Antitumor activity of JS-K [O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and related O 2-aryl diazeniumdiolates in vitro and in vivo

Article abstract of DOI:10.1021/jm060022h

The literature provides evidence that metabolic nitric oxide (NO) release mediates the cytotoxic activities (against human leukemia and prostate cancer xenografts in mice) of JS-K, a compound of structure R₂N-N(O)=NO-Ar for which R₂N

Full text of DOI:10.1021/jm060022h

4356
J. Med. Chem. 2006, 49, 4356-4366
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
Paul J. Shami,† Joseph E. Saavedra,‡ Challice L. Bonifant,§ Jingxi Chu,§ Vidya Udupi,† Swati Malaviya,† Brian I. Carr,|
Siddhartha Kar,| Meifeng Wang,| Lee Jia, Xinhua Ji,∞ and Larry K. Keefer*,§
DiVision of Medical Oncology, Department of Internal Medicine, UniVersity of Utah, Salt Lake City, Utah 84112, Basic Research Program,
SAIC Frederick, Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, and Macromolecular Crystallography Laboratory,
National Cancer Institute at Frederick, Frederick, Maryland 21702, LiVer Cancer Center, UniVersity of Pittsburgh,
Pittsburgh, PennsylVania 15213, and DeVelopmental Therapeutics Program, National Cancer Institute, RockVille, Maryland 20852
ReceiVed January 9, 2006
The literature provides evidence that metabolic nitric oxide (NO) release mediates the cytotoxic activities
(against human leukemia and prostate cancer xenografts in mice) of JS-K, a compound of structure R2N-
N(O)dNO-Ar for which R2N is 4-(ethoxycarbonyl)piperazin-1-yl and Ar is 2,4-dinitrophenyl. Here we
present comparative data on the potencies of JS-K and 41 other O2-arylated diazeniumdiolates as inhibitors
of HL-60 human leukemia cell proliferation, as well as in the NCI 51-cell-line screen for six of them. The
data show JS-K to be the most potent of the 42 in both screens and suggest that other features of its structure
and metabolism besides NO release may contribute importantly to its activity. Results with control compounds
implicate JS-K’s arylating ability, and the surprisingly low IC50 value of the N-(ethoxycarbonyl)piperazine
byproduct of NO release suggests a role for the R2N moiety. In addition to the above-mentioned in vivo
activities, JS-K is shown here to be carcinostatic in a rat liver cancer model.
Introduction
π-isoform is overexpressed in many tumor cells,6 we postulated
that through structural modifications we might be able to
increase the efficiency of the diazeniumdiolate’s metabolism
by GST-π. Even though transition state modeling studies of this
material in the enzyme’s active site suggested that structural
modifications might improve selectivity,1 JS-K was chosen for
further development as an antileukemic drug due to its promising
in vivo activity. On the downside, JS-K was found to have
limited solubility in water and to react at a significant rate with
free glutathione in the absence of GST.
We have continued to synthesize and diversify libraries of
O2-arylated diazeniumdiolates designed to release nitric oxide
upon reaction with glutathione catalyzed by GST. Four sys-
tematic approaches for changing the JS-K structure have been
followed: (a) altering the reactivity of the aryl group without
changing the substituent at N-4 of the piperazine moiety; (b)
modifying the substituent at the N-4 position while leaving the
activated aryl group unchanged; (c) combining approaches a
and b by altering the N-4 substituent and installing a less reactive
aryl group at the O2-position of the diazeniumdiolate; and (d)
replacing the piperazine ring altogether using different secondary
amines as the NO and aryl carriers. As a result, we have found
other prodrug candidates that show notable activity against the
HL-60 cell line and may provide us with important information
toward lead optimization.
Compounds of the O2-aryl diazeniumdiolate family have
shown noteworthy anticancer activity in a variety of model
systems. An example with particular promise is JS-Ka (com-
pound 2a, Scheme 1). It induced monocytic differentiation of
HL-60 human leukemia cells in vitro,1 proved pro-apoptotic
through caspase-3 and -9 activation,2 and blocked angiogenesis
by inhibiting proliferation, migration, and cord formation in
human umbilical vein endothelial cell cultures,3 all at micro-
molar or submicromolar concentrations. Moreover, JS-K halved
the growth rate of both human leukemia and prostate cancer
subcutaneous xenografts in NOD-SCID mice, inducing extensive
necrosis in the tumor mass.1 It also inhibited hepatoma cell
proliferation in vitro4 and increased the cytotoxicity of cisplatin
and arsenite in drug-resistant cells by increasing intracellular
drug concentrations.5
JS-K was designed to be activated for nitric oxide (NO)
release by glutathione S-transferase (GST), and this effect may
account for part of its mechanism of action.1 Because the
* Corresponding author. Phone: 301-846-1467. Fax: 301-846-5946.
E-mail: keefer@ncifcrf.gov.
† Department of Internal Medicine, University of Utah.
‡ SAIC Frederick, National Cancer Institute at Frederick.
§ Laboratory of Comparative Carcinogenesis, National Cancer Institute
at Frederick.
| Liver Cancer Center, University of Pittsburgh.
In the present work, we compare the in vitro anticancer
activity of JS-K with those of numerous structural analogues
and then provide further evidence of its in vivo cytotoxic effects.
Developmental Therapeutics Program, National Cancer Institute.
∞ Macromolecular Crystallography Laboratory, National Cancer Institute
at Frederick.
a Abbreviations: B12, hydroxocobalamine; CDNB, 1-chloro-2,4-dinitro-
benzene; 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; DMSO,
dimethyl sulfoxide; DNP, dinitrophenyl; GI50, 50% growth inhibition; GSH,
glutathione; GST, glutathione S-transferase; JS-K, O2-(2,4-dinitrophenyl)
1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate; LC50, 50%
cell killing; NCI, National Cancer Institute; NO, nitric oxide; NOD-SCID,
nonobese diabetic-severe combined immune deficient; PBS, phosphate-
buffered saline; PTIO, (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-
oxide); TGI, total growth inhibition.
Results
Previous reports on the pharmacological effects of JS-K
(structure 2a, Scheme 1) have been largely consistent with the
interpretation that they are mediated by the nitric oxide (NO)
produced on the drug’s reaction with glutathione (GSH) or other
nucleophiles. NO was detected in good yield on reacting JS-K
with GSH (kGSH ) 1.0 M-1 s-1) as well as on simply
10.1021/jm060022h CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/10/2006

Antitumor ActiVity of JS-K in Vitro and in ViVo
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4357
Scheme 1. Synthesis (up arrows) and Presumed Metabolism (down arrows) of JS-K (compound 2a)a
a IC50 values are for inhibition of HL-60 cell proliferation.
hydrolyzing it in 0.1 M phosphate buffer, pH 7.4 (khydr ) 4 ×
10-5 s-1),1 and JS-K inhibited proliferation of HL-60 human
leukemia cells (IC50 ) 0.2-0.5 µM), which are known to be
especially sensitive to the cytostatic/cytotoxic effects of NO.7
But it seemed possible that other features of JS-K’s chemistry
besides NO release might contribute to its antitumor activity.
In evaluating other possible components of JS-K’s mechanism
of action, we began by determining the growth-inhibitory
activities of JS-K’s other presumed metabolites. These are shown
in Scheme 1, along with the IC50 value for each. Only one of
these had an IC50 value within an order of magnitude of JS-K’s
(i.e., IC50 e 5 µM); ion 1a was 50% inhibitory at 4 µM, again
consistent with an NO-mediated mechanism in that it is known
to generate up to 2 equiv of NO spontaneously in physiological
media.
