as an oil (410 mg, 84%): UV (ethanol)
λ
max (ε) 290 nm (22 mM-1 cm-1); 1H NMR δ 1.29 (t, J ) 7.1 Hz,
3H), 3.28 (s, 6H), 3.61-3.64 (m, 4H), 3.71-3.74 (m, 4H), 4.18
(q, J ) 7.1 Hz, 2H), 7.55 (s, 1H), 8.83 (s, 1H); 13C NMR δ 14.6,
41.8, 50.5, 62.1, 105.3, 125.5, 131.8, 131.9, 153.8, 154.2, 155.0.
Anal. (C15H21N9O10) C, H, N.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-{[(4-n-propoxycarbonyl)piperazin-1-yl]diazen-1-ium-
1,2-diol-2-ato}benzene (4b). The product 4b (478 mg, 95%) was
obtained from 2d (254 mg, 1.00 mmol) and 3a (250 mg, 0.86
mmol). Purification of the crude material was carried out on silica
gel with 10:1 dichloromethane/acetone as the eluant: mp 111-112
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-[(pyrrolidin-1-yl)diazen-1-ium-1,2-diol-2-ato]ben-
zene (4h). A solution of 2j (198 mg, 1.3 mmol) in 15 mL of acetone
was added to a solution of 3a (289 mg, 1 mmol) in 10 mL of
acetone. Purification of the crude material was carried out on silica
gel with 10:1 dichloromethane/acetone as the eluant. The product
1
°C; UV (ethanol) λmax (ε) 290 nm (24 mM-1 cm-1); H NMR δ

7950 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Andrei et al.
4h was obtained as a yellow powder (318 mg, 79%): mp 139-140
) 7.1 Hz, 6H), 2.01-2.19 (m, 4H), 2.28 (t, J ) 5.5 Hz, 1H), 3.58
(q, J ) 7.1 Hz, 4H), 3.71-3.79 (m, 2H), 3.83-3.92 (m, 2H),
4.33-4.39 (m, 1H), 7.60 (s, 1H), 8.87 (s, 1H); 13C NMR δ 11.6,
23.2, 27.0, 46.9, 52.4, 64.0, 64.4, 104.2, 125.6, 131.0, 131.1, 154.4.
Anal. (C15H22N8O9) C, H, N.
1
°C; UV (ethanol) λmax (ε) 293 nm (22 mM-1 cm-1); H NMR δ
2.05-2.09 (m, 4H), 3.27 (s, 6H), 3.74-3.78 (m, 4H), 7.58 (s, 1H),
8.86 (s, 1H);13C NMR δ 23.4, 41.7, 50.5, 104.4, 125.5, 131.1, 131.2,
154.3, 154.7. Anal. (C12H16N8O8) C, H, N.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-[(2-hydroxymethylpyrrolidin-1-yl)diazen-1-ium-1,2-
diol-2-ato]benzene (4i). A solution of 2k (475 mg, 2.6 mmol) in
15 mL of acetone was mixed with a solution of 3a (578 mg, 2
mmol) in 10 mL of acetone. Purification of the crude material was
carried out on silica gel with 7:3 ethyl acetate/hexane as the eluant.
The product 4i was obtained as an oil (290 mg, 72%): UV (ethanol)
1,5-Bis[(2-hydroxymethylpyrrolidin-1-yl)diazen-1-ium-1,2-
diol-2-ato]-2,4-dinitrobenzene (6). Sodium 2-(hydroxymethylpyr-
rolidin-1-yl)diazen-1-ium-1,2-diolate, 2k (745 mg, 4.07 mmol), was
dissolved in 5 mL of cold (4 °C) water and placed in a round-
bottom flask that had been immersed in an ice-water bath. A
solution of 830 mg (4.07 mmol) of 1,5-difluoro-2,4-dinitrobenzene
in tetrahydrofuran was prepared, and to this was added 5 mL of
tert-butyl alcohol. The resulting solution was added to the cold
reagent and stirred at 0 °C for 15 min. The ice bath was removed,
and the resulting yellow slurry was stirred overnight. Sodium
bicarbonate solution (5%) was added, and the resulting precipitate
was collected by filtration to give 645 mg of a yellow solid. Flash
chromatography on silica gel using 5:1 dichloromethane/ethyl
acetate followed by 10:1 dichloromethane/methanol gave 243 mg
(12%) of product: mp 131-132 °C; UV (ethanol) λmax (ε) 300 nm
λ
max (ε) 295 nm (21 mM-1 cm-1); 1H NMR δ 2.00-2.20 (m, 5H),
3.28 (s, 6H), 3.71-3.77 (m, 2H), 3.83-3.92 (m, 2H), 4.33-4.37
(m, 1H), 7.58 (s, 1H), 8.86 (s, 1H); 13C NMR δ 23.2, 27.0, 41.7,
52.4, 64.2, 64.5, 104.3, 125.6, 131.0, 131.1, 154.3, 154.4. Anal.