Importantly, the known arylating agent 1-chloro-2,4-dinitro-
benzene (CDNB)8 had an IC50 of 1.4 µM. This control was
included so that we could estimate the potential contribution of
simple arylation by JS-K as a cytotoxic mechanism. Diazenium-
diolate ions are reported to be similar in nucleofugality to
chloride,9 suggesting that CDNB should be a reasonable tool
for addressing this question. The low IC50 value observed (1.4
µM) suggests that arylation of cellular nucleophiles may well
be an important contributor to JS-K’s activity.

4358 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Shami et al.
It was interesting, and somewhat surprising, that the byproduct
of ultimate NO release, N-(ethoxycarbonyl)piperazine (1b), was
about 10-fold more potent in the HL-60 screen than the
anthelminthic piperazine (1c) and, indeed, with an IC50 of 8.6
µM, almost half as active as its diazeniumdiolated derivative,
ion 1a. Furthermore, the BOC analogue N-(tert-butoxycarbonyl)-
piperazine and its ionic diazeniumdiolate derivative had similar
activities (IC50 values of 9.5 and 9.0 µM, respectively). This
suggested that the N-acylated piperazine moiety may also
contribute to JS-K’s activity.
We prepared and tested a series of analogues represented by
structure 2. Four of these (including JS-K) had IC50 values e5
µM, with the methyl (2b), ethyl (2a), n-propyl (2c), n-butyl
(2d) series giving values of 3.5, 0.5, 3.7, and 3.5 µM,
respectively.
Analogues with structure 3 explored further variations on the
nature of the substituent attached to the terminal amino group
of JS-K’s piperazine ring. Secondary amine 3a, prepared as the
hydrochloride salt to prevent its self-destruction by SNAr
displacement of its diazeniumdiolate substituent by a neighbor-
ing molecule’s basic nitrogen center, had IC50 ) 5.0 µM.
Acetylated analogue 3j had IC50 ) 3.9 µM. Very surprisingly,
N-aryl derivatives 3g and 3h were also within the 0.5-5 µM
potency range, with values of 1.5 and 3.6 µM, respectively.
The results of the previous paragraph, together with the high
IC50 values associated with such bulky N-subsitituents as benzyl
(2e) and tert-butyl (2f) (17 and 8.1 µM, respectively), had led
us to expect that activity was a rather sensitive function of the
N-substituent’s steric requirement, optimized among the deriva-
tives tested thus far by ethoxycarbonyl structure 2a. Apparently,
phenyl and pyrimidin-2-yl are also substituents favorable for
activity in this system.
Analogues of structure 4 involved further perturbations of
JS-K’s amino group. Reducing its size to the minimum, as in
the dimethylamino group of 4a, yielded a reasonably potent
analogue with IC50 ) 2.4 µM. Diethylamino was similar, at
2.2 µM. 3-Carboxamidopiperidine derivative 4d had IC50 ) 4.4
µM. Compound 4e explored the effect of expanding JS-K’s
piperazine ring by one methylene group; the resulting IC50 was
3.4 µM.
0.5-5 µM. Attaching a second diazeniumdiolate group to the
5 position of JS-K’s aryl ring to make the symmetrical
compound 6b yielded the second-most active compound seen
in this HL-60 screen, with IC50 ) 0.8 µM. The 5-fluoro analogue
6a, from which 6b was prepared, had IC50 ) 1.0 µM, as did
symmetrically diazeniumdiolated derivative 10. 5-Fluoro deriva-
tive 11a was also within the reasonably active range, with IC50
) 3.5 µM. It appears that placement of a diazeniumdiolate group
both ortho and para to nitro substituents is a requisite for
maximum (e5 µM) potency on the part of the compounds
screened for HL-60 antiproliferative activity in the study
reported here.
Results in the NCI 51-Cell-Line Screen. JS-K and its
O-alkyl variants 2b-2e as well as its homopiperazine analogue
4e were subjected to the National Cancer Institute (NCI) 51-
cell-line screen. The overall dose-response curves revealed that
the renal cancer subpanel was the most sensitive to these
Structures 5-11 examined the consequences of altering the
nature of JS-K’s O2-aryl substituent. No compound in which
the 2,4-dinitrophenyl ring was replaced by any other substituent
(phenyl, pyridyl, or pyrimidinyl) fell within the IC50 range of

Antitumor ActiVity of JS-K in Vitro and in ViVo
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4359
analogues. In general, the five analogues tested produced 50%
growth inhibition (GI50) and 50% cell killing (LC50) of seven
cell lines of the human renal cancer subpanel at concentrations
of about 10 and <100 µM, respectively. When concentrations
of these analogues increased to 100 µM, 100% cell killing was
observed with 786-0, TK-10, and CAKI-1 cell lines, whereas
the central nervous system and prostate cancer subpanels were
ranked next. It was somewhat surprising that, unlike JS-K that
potently and selectively inhibited leukemia HL-60 cells at
GI50 ) 26 nM, TGI (total growth inhibition) ) 68 nM, and
LC50 ) 3.5 µM,1,10 all five analogues showed relatively poor
potency in inhibiting the leukemia subpanel. They were unable
to produce 50% cell killing of all six leukemia cell lines,
including the HL-60 cells, at a concentration of 100 µM. The
five analogues showed total growth inhibition of the HL-60 cells
at 8.3 µM (2b), 8.1 µM (2c), 57.5 µM (2d), and >100 µM
(4e).
killing of about a third of the 51 cell lines in the concentration
range of 1-10 µM and 100% cell killing for a few cell lines at
100 µM.
In Vivo Activity against Rat Hepatoma. Previously we have
found that JS-K efficiently inhibited the growth of the human
hepatoma cell line Hep3B in culture.4 Both the MEK inhibitor
2′-amino-3′-methoxyflavone and the nitric oxide scavenger
PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide)
antagonized this growth inhibition. This suggested that genera-
tion of nitric oxide and ERK phosphorylation might be involved
in JS-K-mediated growth inhibition of the Hep3B hepatoma
cells. We therefore tested the effects of JS-K and its inhibitors
in vivo on rat liver tumor growth.
Analysis of the results from the NCI 51 human cancer cell
lines further demonstrated that, as a whole, 2e (benzyl) and 2d
(n-butyl) exhibited similar inhibition patterns against the 51 cell
lines, which were weaker than analogues 2b, 2c, and 4e (Table
1). For instance, both 2d and 2e needed concentrations of more
than 5 and 23 µM in order to reach 50% growth inhibition and
total growth inhibition, respectively, of the 51 cell lines, whereas
2b (methyl), 2c (n-propyl), and 4e were able to cause 50%
growth inhibition and total growth inhibition, respectively, of
the 51 cell lines at concentrations as low as 1-2 and 10 µM.
These three analogues were even able to produce 50% cell
Rat hepatoma cell line JM-1 was grown in culture. A million
cells suspended in 1 mL of phosphate-buffered saline (PBS)

4360 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Shami et al.
Table 1. Cytotoxic Concentrations (Means ( SD) of JS-K and Related
O2-Aryl Diazeniumdiolates against the NCI 51 Human Cancer Cell
Lines
Discussion
Discovery of JS-K (2a) as a promising anticancer lead
occurred when a panel of randomly chosen O2-arylated di-
azeniumdiolates was screened for activity against the NO-
sensitive HL-60 human leukemia cell line. Of special interest
in the earlier study was the in vivo activity of JS-K against not
only HL-60 but also PPC-1 human prostate cancer xenografts
in NOD-SCID mice.1 The latter cells were not known to be
especially NO sensitive, suggesting that JS-K’s antitumor effect
might involve more than NO-mediated cytotoxicity.
cytotoxic concentrations (µM)
analogue
GI50
TGI
LC50
2a (JS-K)
1.32 ( 0.03
1.66 ( 0.24
2.04 ( 0.27
6.31 ( 1.70
7.08 ( 1.62
1.51 ( 0.04
6.61 ( 0.07
11.75 ( 0.59
10.23 ( 0.65
23.44 ( 3.47
30.90 ( 3.80
11.75 ( 0.20
29.51 ( 3.31
51.29 ( 2.57
40.74 ( 4.36
63.10 ( 6.03
77.63 ( 28.18
51.29 ( 1.55
2b
2c
2d
2e
4e
To gain further insight into JS-K’s mechanism of action, as
well as to search for additional promising lead compounds
among its analogues, we have prepared 41 different O2-aryl
diazeniumdiolates and compared them with JS-K for the ability
to inhibit proliferation of HL-60 cells in vitro. Five of these
were further subjected to the NCI 51-cell-line screen. JS-K
emerged as the most potent of the 42 by every criterion
examined.
As far as JS-K’s mechanism of action is concerned, the 4
µM IC50 of JS-K’s NO-releasing metabolite 1a supported the
importance of NO-mediated cytotoxicity. This is further sup-
ported by our previous work showing that pretreatment of HL-
60 cells with the NO-quencher hydroxocobalamin (B12) pro-
tected them from JS-K’s cytotoxic effects.2 However, results
presented here suggest that other components of the JS-K
molecule may contribute to its cytotoxicity. Indeed, the dem-
onstrated ability of JS-K to arylate glutathione (GSH) and other
cellular nucleophiles also appears to play a substantial role. JS-K
has been shown to be similar in electrophilicity to 1-chloro-
2,4-dinitrobenzene,9 with each one easily transferring its di-
nitrophenyl (DNP) ring to GSH. The 1.4 µM IC50 of the chloro
compound showed it to be only slightly less potent than JS-K.
These data are in agreement with the notion that JS-K acts
through a two-step mechanism: (1) arylation of GSH or other
important nucleophilic biomolecules, irreversibly altering their
normal function; and (2) spontaneous NO release after the DNP
group is no longer present to stabilize the diazeniumdiolate
moiety. One could further speculate that with this drug design,
GSH arylation makes cells more susceptible to NO attack by
rendering thiol groups that act as NO “sinks” unavailable. This
is supported by our previous observation that loading HL-60
cells with the GSH precursor N-acetyl-L-cysteine protects them
from JS-K’s cytotoxic effects.2 The latter observation and the
protection of HL-60 cells from JS-K by B12 support a central
role for NO in JS-K’s cytotoxic effect. This action is com-
pounded by the arylation reactions of the DNP component of
the molecule. It is hard to predict which pathways affected by
JS-K lead to cytotoxicity. Nitric oxide modifies intracellular
targets that affect cell growth through multiple mechanisms.
These include protein thiol nitrosylation, protein tyrosine
nitration (by generating peroxynitrite in the presence of super-
oxide), ADP ribosylation, inhibition of mitochondrial respiration,
inhibition of ribonucleotide reductase, and induction of DNA
strand breaks.24
Figure 1. Suppression of tumor growth by JS-K after implanting JM-1
hepatoma cells intrahepatically in rats and then injecting JS-K in
dimethyl sulfoxide (DMSO) intraperitoneally 1 week later and every
other day thereafter for 2 weeks at the indicated doses. The MEK
inhibitor 2′-amino-3′-methoxyflavone (PD) and the NO scavenger PTIO
were administered intravenously an hour before JS-K, reversing JS-
K’s antitumor effect as indicated. The data represent means ( SD, n
) 4.
were injected under direct vision into the livers of syngeneic
Fischer rats through their mesenteric vein. The cells were
allowed to grow in the rat livers for 1 week. Then the rats were
treated with five intraperitoneal injections every other day of
JS-K at a dose of 3 or 30 mg/kg. Animals were sacrificed 3
weeks after JM-1 cell injection. Liver tumors were excised and
weighed. Two groups of rats were also intravenously (via tail
vein) pretreated with either the MEK inhibitor 2′-amino-3′-
methoxyflavone or the nitric oxide scavenger PTIO 1 h before
each of the JS-K treatments.
As shown in Figure 1, the total tumor weight was found to
decrease with increasing dose of JS-K in vivo. Pretreatment of
the animals with either the MEK inhibitor 2′-amino-3′-methoxy-
flavone or the nitric oxide scavenger PTIO antagonized the
tumor growth-inhibitory effect of JS-K. This implied the
involvement of both ERK and NO in the growth-inhibitory
mechanisms of JS-K action. Exactly how NO might activate
MAPKs is currently the subject of intense investigation. It has
been shown to include activation of JNK, ERK, and p3811-14
as well as inhibition of protein tyrosine phosphatases.15-17 The
transient activation of the MAPK pathway has been long
associated with the growth-stimulatory action of a variety of
mitogens. Recently, it has become clear that prolonged activation
of components of the same pathway is an important aspect of
several mediators of growth inhibition.18,19 The prolonged
activation of MAPK is proposed in turn to induce AP-1. NO
has also been found to induce AP-1 in other cell systems20,21
and AP-1 induction is associated with apoptosis and growth
inhibition in other culture conditions.22,23
The possible involvement of a carbamoylation pathway was
suggested by the surprising potency of carbamoylated piperazine
1b (Scheme 1). At 8.6 µM, it was almost half as active as the
corresponding diazeniumdiolate, 1a. This possibility was further
investigated by modifying the alkyl group of the potential
carbamoylating moiety. Both shortening and lengthening the
C2 chain in the series 2a-2d decreased the potency by at least
7-fold. Further reductions in potency were seen with the benzyl
and tert-butyl derivatives, 2e and 2f.

Antitumor ActiVity of JS-K in Vitro and in ViVo
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4361
described,9 as were 1a,25 sodium 1-(N,N-diethylamino)diazen-1-
ium-1,2-diolate (DEA/NO),26,27 sodium 1-(N,N-dimethylamino)-
diazen-1-ium-1,2-diolate (DMA/NO),9 3f,3 and 11d-11g.28
General Procedures for the Arylation of Diazeniumdiolates.
Method A. A solution of 11 mmol of a diazeniumdiolate anion in
20 mL of 5% aqueous sodium bicarbonate is cooled to 0 °C under
nitrogen. A solution containing 10 mmol of the activated fluoro-
or chloroarene in 10 mL of tert-butyl alcohol is added slowly. A
precipitate normally forms upon addition. The mixture is allowed
to warm gradually to room temperature and then stirred overnight.
The product is extracted with dichloromethane and washed
subsequently first with cold dilute hydrochloric acid and then with
sodium bicarbonate solution. The organic layer is dried over sodium
sulfate, filtered through a layer of magnesium sulfate, and evapo-
rated under vacuum to give the crude product. Purification is carried
out by recrystallization, flash chromatography, or preparative HPLC.
Method B. To a solution of 7.5 mmol of the diazeniumdiolate
in 10 mL of DMSO at 0 °C under a steady stream of nitrogen is
added dropwise 6 mmol of the activated aryl compound. The
reaction is allowed to warm to room temperature and stirred for
24-72 h. The reaction is quenched with water, extracted with ether,
dried over sodium sulfate, and evaporated. The product is either
recrystallized or purified by flash chromatography.