(C13H18N8O9) H, N. C: calcd 36.28; found 35.87.
1-[1-(N,N-Dimethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-
dinitro-5-[1-(N,N-diethylamino)diazen-1-ium-1,2-diol-2-ato]ben-
zene (4j). The product 4j (368 mg, 91%) was obtained from 2b
(220 mg, 1.4 mmol) and 3a (300 mg, 1 mmol). Purification of the
crude material was carried out by recrystallization from ethyl
acetate/hexane (7:3): mp 130-131 °C; UV (ethanol) λmax (ε) 292
1
(22.5 mM-1 cm-1); H NMR δ 1.99-2.21 (m, 10H), 3.76-3.81
(m, 4H), 3.83-3.91 (m, 4H), 4.34-4.39 (m, 2H), 7.54 (s, 1H),
8.88 (s, 1H); 13C NMR δ 23.1, 27.2, 52.4, 64.3, 64.4, 103.8, 125.7,
130.9, 154.6. Anal. (C16H22N8O10) C, H, N.
1
nm (25 mM-1 cm-1); H NMR δ 1.26 (t, J ) 7.0 Hz, 6H), 3.27
(s, 6H), 3.58 (q, J ) 7.0 Hz, 4H), 7.59 (s, 1H), 8.88 (s, 1H); 13C
NMR δ 11.6, 41.7, 46.9, 104.5, 125.6, 131.2, 131.1, 154.4, 154.5.
Anal. (C12H18N8O8) C, H, N.
1,5-Bis[(2-carboxylatopyrrolidin-1-yl)diazen-1-ium-1,2-diol-
2-ato]-2,4-dinitrobenzene (7). To a solution of 6 (160 mg, 0.33
mmol) in 2 mL of acetonitrile and 2 mL of ethyl acetate were added
564 mg (2.64 mmol) of sodium periodate and 4 mL of water,
followed by 4.6 mg (0.02 mmol) of ruthenium trichloride. The
mixture was stirred at room temperature for 2 h. The reaction
mixture was filtered, diluted with ethyl acetate, and washed with
water. The bis-carboxylic acid was extracted from the organic layer
with 5% sodium bicarbonate. The aqueous layer was acidified with
dilute HCl and extracted with ethyl acetate, dried over sodium
sulfate, filtered, and evaporated to give a solid. The solid was taken
up in dichloromethane, giving a cloudy solution that was filtered
through magnesium sulfate and concentrated under vacuum to give
100 mg (59%) of product: mp 135-136 °C; UV (EtOH) λmax (ε)
295 nm (23 mM-1 cm-1); 1H NMR δ 2.02-2.05 (m, 6H),
2.33-2.39 (m, 2H), 3.90 (broad, 4H), 4.71-4.73 (m, 2H), 7.55 (s,
1H), 8.88 (s, 1H), 13.17 (b, 1H); 13C NMR δ 22.0, 27.9, 50.5,
1-[1-(N,N-Diethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-dini-
tro-5-{[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-
2-ato}benzene (5a). A solution of 2c (390 mg, 1.79 mmol) in 10
mL of acetone was added to a solution of 3b (480 mg, 1.51 mmol)
in 10 mL of acetone. Purification of the crude material was carried
out on silica gel with 7:3 ethyl acetate/hexane as the eluant. The
product 5a was obtained as an oil (400 mg, 77%): UV (ethanol)
λ
max (ε) 291 nm (24 mM-1 cm-1); 1H NMR δ 1.27 (t, J ) 7.1 Hz,
6H), 1.29 (t, J ) 7.2 Hz, 3H), 3.58-3.66 (m, 8H), 3.71-3.75 (m,
4H), 4.18 (q, J ) 7.1 Hz, 2H), 7.57 (s, 1H), 8.86 (s, 1H); 13C
NMR δ 11.5, 14.5, 42.1, 46.8, 50.4, 62.0, 104.7, 125.5, 131.2, 131.5,
153.9, 154.4, 154.9. Anal. (C17H25N9O10) C, H, N.