Evidence for the possible importance of carbamoylation is
weak overall, inasmuch as removing the group altogether, as
in decarbamoylated derivative 3a, led to an IC50 of 5.0 µM,
comparable to those of 2b-2f. The same was true for acyl
analogues 3c-3f and 3i-3k. Comparable activities were also
seen when the piperazine ring was replaced by pyrrolidine (4f),
piperidine derivatives (4c and 4d), or homopiperazine (4e) or
even by the simple acyclic dimethylamino (4a) or diethylamino
(4b) groups.
An alternative suggestion from these data is that the activity
of these compounds depends at least in part on the precise steric
arrangement of the amino moiety to which the diazeniumdiolate
group is bound. JS-K just happens to be optimal in this regard
among the agents tested thus far. However, either decreasing
the amino group’s steric requirement as described above or in-
creasing it dramatically, as with the N-phenyl and N-(pyrimidin-
2-yl)piperazine derivatives 3g and 3h (IC50 values of 1.5 and
3.6, respectively), can also sustain significant activity.
A further conclusion arising from our data is that substitution
of the aryl ring with nitro groups both ortho and para to the
diazeniumdiolate function is required for significant activity.
Removal of a nitro group, or replacement of one or both nitro
groups by trifluoromethyl or a heterocyclic nitrogen, as in 5a-
5d, 7a, 8, and 9, lowered the in vitro potency by at least 2 orders
of magnitude. The only exception was 7b, whose relatively low
IC50 of 8.0 µM may have been attributable at least in part to
the presence of the N-phenylpiperazine group, as postulated
above for 3g. However, the N-phenylpiperazine effect could
apparently not compensate for the lowered electrophilicity of
the 2-nitro-4-trifluoromethylbenzene ring in compound 9, which
proved inactive at the 100 µM level.
A referee has suggested the further intriguing possibilities
that nucleophilic metabolite 1b might react with JS-K to activate
it for NO release and that the N-nitroso derivative of 1b might
play an important mechanistic role. These are interesting ideas
that we had not considered, but at the low concentrations/doses
administered in our study, such secondary reactions would seem
to be relatively unlikely to contribute significantly to the
observed pharmacological activities.
While much remains to be elucidated about the mechanism
of JS-K’s antitumor effects, its demonstrated in vitro and in
vivo activities continue to recommend it as a worthy lead in
anticancer drug discovery. Of particular note is its activity
against human prostate cancer xenografts not known for being
susceptible to the cytotoxic potential of NO.
Method C. A solution of 1.76 mmol of the activated bromo-,
fluoro-, or chloroarene reagent in 3 mL of DMSO is added to a
slurry (1.76 mmol) of the diazeniumdiolate in 3 mL of tetrahydro-
furan at room temperature under nitrogen. The mixture is stirred
for 72 h. The resulting homogeneous solution is treated with 100
mL of water. The precipitate is extracted with ether. The organic
layer is dried and evaporated, whereupon the product is purified
as described in method A.
Sodium 1-[4-(Methoxycarbonyl)piperazin-1-yl]diazen-1-ium-
1,2-diolate (JS-36-143). A solution of 12.93 g (0.899 mol) of
N-(methoxycarbonyl)piperazine29 in 19.6 mL of 25% sodium
methoxide in methanol and 5 mL of additional methanol was placed
in a Parr bottle and charged with NO as described above. The
product began to form within 2 h but was allowed to stir overnight
at room temperature to give 12.3 g (61%) of the diazeniumdiolate
sodium salt: mp 183-184 °C; 1H NMR (D2O/-OD) δ 3.10-3.16
(m, 4 H), 3.36 (s, 3 H), 3.66-3.72 (m, 4 H); 13C NMR (D2O/-
OD) δ 45.49, 54.09, 56.08, 160.14; UV (10 mM NaOH) λmax (ꢀ)
250 nm (9.1 mM-1 cm-1); half-life 5.1 min in pH 7.4 buffer at 37
°C. Anal. Calcd for C6H11N4O4Na: C, 31.86; H, 4.90; N, 24.77;
Na, 10.17. Found: C, 31.94; H, 4.88; N, 24.45; Na, 10.21.
O2-(2,4-Dinitrophenyl) 1-[4-(Methoxycarbonyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (2b) (JS-36-152). A solution of 3.76
g (0.0166 mol) of sodium 1-[4-(methoxycarbonyl)piperazin-1-yl]-
diazen-1-ium-1,2-diolate in 30 mL of 5% aqueous sodium bicar-
bonate was treated with 1 equiv of 2,4-dinitrofluorobenzene in tert-
butyl alcohol according to method A, giving 5.38 g of a glass. The
crude material was dissolved in 10 mL of 5:1 dichloromethane:
ethyl acetate, whereupon crystalline product formed on standing.
The product 2b was collected by filtration and washed with ether
to give 1.8 g of analytically pure material. An additional 780 mg
of product was obtained upon flash chromatography of the mother
liquor on silica gel on elution with 5:1 dichloromethane:ethyl
With this in mind, we have examined the action of JS-K
against an orthotopic rat liver tumor model. As shown in Figure
1, JS-K significantly slowed the growth of these tumors. In
agreement with an NO-mediated mechanism of action, the NO
scavenger PTIO blunted JS-K’s antitumor effect. The MEK
inhibitor 2′-amino-3′-methoxyflavone also reversed JS-K’s
activity. Ongoing and future studies are aimed at further refining
our mechanistic understanding of the pathways responsible for
JS-K’s promising antitumor effects.
1
acetate: mp 144-145 °C; H NMR (CDCl3) δ 3.63-3.69 (m, 4
H), 3.65-3.71 (m, 4 H), 3.75 (s, 3 H), 7.66 (d, 1 H, J ) 9.25 Hz),
8.43-8.49 (dd, 1 H, J ) 2.74 and 9.25 Hz), 8.88 (d, 1 H, J ) 2.65
Hz); 13C NMR (CDCl3) δ 42.27, 50.52, 53.10, 117.71, 122.18,
129.08, 137.38, 142.47, 153.67, 155.39; UV (ethanol) λmax (ꢀ) 299
nm (12 mM-1 cm-1). Anal. Calcd for C12H14N6O8: C, 38.92; H,
3.81; N, 22.70. Found: C, 39.06; H, 3.85; N, 22.51.
Experimental Section
NO was purchased from Matheson Gas Products (Montgomery-
ville, PA). Starting materials were purchased from Aldrich Chemical
Co. (Milwaukee, WI) unless otherwise indicated. Proton NMR
spectra were obtained in chloroform-d, acetone-d6, or DMSO-d6.
Chemical shifts (δ) are reported in parts per million (ppm)
downfield from tetramethylsilane. Elemental analyses were per-
formed by Atlantic Microlab, Inc. (Norcross, GA) or Midwest
Microlab, LLC (Indianapolis, IN). Compounds 2a, 3g, 3h, 4a-
4d, 5b, 5c, 7a, 7b, and 8-10 were prepared as previously
Sodium 1-[4-(n-Propoxycarbonyl)piperazin-1-yl]diazen-1-
ium-1,2-diolate (JS-36-166). A solution of 7.04 g (0.041 mol) of
N-(n-propoxycarbonyl)piperazine30 in 8.9 mL (0.041 mol) of 25%
methanolic sodium methoxide and 4 mL of methanol was exposed
to nitric oxide as described previously to give 6 g (58%) of the
diazeniumdiolate sodium salt: mp 191-192 °C; 1H NMR (D2O/-
OD) δ 0.94 (t, 3 H, J ) 7.42 Hz), 1.63-1.72 (m, 2 H), 3.12-3.14
(m, 4 H), 3.70 (b, 4 H), 4.08 (t, 2 H, J ) 6.54 Hz); 13C NMR

4362 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Shami et al.
(D2O/-OD) δ 12.40, 24.58, 45.42, 54.12, 71.06, 159.82; UV (10
mM NaOH) λmax (ꢀ) 250 nm (7.9 mM-1 cm-1); half-life 4.7 min
in pH 7.4 buffer at 37 °C. Anal. Calcd for C8H15N4O4Na: C, 37.80;
H, 5.95; N, 22.04; Na, 9.04. Found: C, 37.68; H, 5.77; N, 21.79;
Na, 9.15.
sodium 1-[4-(tert-butoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-
diolate25 in 5% sodium bicarbonate at 0 °C under a steady stream
of nitrogen gas was added 0.9 equiv of 2,4-dinitrofluorobenzene
in tert-butyl alcohol (method A). The reaction was warmed to room
temperature and stirred for 24 h. Precipitation occurred at such a
rapid rate that stirring was impeded throughout the course of the
reaction. The yellow mixture was filtered and recrystallized in
EtOH: mp 115-117 °C; 1H NMR (CDCl3) δ 1.49 (s, 9 H), 3.60-
3.76 (m, 8 H), 7.67 (d, 1 H, J ) 9.2 Hz), 8.43-8.46 (dd, 1 H, J )
2.65 and 9.24 Hz), 8.89 (d, 1 H, J ) 2.7); 13C NMR (CDCl3) δ
28.29, 42.07, 50.56, 80.91, 117.67, 122.17, 129.07, 137.32, 142.41,
153.72, 154.10; UV (EtOH) λmax (ꢀ) 299 nm (13.8 mM-1 cm-1).
Anal. Calcd for C15H20N6O8: C, 43.69; H, 4.89; N, 20.38. Found:
C, 43.75; H, 4.92; N, 20.44.
O2-(2,4-Dinitrophenyl) 1-[4-(n-Propoxycarbonyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (2c) (JS-36-172). A solution of 2.35
g (0.0093 mol) of sodium 1-[4-(n-propoxycarbonyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate in 5% aqueous sodium bicarbonate was
treated with 2,4-dinitrofluorobenzene in tert-butyl alcohol according
to method A. The product was recrystallized from ether:petroleum
1
ether to give 1.82 g of 2c: mp 134-135 °C; H NMR (CDCl3) δ
0.96 (t, 3 H, J ) 7.32 Hz); 1.64-1.73 (m, 2 H), 3.62-3.65 (m, 4
H), 3.73-3.75 (m, 4 H), 4.09 (t, 2 H, J ) 6.73 Hz); 7.67 (d, 1 H,
J ) 9.27 Hz); 6.45-8.48 (dd, 1 H, J ) 2.73 and 9.27 Hz), 8.88 (d,
1 H, J ) 2.73 Hz); 13C NMR (CDCl3) δ 10.33, 22.24, 42.17, 50.52,
67.65, 117.69, 122.15, 129.07, 137.35, 142.45, 153.67, 155.05; UV
(ethanol) λmax (ꢀ) 252 nm (9.3 mM-1 cm-1). Anal. Calcd for
C8H18N6O8: C, 42.21; H, 4.55; N, 21.10. Found: C, 41.95; H, 4.51;
N, 20.85.
O2-(2,4-Dinitrophenyl) 1-[4-(Succinimidoxycarbonyl)piper-
azin-1-yl]diazen-1-ium-1,2-diolate (2g) (CB-4-140). To a mixture
of 1.1 g (3 mmol) of 3a (see next paragraph) in 15 mL of
dichloromethane was added 510 µL (3.7 mmol) of triethylamine
to generate the free base. A partial solution of 772 mg (3.02 mmol)
of N,N′-disuccinimidyl carbonate in 15 mL of dichloromethane was
added, followed by 510 µL (3.7 mmol) of additional triethylamine.
The resulting reaction mixture was stirred at room temperature for
15 h. The mixture was concentrated on a rotary evaporator and the
residue was extracted with dichloromethane. The organic solution
was washed with 10% hydrochloric acid, followed by 5% sodium
bicarbonate solution, and then dried over sodium sulfate, filtered
through magnesium sulfate, and evaporated to give a solid. The
product was recrystallized from ethanol:acetonitrile to give 143 mg
Sodium 1-[4-(n-Butoxycarbonyl)piperazin-1-yl]diazen-1-ium-
1,2-diolate (JS-37-9). A solution of 5.486 g (0.0295 mol) of N-(n-
butoxycarbonyl)piperazine29 in 6.52 mL (0.03 mol) of 25% sodium
methoxide in methanol, 10 mL of additional methanol, and 10 mL
of ether was placed in a Parr bottle and charged with NO as
described above to give the diazeniumdiolate sodium salt: mp 180
°C (dec); 1H NMR (D2O/-OD) δ 0.92 (t, 3 H, J ) 7.4 Hz), 1.35-
1.44 (m, 2 H), 1.61-1.68 (m, 2 H), 3.11-3.14 (m, 4 H), 3.69 (b,
4 H), 4.14 (t, 2 H, J ) 6.5 Hz); 13C NMR (D2O/ -OD) 15.81,
21.37, 33.14, 45.44, 54.14, 69.34, 159.87; UV (10 mM NaOH) λmax
(ꢀ) 251 nm (9.3 mM-1 cm-1); half-life of 5.7 min in pH 7.4 buffer
at 37 °C. Anal. Calcd for C9H17N4NaO4‚1/3CH3OH‚1/3H2O: C,
39.34; H, 6.72; N, 19.66; Na, 8.07. Found: C, 39.30; H, 7.65; N,
19.81; Na, 8.01.
1
of pure 2g: mp 162-164 °C; H NMR (DMSO-d6) δ 2.81 (s, 4
H), 3.70-3.79 (m, 8 H), 7.96 (d, 1 H, J ) 9.3 Hz), 8.54-8.58 (dd,
1 H, J ) 2.75 and 9.31 Hz), 8.88 (d, 1 H, J ) 2.7 Hz); 13C NMR
(DMSO-d6) δ 25.28, 42.47, 42.86, 49.27, 118.19, 121.78, 129.71,
136.90, 142.24, 150.05, 152.73, 170.49; UV (acetonitrile) λmax (ꢀ)
300 nm (14.2 mM-1 cm-1). Anal. Calcd for C15H15N7O10: C, 39.74;
H, 3.34; N, 21.63. Found: C, 39.85; H, 3.53; N, 21.61.
O2-(2,4-Dinitrophenyl) 1-[4-(n-Butoxycarbonyl)piperazin-yl]-
diazen-1-ium-1,2-diolate (2d) (JS-37-24). A solution of 2.28 g
(0.0085 mol) of sodium 1-[4-(n-butoxycarbonyl)piperazin-1-yl]-
diazen-1-ium-1,2-diolate in aqueous sodium bicarbonate was aryl-
ated with 2,4-dinitrofluorobenzene according to method A to give
2.5 g (71%) of 2d. The isolated powder was recrystallized from
ether:petroleum ether to give 1.75 g of pure material: mp 109-
110 °C; 1H NMR (CDCl3) δ 0.95 (t, 3 H, J ) 7.05 Hz), 1.35-1.45
(m, 2 H), 1.61-1.68 (m, 2 H), 3.62-3.65 (m, 4 H), 3.70-3.75
(m, 4 H), 4.13 (t, 2 H, J ) 6.64 Hz), 7.68 (d, 1 H, J ) 9.27 Hz),
8.45-8.48 (dd, 1 H, J ) 2.73 and 9.27 Hz), 8.88 (d, 1 H, J ) 2.73
Hz); 13C NMR (CDCl3) δ 13.69, 19.09, 30.92, 42.16, 50.49, 55.94,
117.67, 122.12, 129.07, 137.31, 142.42, 153.66, 155.04; UV
(ethanol) λmax (ꢀ) 299 nm (14.5 mM-1 cm-1). Anal. Calcd for
C15H20N6O8: C, 43.69; H, 4.89; N, 20.38. Found: C, 43.62; H,
4.84; N, 20.11.