1-[4-(Ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-
ato]-2,4-dinitro-5-{[4-(N,N-diethylaminocarbonyl)piperazin-1-
yl]diazen-1-ium-1,2-diol-2-ato}benzene (5b). A solution of 3c (172
mg, 0.42 mmol) in 10 mL of acetone was added to a solution of 2f
(154 mg, 0.60 mmol) in 10 mL of acetone. Purification of the crude
material was carried out by recrystallization from ethanol to give
5b (215 mg, 81%): mp 94-95 °C; UV (ethanol) λmax (ε) 288 nm
61.8, 102.6, 125.7, 130.3, 154.1, 171.8. Anal. (C16H18N8O12
0.2EtOAc) C, H, N.
·
Nitric Oxide Release. Chemiluminescence detection and quan-
tification of NO evolving from the reactions of PABA/NO and its
analogues were conducted by preparing 10 mM stock solutions of
these compounds in pH 7.4 phosphate buffer. A pH 7.4 0.1 M
phosphate buffer (3 mL) containing 50 µM diethyltriaminepen-
taacetic acid and GSH (4 mM) was sparged into the chemilumi-
nescence detector with inert gas until a steady detector response
was established. The stock solution of the diazeniumdiolate prodrug
(20-100 µL, 0.31-1.87 µM) was injected into this GSH solution,
and the NO release profile was followed at 37 °C. Nitric oxide
release was monitored until baseline signal was achieved (25 min
to 5 h). The resulting curve was integrated to quantify the amount
of NO released per mole of compound.
Intracellular NO Release. The intracellular level of nitric oxide
after bis(diazeniumdiolate) prodrug treatment was quantified using
the NO-sensitive fluorophore 4-amino-5-methylamino-2′,7′-difluo-
rofluorescein diacetate (DAF-FM diacetate) (Invitrogen, Carlsbad,
CA). HL-60 cells were loaded with 2.5 µM DAF-FM diacetate in
Hanks’ balanced salt solution (HBSS) at 37 °C and 5% CO2. After
30 min of incubation the cells were collected by centrifugation,
rinsed with HBSS to remove excess of probe, and resuspended in
fresh HBSS. The compounds were added to the cells at 5 µM final
concentration. After 40 min of incubation the fluorescence of the
benzotriazole derivative formed on DAF-FM’s reaction with aerobic
NO was analyzed using a Perkin-Elmer LS50B luminescence
spectrometer with the excitation source at 495 nm and emission at
515 nm.
1
(26 mM-1 cm-1); H NMR δ 1.16 (t, J ) 6.8 Hz, 6H), 1.29 (t, J
) 7.0 Hz, 3H), 3.26 (q, J ) 7.0 Hz, 4H), 3.42-3.45 (m, 4H),
3.60-3.68 (m, 8H), 3.72-3.74 (m, 4H), 4.19 (q, J ) 6.8 Hz, 2H),
7.55 (s, 1H), 8.81 (s, 1H); 13C NMR δ 13.4, 14.5, 41.7, 45.6, 50.2,
50.5, 62.0, 105.9, 125.4, 132.1, 153.7, 153.8, 154.9, 163.5. Anal.
(C22H33N11O11) C, H, N.
1-[1-(N,N-Diethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-dini-
tro-5-{[(4-(N,N-diethyl-aminocarbonyl)piperazin-1-yl]diazen-1-
ium-1,2-diol-2-ato}benzene (5c). Product 5c (490 mg, 90%) was
obtained from 2f (229 mg, 0.9 mmol) and 3b (240 mg, 0.75 mmol).