O2-(2,4-Dinitrophenyl) 1-(Piperazin-1-yl)diazen-1-ium-1,2-di-
olate, Hydrochloride Salt (3a) (CB-4-35). To a solution of 1.27
g (0.0031 mol) of 2f in 150 mL of ethyl acetate was added 6 mL
of concentrated hydrochloric acid. The resulting yellow solution
was stirred at room temperature for 2 h, during which time a white
precipitate was evident. The mixture was filtered and washed with
ether to yield a clean product (944 mg, 87% yield) which could be
used for subsequent reactions. Recrystallization was achieved in
1:1 methanol:H2O: mp 184-185 °C; 1H NMR (DMSO-d6) δ 3.34-
3.38 (m, 4 H), 3.85-3.89 (m, 4 H), 7.96 (d, 1 H, J ) 9.2 Hz),
8.56-8.59 (ddd, 1 H, J ) 0.8, 2.8, and 4.3 Hz), 8.88-8.89 (dd, 1
H, J ) 0.8 and 2.8 Hz); 13C NMR (CD3OD) δ 43.67, 119.24,
122.86, 130.33, 138.88, 144.26, 154.45; UV (ethanol) λmax (ꢀ) 297
nm (12.2 mM-1 cm-1). Anal. Calcd for C10H13ClN6O6‚1/6 H2O:
C, 34.15; H, 3.82; N, 23.90. Found: C, 34.52; H, 3.92; N, 23.42.
O2-(2,4-Dinitrophenyl) 1-[4-(Benzyloxycarbonyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (2e) (JS-36-187). A solution of 4.7 g
(0.016 mol) of sodium 1-[4-benzyloxycarbonyl)piperazin-1-yl]-
diazen-1-ium-1,2-diolate25 in aqueous sodium bicarbonate was
arylated with 2,4-dinitrofluorobenzene according to method A to
give 5.4 g of crude 2e as an amber oil. The oil was chromatographed
on a dry-packed silica gel column and eluted with dichloromethane.
The fractions containing the desired product were pooled and
concentrated under vacuum to produce a thick oil that crystallized
on standing. After trituration with ether, the solid was collected by
filtration and dried to give 2.16 g of product 2e: mp 118-120 °C;
1H NMR (CDCl3) δ 3.63 (b, 4 H), 3.75-3.77 (m, 4 H), 5.16 (s, 2
H), 7.36 (s, 5 H), 7.66 (d, 1 H, J ) 9.27 Hz), 8.44-8.47 (dd, 1 H,
J ) 2.73 and 9.27 Hz), 8.86 (d, 1 H, J ) 2.73 Hz); 13C NMR
(CDCl3) δ 42.29, 50.44, 67.72, 117.63, 122.09, 126.31, 128.06,
128.56, 129.08, 137.25, 136.01, 142.39, 153.62, 154.71; UV
(ethanol) λmax (ꢀ) 299 nm (11 mM-1 cm-1). Anal. Calcd for
C18H18N6O8: C, 48.43; H, 4.06; N, 18.83. Found: C, 48.61; H,
4.14; N, 18.49.
O2-(2,4-Dinitrophenyl) 1-(4-Methylpiperazin-1-yl)diazen-1-
ium-1,2-diolate (3b) (JS-29-86). A solution of 50 g (0.5 mol) of
1-methylpiperazine in 100 mL of 25% sodium methoxide (0.46
mol) in methanol and 50 mL of ether was placed in a Parr bottle,
degassed, charged with 40 psi of nitric oxide, and stirred overnight
at room temperature. The pressure was released and the product
was filtered, washed with ether, and dried under vacuum to give
7.54 g of sodium 1-(4-methylpiperazin-1-yl)diazen-1-ium-1,2-
diolate: 1H NMR (D2O) δ 2.29 (s, 3 H), 2.69 (b, 4 H), 3.16-3.19
(m, 4 H); a peak at 3.36 was detected, indicating the presence of
sodium methoxide, but the material was used without further
purification in the subsequent arylation reaction. The arylation was
carried out with 1,5-difluoro-2,4-dinitrobenzene according to
1
method A: mp 149-150 °C; H NMR (CDCl3) δ 2.36 (s, 3 H),
2.36-2.66 (m, 4 H), 3.67-3.70 (m, 4 H), 7.66 (d, 1 H, J ) 2.7
Hz), 8.44-8.48 (dd, 1 H, J ) 2.8, 12.0 Hz) 8.88 (d, 1 H, J ) 9.3
Hz); 13C NMR (CDCl3) δ 45.47, 50.43, 53.28, 117.59, 122.17,
129.07, 137.23, 142.23, 153.95; UV (ethanol) λmax (ꢀ) 300 nm (16
mM-1 cm-1). Anal. Calcd for C11H14N6O6: C, 40.49; H, 4.33; N,
25.76. Found: C, 40.09; H, 4.29; N, 25.32.
O2-(2,4-Dinitrophenyl) 1-[4-(tert-Butoxycarbonyl)piperazin-
1-yl]diazen-1-ium-1,2-diolate (2f) (CB-3-97). To a solution of

Antitumor ActiVity of JS-K in Vitro and in ViVo
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4363
O2-(2,4-Dinitrophenyl) 1-(4-Nicotinylpiperazin-1-yl)diazen-1-
ium-1,2-diolate (3c) (CB-3-125). To a solution of 3a in CH2Cl2
was added a slight molar excess of triethylamine to liberate the
free base. An equivalent molar amount of nicotinyl chloride was
added, followed by another aliquot of triethylamine. The reaction
mixture was stirred at room temperature for 30 min, at which time
the cloudy solution was washed with water. The organic solution
was collected, dried over sodium sulfate, filtered through mag-
nesium sulfate, and evaporated to a yellow film. Recrystallization
was achieved with ethanol to yield a beige crystalline material:
O2-(2,4-Dinitrophenyl) 1-(4-Acetylpiperazin-1-yl)diazen-1-
ium-1,2-diolate (3j) (CB-4-132). A solution of 587 mg (2.8 mmol)
of CB-4-130, the diazeniumdiolate of 1-acetyl-piperazine, in dilute
aqueous sodium bicarbonate was arylated with 1 equiv of 2,4-
dinitrofluorobenzene according to method A. The isolated product
was recrystallized from ethanol:acetonitrile to give 763 mg (68%)
1
of pure 3j: mp 153-154 °C; H NMR (DMSO-d6) δ 2.06 (s, 3
H), 3.61-3.68 (m, 8 H), 7.93 (d, 1 H, J ) 9.3 Hz), 8.54-8.58 (dd,
1 H, J ) 2.75 and 9.38 Hz), 8.87 (d, 1 H, J ) 2.7 Hz); 13C NMR
(DMSO-d6) δ 43.88, 49.81, 50.03, 118.09, 121.79, 129.72, 136.87,
142.18, 152.76, 168.45; UV (acetonitrile) λmax (ꢀ) 394 nm (16.1
mM-1 cm-1). Anal. Calcd for C12H14N6O7: C, 40.68; H, 3.98; N,
23.72. Found: C, 40.89; H, 4.05; N, 23.62.
1
mp 150-152 °C; H NMR (CDCl3) δ 3.51-4.28 (b, 8 H), 7.40-
7.45 (m, 1 H), 7.66 (d, 1 H, J ) 9.2 Hz), 7.79-7.83 (m, 1 H),
8.44-8.48 (dd, 1 H, J ) 2.8 and 9.2 Hz), 8.71-8.75 (m, 2 H),
8.89 (d, 1 H, J ) 2.7 Hz); 13C NMR (CDCl3) δ 50.71, 61.22,
117.80, 122.19, 123.71, 129.08, 130.35, 135.22, 137.49, 142.63,
147.96, 151.56, 153.50, 167.94; UV (acetonitrile) λmax (ꢀ) 300 nm
(16.5 mM-1 cm-1). Anal. Calcd for C16H15N7O7: C, 46.05; H, 3.62;
N, 23.49. Found: C, 46.18; H, 3.73; N, 23.42.
O2-(2,4-Dinitrophenyl) 1-[4-(N,N-Dimethylaminosulfonyl)pip-
erazin-1-yl]diazen-1-ium-1,2-diolate (3k) (JS-41-92). To a slurry
of 108 mg (0.31 mmol) of 3a in 5 mL of dichloromethane was
added 33 µL (0.31 mmol) of dimethyl sulfamoyl chloride. To the
resulting slurry was added 139 µL (0.64 mmol) of triethylamine.
The homogeneous solution that formed was stirred for 3 h. The
solution was then washed with 1 M hydrochloric acid, dried over
sodium sulfate, filtered through magnesium sulfate, and evaporated
to give 114 mg of an oil that crystallized on standing. The product
was recrystallized from ether:petroleum ether: mp 146-147 °C;
1H NMR (CDCl3) δ 2.87 (s, 6 H), 3.48-3.52 (m, 4 H), 3.71-3.74
(m, 4 H), 7.66 (d, 1 H, J ) 9.24 Hz), 8.46-8.49 (dd, 1 H, J )
2.75 and 9.23 Hz), 8.89 (d, 1 H, J ) 2.74 Hz); 13C NMR (CDCl3)
δ 38.22, 44.90, 50.18, 117.79, 122.19, 129.08, 137.46, 142.56,
153.6; UV (ethanol) λmax (ꢀ) 299 nm (10.2 mM-1 cm-1). Anal.
Calcd for C12H17N7O8S: C, 34.37; H, 4.09; N, 23.38; S, 7.65.
Found: C, 34.22; H, 4.07; N, 22.98; S, 7.75.
O2-(2,4-Dinitrophenyl) 1-{4-[2-(4-{2-Methylpropyl}phenyl)-
propionyl]piperazin-l-yl}diazen-1-ium-1,2-diolate (3d) (CB-3-
132). To a solution of 112 mg of 3a in dichloromethane was added
2.4 equiv of triethylamine. The solution was cooled to 0 °C with
stirring, and an equimolar amount of 4-isobutyl-R-methylphenyl-
acetic acid chloride was added. The reaction was carried out as
1
described above to yield pure 3d (232 mg): mp 54-58 °C; H
NMR (CDCl3) δ 0.86 (d, 6 H, J ) 6.6 Hz), 1.41 (d, 3 H, J ) 6.8
Hz), 1.76-1.90 (m, 1 H), 2.44 (d, 2 H, J ) 7.2 Hz), 2.75-4.12
(m, 8 H), 3.84 (q, 1 H, J ) 6.8 Hz), 7.12 (s, 4 H), 7.58 (d, 1 H, J
) 9.2 Hz), 8.40-8.46 (dd, 1 H, J ) 2.8 and 9.3 Hz), 8.87 (d, 1 H,
J ) 2.7 Hz); 13C NMR (CDCl3) δ 20.58, 22.31, 30.13, 40.23, 43.25,
43.71, 44.93, 50.01, 50.46, 117.59, 122.16, 126.75, 129.01, 129.94,
137.33, 138.57, 140.72, 142.43, 153.62, 172.26; UV (EtOH) λmax
(ꢀ) 300 nm (20.9 mM-1 cm-1). Anal. Calcd for C23H28N6O7: C,
55.19; H, 5.64; N, 16.79. Found: C, 55.59; H, 5.87; N, 16.34.
Sodium 1-[4-(Ethoxycarbonyl)-1,4-diazacycloheptan-1-yl]di-
azen-1-ium-1,2-diolate (JS-35-167). A solution of 4 g (0.023 mol)
of 1-ethoxycarbonyl-1,4-diazacycloheptane31 in 4.6 mL of 30%
methanolic sodium methoxide, 5 mL of methanol, and 15 mL of
ether was placed in a Parr bottle. The homogeneous solution was
purged with nitrogen, charged with 50 psi of nitric oxide, and stirred
at room temperature overnight. The pressure was released, and the
product was collected by filtration, washed with copious amounts
of ether, and dried under vacuum to give 1.36 g of diazeniumdiolate
O2-(2,4-Dinitrophenyl) 1-[4-(2-Acetoxybenzoyl)piperazin-1-
yl]diazen-1-ium-1,2-diolate (3e) (CB-4-69). Compound 3a was
acylated with an equimolar amount of acetylsalicylic acid chloride
as described above. The product was recrystallized from ether:
petroleum ether:acetone to yield pale yellow crystals: mp 149-
1
sodium salt: mp 149-150 °C; H NMR (D2O/-OD) δ 1.28 (t, 3
1
150 °C; H NMR (DMSO-d6) δ 2.25 (s, 3 H), 3.42 (s, b, 2 H),
H, J ) 7.0 Hz), 2.78-2.82 (m, 2 H), 3.19-3.23 (m, 2 H), 3.27-
3.63-3.85 (m, 6 H), 7.25-7.27 (m, 1 H), 7.35-7.39 (m, 1 H),
7.42-7.45 (m, 1 H), 7.50-7.54 (m, 1 H), 7.94 (d, 1 H, J ) 9.3
Hz), 8.54-8.58 (dd, 1 H J ) 2.7 and 9.3 Hz), 8.87 (d, 1 H, J )
2.7 Hz); 13C NMR (DMSO-d6) δ 168.72, 165.24, 152.52, 146.47,
142.07, 136.76, 130.45, 129.58, 128.65, 127.70, 126.02, 123.08,
121.64, 118.01, 49.85, 49.74, 44.79, 30.66; UV (acetonitrile) λmax
(ꢀ) 300 nm (22.1 mM-1 cm-1). Anal. Calcd for C19H18N6O9: C,
48.11; H, 3.82; N, 17.72. Found: C, 48.20; H, 4.02; N, 17.14.
3.29 (m, 2 H), 3.43-3.69 (m, 4 H), 4.17 (q, 2 H, J ) 7.0 Hz); 13
C
NMR (D2O/-OD) δ 15.51, 26.97, 45.80, 46.60, 56.19, 56.62, 64.21,
159.65; UV (10 mM NaOH) λmax (ꢀ) 250 nm (7.88 mM-1 cm-1);
half-life 45 s in pH 7.4 buffer at 37 °C.