Purification of the crude material was carried out on silica gel with
10:1 dichloromethane/acetone as the eluant to give 5c as a solid:
mp 98-99 °C; UV (ethanol) λmax (ε) 291 nm (25 mM-1 cm-1);
1H NMR δ 1.15 (t, J ) 6.8 Hz, 6H), 1.27 (t, J ) 7.1 Hz, 6H), 3.26
(q, J ) 7.1 Hz, 4H), 3.43 (m, 4H), 3.60 (q, J ) 6.8 Hz, 4H),
3.63-3.67 (m, 4H), 7.58 (s, 1H), 8.87 (s, 1H); 13C NMR δ 11.5,
13.1, 41.7, 45.6, 46.7, 50.1, 104.5, 125.5, 131.0, 131.3, 154.0, 154.5,
163.5. Anal. (C19H30N10O9) C, H, N.
1-[1-(N,N-Diethylamino)diazen-1-ium-1,2-diol-2-ato]-2,4-dini-
tro-5-{[(2-hydroxymethyl)-pyrrolidin-1-yl]diazen-1-ium-1,2-diol-
2-ato}benzene (5d). Product 5d (400 mg, 87%) was obtained from
2k (183 mg, 1.00 mmol) and 3b (412 mg, 1.3 mmol). Purification
of the crude material was carried out on silica gel with 10:1
dichloromethane/acetone as the eluant: mp 116-117 °C; UV
(ethanol) λmax (ε) 297 nm (21 mM-1 cm-1); 1H NMR δ 1.25 (t, J

Aryl Bis(diazeniumdiolates)
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7951
(5) Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Nitric oxide: physiology,
pathophysiology, and pharmacology. Pharmacol. ReV. 1991, 43, 109–
142.
(6) Murad, F. Discovery of some of the biological effects of nitric oxide
and its role in cell signaling (Nobel lecture). Angew. Chem., Int. Ed.
1999, 38, 1856–1868.
(7) Bonavida, B.; Khineche, S.; Huerta-Yepez, S.; Garba´n, H. Therapeutic
potential of nitric oxide in cancer. Drug Resist. Updates 2006, 9, 157–
173.
(8) Magrinat, G.; Mason, S. N.; Shami, P. J.; Weinberg, J. B. Nitric oxide
modulation of human leukemia cell differentiation and gene expression.
Blood 1992, 80, 1880–1884.
(9) Shami, P. J.; Moore, J. O.; Gockerman, J. P.; Hathorn, J. W.;
Misukonis, M. A.; Weinberg, J. B. Nitric oxide modulation of the
growth and differentiation of freshly isolated acute non-lymphocytic
leukemia cells. Leuk. Res. 1995, 19, 527–533.
(10) Shami, P. J.; Sauls, D. L.; Weinberg, J. B. Schedule and concentration-
dependent induction of apoptosis in leukemia cells by nitric oxide.
Leukemia 1998, 12, 1461–1466.
(11) Weinberg, J. B.; Misukonis, M. A.; Shami, P. J.; Mason, S. N.; Sauls,
D. L.; Dittman, W. A.; Wood, E. R.; Smith, G. K.; McDonald, B.;
Bachus, K. E.; Haney, A. F.; Granger, D. L. Human mononuclear
phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS
mRNA, iNOS protein, biopterin, and nitric oxide production by blood
monocytes and peritoneal macrophages. Blood 1995, 86, 1184–1195.
(12) Keefer, L. K.; Nims, R. W.; Davies, K. M.; Wink, D. A. “NONOates”
(1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors:
convenient nitric oxide dosage forms. Methods Enzymol. 1996, 268,
281–293.
Cell Culture and Cytotoxicity Assays. Cell lines HL-60, U-937,
H441, and A549 were obtained from American Type Culture
Collection (ATCC, Manassas, VA), and HCT-116, HCT-15, HT29,
OVCAR-3, OVCAR-5, and PC-3 were from the NCIsFrederick
DCTD tumor/cell line repository (Frederick, MD). 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.
The CellTiter 96 nonradioactive cell proliferation assay (MTT
assay; Promega, Madison, WI), performed according to the
manufacturer’s protocol, was used to measure cell proliferation.
Cells were seeded in 96-well plates at a density of 104 per well
and allowed to grow for 24 h before addition of the drugs.
Bis(diazeniumdiolates) were prepared as 10 mM stock solution in
dimethyl sulfoxide (Sigma, St. Louis, MO). Increasing drug
concentrations in 10 µL of phosphate-buffered saline (PBS) were
added to 100 µL of the culture medium and incubated at 37 °C for
72 h. Each compound concentration was represented in six repeats,
and the screening was performed as at least two independent
experiments.
S-Glutathionylating Activities of Bis(diazeniumdiolates). HL-
60 cells in complete growth medium (RPMI 1640 supplemented
with 10% fetal bovine serum, glutamine, and penicillin/streptomy-
cin) were treated with 1 µM compound, or an equal volume of
dimethyl sulfoxide as a control, for 15 min. Cells were collected
by centrifugation, washed with PBS, and prepared for immunob-
lotting by lysing in lysis buffer (25 mM Hepes buffer, pH 7.2,
containing 150 mM NaCl, 10 mM MgCl2, 1% Nonidet P40, 0.25%
sodium deoxycholate, 10% glycerol, 2.5 mM EDTA, and protease
inhibitor cocktail (Roche Applied Science, Indianapolis, IN).
Proteins in cell lysates were separated under nonreducing conditions
on 4-12% NuPage bis-Tris gels (Invitrogen Life Technologies,
Carlsbad, CA) and transferred to PVDF membranes (Invitrogen)
at 30 V for 1.5 h. After overnight blocking in 5% nonfat milk in
Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the
membranes were probed with anti-glutathione monoclonal antibod-
ies (1:1000, Virogen, Watertown, MA) in 5% milk in TBS-T for
3 h at room temperature. After repeated washings in TBS-T, the
blots were incubated for 60 min at room temperature with antimouse
secondary antibody, 1:1000, conjugated with horseradish peroxidase
(GE Healthcare, Piscataway, NJ), in 5% milk in TBS-T, and then,
after washing, developed using the chemiluminescence ECL kit (GE
Healthcare, Piscataway, NJ). To show equal loading, the blots were
stripped with stripping buffer (Thermo Scientific, Rockford, IL)
and reprobed with anti-ꢀ-actin rabbit polyclonal antibodies (Abcam,
Inc., Cambridge, MA).
(13) Keefer, L. K. Nitric oxide (NO)- and nitroxyl (HNO)-generating
diazeniumdiolates (NONOates): emerging commercial opportunities.
Curr. Top. Med. Chem. 2005, 5, 625–636.
(14) Scatena, R.; Bottoni, P.; Martorana, G. E.; Giardina, B. Nitric oxide
donor drugs: an update on pathophysiology and therapeutic potential.
Expert Opin. InVest. Drugs 2005, 14, 835–846.
(15) King, S. B. Mechanisms and novel directions in the biological
applications of nitric oxide donors. Free Radical Biol. Med. 2004,
37, 735–736.
(16) Thatcher, G. R. J. An introduction to NO-related therapeutic agents.
Curr. Top. Med. Chem. 2005, 5, 597–601.
(17) Kiziltepe, T.; Hideshima, T.; Ishitsuka, K.; Ocio, E. M.; Raje, N.;
Catley, L.; Li, C. Q.; Trudel, L. J.; Yasui, H.; Vallet, S.; Kutok, J. L.;
Chauhan, D.; Mitsiades, C. S.; Saavedra, J. E.; Wogan, G. N.; Keefer,
L. K.; Shami, P. J.; Anderson, K. C. JS-K, a GST-activated nitric
oxide generator, induces DNA double-strand breaks, activates DNA
damage response pathways, and induces apoptosis in vitro and in vivo
in human multiple myeloma cells. Blood 2007, 110, 709–718.
(18) Liu, J.; Li, C.; Qu, W.; Leslie, E.; Bonifant, C. L.; Buzard, G. S.;
Saavedra, J. E.; Keefer, L. K.; Waalkes, M. P. Nitric oxide prodrugs
and metallochemotherapeutics: JS-K and CB-3-100 enhance arsenic
and cisplatin cytolethality by increasing cellular accumulation. Mol.
Cancer Ther. 2004, 3, 709–714.
(19) Ren, Z.; Kar, S.; Wang, Z.; Wang, M.; Saavedra, J. E.; Carr, B. I.
JS-K, a novel non-ionic diazeniumdiolate derivative, inhibits Hep 3B
hepatoma cell growth and induces c-Jun phosphorylation via multiple
MAP kinase pathways. J. Cell. Physiol 2003, 197, 426–434.
(20) Shami, P. J.; Saavedra, J. E.; Bonifant, C. L.; Chu, J.; Udupi, V.;
Malaviya, S.; Carr, B. I.; Kar, S.; Wang, M.; Jia, L.; Ji, X.; Keefer,
L. K. Antitumor activity of JS-K [O2-(2,4-dinitrophenyl) 1-[(4-
ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and related
O2-aryl diazeniumdiolates in vitro and in vivo. J. Med. Chem. 2006,
49, 4356–4366.
Acknowledgment. This research was supported by the
Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research, as well as by National
Cancer Institute Contract N01-CO-12400 to SAIC-Frederick.
(21) Shami, P. J.; Saavedra, J. E.; Wang, L. Y.; Bonifant, C. L.; Diwan,
B. A.; Singh, S. V.; Gu, Y.; Fox, S. D.; Buzard, G. S.; Citro, M. L.;
Waterhouse, D. J.; Davies, K. M.; Ji, X.; Keefer, L. K. JS-K, a
glutathione/glutathione S-transferase-activated nitric oxide donor of
the diazeniumdiolate class with potent antineoplastic activity. Mol.
Cancer Ther. 2003, 2, 409–417.
(22) Udupi, V.; Yu, M.; Malaviya, S.; Saavedra, J. E.; Shami, P. J. JS-K,
a nitric oxide prodrug, induces cytochrome c release and caspase
activation in HL-60 myeloid leukemia cells. Leuk. Res. 2006, 30, 1279–
1283.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) Ignarro, L. J.; Cirino, G.; Casini, A.; Napoli, C. Nitric oxide as a
signaling molecule in vascular system: An overview. J. CardioVasc.
Pharmacol. 1999, 34, 879–886.
(23) Saavedra, J. E.; Srinivasan, A.; Buzard, G. S.; Davies, K. M.;
Waterhouse, D. J.; Inami, K.; Wilde, T. C.; Citro, M. L.; Cuellar, M.;
Deschamps, J. R.; Parrish, D.; Shami, P. J.; Findlay, V. J.; Townsend,
D. M.; Tew, K. D.; Singh, S.; Jia, L.; Ji, X.; Keefer, L. K. PABA/NO
as an anticancer lead: analogue synthesis, structure revision, solution
chemistry, reactivity toward glutathione, and in vitro activity. J. Med.
Chem. 2006, 49, 1157–1164.
(24) Findlay, V. J.; Townsend, D. M.; Saavedra, J. E.; Buzard, G. S.; Citro,
M. L.; Keefer, L. K.; Ji, X.; Tew, K. D. Tumor cell responses to a
novel glutathione S-transferase-activated nitric oxide-releasing prodrug.
Mol. Pharmacol. 2004, 65, 1070–1079.
(2) Ignarro, L. J. Nitric oxide: a unique endogenous signaling molecule
in vascular biology (Nobel lecture). Angew. Chem., Int. Ed. 1999, 38,
1882–1892.
(3) Moncada, S.; Herman, A. G.; Vanhoutte, P. Endothelium-derived
relaxing factor is identified as nitric oxide. Trends Pharmacol. Sci.
1987, 8, 365–368.
(4) Moncada, S.; Radomski, M. W.; Palmer, R. M. J. Endothelium-derived
relaxing factor: identification as nitric oxide and role in the control of
vascular tone and platelet function. Biochem. Pharmacol. 1988, 37,
2495–2501.

7952 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Andrei et al.
(25) Townsend, D. M.; Findlay, V. J.; Fazilev, F.; Ogle, M.; Fraser, J.;
Saavedra, J. E.; Ji, X.; Keefer, L. K.; Tew, K. D. A glutathione
S-transferase π-activated prodrug causes kinase activation concurrent
with S-glutathionylation of proteins. Mol. Pharmacol. 2006, 69, 501–
508.
(26) Armstrong, R. N. Glutathione S-transferases: reaction mechanism,
structure, and function. Chem. Res. Toxicol. 1991, 4, 131–140.
(27) Armstrong, R. N. Glutathione S-transferases: structure and mechanism
of an archetypical detoxication enzyme. AdV. Enzymol. Relat. Areas
Mol. Biol. 1994, 69, 1–44.
(28) Armstrong, R. N. Structure, catalytic mechanism, and evolution of
the glutathione transferases. Chem. Res. Toxicol. 1997, 10, 2–18.
(29) Giustarini, D.; Rossi, R.; Milzani, A.; Colombo, R.; Dalle-Donne, I.
S-Glutathionylation: from redox regulation to human diseases. J. Cell.
Mol. Med. 2008, 8, 201–212.
(30) Ghezzi, P.; Di Simplicio, P. Glutathionylation pathways in drug
response. Curr. Opin. Pharmacol. 2007, 7, 398–403.
(31) Gallogly, M. M.; Mieyal, J. J. Mechanisms of reversible protein
gluathionylation in redox signaling and oxidative stress. Curr. Opin.
Pharmacol. 2007, 7, 381–391.
(32) Tew, K. D. Redox in redux: emergent roles for glutathione S-
transferase P (GSTP) in regulation of cell signaling and S-glutathio-
nylation. Biochem. Pharmacol. 2007, 73, 1257–1269.
(33) Dalle-Donne, I.; Rossi, R.; Giustarini, D.; Colombo, R.; Milzani, A.
S-Glutathionylation in protein redox regulation. Free Radical Biol.
Med. 2007, 43, 883–898.
(34) Martinez-Ruiz, A.; Lamas, S. Signalling by NO-induced protein
S-nitrosylation and S-glutathionylation: convergences and divergences.
CardioVasc. Res. 2007, 75, 220–228.
(37) Wang, J.; Tekle, E.; Oubrahim, H.; Mieyal, J. J.; Stadtman, E. R.;
Chock, P. B. Stable and controllable RNA interference: investigating
the physiological function of glutathionylated actin. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 5103–5106.
(38) Wang, J.; Boja, E. S.; Tan, W.; Tekle, E.; Fales, H. M.; English, S.;
Mieyal, J. J.; Chock, P. B. Reversible glutathionylation regulates actin
polymerization in A431 cells. J. Biol. Chem. 2001, 276, 47763–47766.
(39) Chakrapani, H.; Goodblatt, M. M.; Udupi, V.; Malaviya, S.; Shami,
P. J.; Keefer, L. K.; Saavedra, J. E. Synthesis and in vitro anti-leukemic
activity of structural analogues of JS-K, an anti-cancer lead compound.
Bioorg. Med. Chem. Lett. 2008, 18, 950–953.
(40) Saavedra, J. E.; Srinivasan, A.; Bonifant, C. L.; Chu, J.; Shanklin,
A. P.; Flippen-Anderson, J. L.; Rice, W. G.; Turpin, J. A.; Davies,
K. M.; Keefer, L. K. The secondary amine/nitric oxide complex ion
R2N[N(O)NO]- as nucleophile and leaving group in SNAr reactions.
J. Org. Chem. 2001, 66, 3090–3098.
(41) Drago, R. S.; Karstetter, B. R. The reaction of nitrogen(II) oxide with
various primary and secondary amines. J. Am. Chem. Soc. 1961, 83,
1819–1822.
(42) Saavedra, J. E.; Booth, M. N.; Hrabie, J. A.; Davies, K. M.; Keefer,
L. K. Piperazine as a linker for incorporating the nitric oxide-releasing
diazeniumdiolate group into other biomedically relevant functional
molecules. J. Org. Chem. 1999, 64, 5124–5131.
(43) Chakrapani, H.; Showalter, B. M.; Citro, M. L.; Keefer, L. K.;
Saavedra, J. E. Nitric oxide prodrugs: diazeniumdiolate anions of
hindered secondary amines. Org. Lett. 2007, 9, 4551–4554.
(44) Saavedra, J. E.; Billiar, T. R.; Williams, D. L.; Kim, Y.-M.; Watkins,
S. C.; Keefer, L. K. Targeting nitric oxide (NO) delivery in vivo.
Design of a liver-selective NO donor prodrug that blocks tumor
necrosis factor-R-induced apoptosis and toxicity in the liver. J. Med.
Chem. 1997, 40, 1947–1954.
(35) Dalle-Donne, I.; Milzani, A.; Gagliano, N.; Colombo, R.; Giustarini,
D.; Rossi, R. Molecular mechanisms and potential clinical significance
of S-glutathionylation. Antioxid. Redox Signaling 2008, 10, 445–473.
(36) Chakrapani, H.; Wilde, T. C.; Citro, M. L.; Goodblatt, M. M.; Keefer,
L. K.; Saavedra, J. E. Synthesis, nitric oxide release, and anti-leukemic
activity of glutathione-activated nitric oxide prodrugs: structural
analogues of PABA/NO, an anti-cancer lead compound. Bioorg. Med.
Chem. 2008, 16, 2657–2664.
(45) Chakrapani, H.; Showalter, B. M.; Kong, L.; Keefer, L. K.; Saavedra,
J. E. V-PROLI/NO, a prodrug of the nitric oxide donor, PROLI/NO.
Org. Lett. 2007, 9, 3409–3412.
JM800831Y