O2-(2,4-Dinitrophenyl) 1-[(4-Ethoxycarbonyl)-2,4-diazacyclo-
heptan-1-yl]diazen-1-ium-1,2-diolate (4e) (JS-36-25). To a solu-
tion of 1.15 g (0.0045 mol) of sodium 1-[4-(ethoxycarbonyl)-1,4-
diazacycloheptan-1-yl]diazen-1-ium-1,2-diolate in 15 mL of 5%
aqueous sodium bicarbonate was added a slurry of 0.527 mL
(0.0042 mol) of 2,4-dinitrofluorobenzene in 8 mL of tert-butyl
alcohol as described in method A. The reaction mixture turned
bright yellow, and a precipitate formed during stirring for 1 h. No
change was observed beyond 1 h of stirring. The mixture was
extracted with dichloromethane and washed subsequently with cold
dilute hydrochloric acid and sodium bicarbonate. The organic
solution was dried over sodium sulfate, filtered through magnesium
sulfate, and evaporated under vacuum to give 1.42 g (79%) of 4e
as a glass that crystallized upon trituration with ether:petroleum
ether: mp 110-111 °C; 1H NMR (CDCl3) δ 1.26 (t, 3 H, J ) 7.1
Hz), 2.05-2.13 (m, 2 H), 3.45-3.55 (m, 2 H), 3.73-3.82 (m, 2
H), 3.92-3.98 (m, 4 H), 4.10-4.27 (q, 2 H, J ) 7.1 Hz), 7.59-
7.69 (m, 1 H), 8.43-8.48 (dd, 1 H, J ) 2.7 and 9.2 Hz), 8.88 (d,
1 H, J ) 2.75 Hz); 13C NMR (CDCl3) δ 14.63, 24.88, 25.71, 45.40,
45.97, 49.71, 51.68, 61.78, 117.26, 117.50, 122.12, 129.18, 141.91,
154.23; UV (ethanol) λmax (ꢀ) 308 nm (11.85 mM-1 cm-1). Anal.
Calcd for C14H18N6O8: C, 42.21; H, 4.55; N, 21.10. Found: C,
42.16; H, 4.48; N, 20.99.
O2-(2,4-Dinitrophenyl) 1-[4-(N-Acetylglycinyl)piperazin-1-yl]-
diazen-1-ium-1,2-diolate (3i) (CB-4-9). Compound 3a was acyl-
ated with an equimolar amount of acetylglycine succinimidyl ester
1
as described above for similar acylations: mp 131-133 °C; H
NMR (CDCl3) δ 1.88 (s, 3 H), 3.64 (b, 4 H), 3.68 (b, 4 H), 3.99
(d, 2 H, J ) 5.6 Hz), 7.94 (d, 1 H, J ) 9.4 Hz), 7.98-8.02 (m, 1
H), 8.55-8.58, 1 H, J ) 2.83 and 9.36 Hz), 8.87 (d, 1 H, J ) 2.7
Hz); 13C NMR (CDCl3) δ 22.40, 42.32, 49.77, 49.90, 118.14,
121.78, 129.71, 136.90, 142.20, 152.73, 167.37, 169.36; UV
(acetonitrile) λmax (ꢀ) 300 nm (14.2 mM-1 cm-1). Anal. Calcd for
C14H17N7O8: C, 40.88; H, 4.17; N, 23.84. Found: C, 40.75; H,
4.22; N, 23.62.
Sodium 1-(4-Acetylpiperazin-1-yl)diazen-1-ium-1,2-diolate (CB-
4-130). A solution of 2.65 g (20.7 mmol) of 1-acetylpiperazine in
5 mL of methanol and 50 mL of ether was placed in a Parr bottle
and treated with 4.73 mL of 25% sodium methoxide in methanol.
The solution was flushed with nitrogen and charged with 40 psi of
nitric oxide. After 24 h, the pressure was released and the white
solid was collected by filtration, washed with ether, and dried under
vacuum to give 17.6 g of product: NMR (D2O) δ 2.18 (s, 3 H),
3.13-3.20 (m, 4 H), 3.75-3.77 (m, 4 H). The product was used
without further purification for the arylation step.
O2-(2-Nitrophenyl) 1-(N,N-Diethylamino)diazen-1-ium-1,2-
diolate (5a) (JS-30-4). To a solution of 2.79 g (0.018 mol) of
sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO)

4364 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Shami et al.
in 9 mL of DMSO was added a solution of 1.6 mL (0.015 mol) of
2-nitrofluorobenzene in 20 mL of tetrahydrofuran according to
method B. The mixture was stirred at room temperature for 48 h.
The solution was poured into ice-water and extracted with ether.
The ether extract was dried over sodium sulfate, filtered, and
evaporated to give 2.88 g of an oil containing mostly diethyl-
nitrosamine and 5a. The product mixture was chromatographed on
silica gel using 2:1 dichloromethane:hexane as the eluant to give
548 mg of crystalline 5a: mp 34-35 °C; 1H NMR (CDCl3) δ 1.20
(t, 6 H, J ) 7.1 Hz), 3.38 (q, 4 H, J ) 7.1 Hz), 7.25-7.26 (m, 1
H), 7.49-7.52 (m, 1 H), 7.57-7.53 (m, 1 H), 7.96-8.00 (dd, 1 H,
J ) 1.6 and 8.16 Hz); 13C NMR (CDCl3) δ 11.47, 47.68, 119.01,
124.51, 125.76, 134.32, 139.16, 149.50; UV (ethanol) λmax (ꢀ) 238
nm (8.8 mM-1 cm-1). Anal. Calcd for C10H14N4O4: C, 47.24; H,
5.55; N, 22.04. Found: C, 47.45; H, 5.69; N, 22.23.
O2-(2-Methylmercaptopyrimidin-4-yl) 1-(N,N-Diethylamino)-
diazen-1-ium-1,2-diolate (5d) (JS-30-9). Sodium 1-(N,N-diethyl-
amino)diazen-1-ium-1,2-diolate (DEA/NO) (3.84 g, 0.025 mol) in
DMSO was arylated with 2.3 mL (0.02 mol) of 4-chloro-2-
methylmercaptopyrimidine in tetrahydrofuran according to method
C to give 4.8 g of a yellow-orange oil. Purification of the crude
material was carried out on silica gel with 2:1 dichloromethane:
hexane as the eluant. The product 5d was obtained as an oil: 1H
NMR (CDCl3) δ 1.23 (t, 6 H, J ) 7.19 Hz), 2.56 (s, 3 H), 3.51 (q,
4 H, J ) 7.19 Hz), 6.74 (d, 1 H, J ) 5.6 Hz), 8.42 (d, 1 H, J ) 5.6
Hz); 13C NMR (CDCl3) δ 11.35, 14.10, 47.15, 100.81, 158.97,
167.82, 173.18; UV (ethanol) λmax (ꢀ) 301 nm (17.3 mM-1 cm-1).
Anal. Calcd for C9H15N5O2S: C, 42.01; H, 5.88; N, 27.22; S, 12.46.
Found: C, 42.24; H, 6.06; N, 27.44; S, 12.45.
O2-(2,4-Dinitro-5-hydroxyphenyl) 1-(N,N-Dimethylamino)-
diazen-1-ium-1,2-diolate (11b) (JS-42-25). To a partial solution
of 510 mg (1.76 mmol) of 11a in 30 mL of tert-butyl alcohol was
added 10 mL of 3 M aqueous sodium hydroxide, resulting in a
homogeneous solution. No starting material remained after 1.5 h.
At this time the reaction mixture was made acidic with 3 M
hydrochloric acid and concentrated on a rotary evaporator. The
aqueous solution was extracted with dichloromethane and the acidic
component (phenol) was extracted into 5% sodium bicarbonate
solution. The aqueous layer was acidified with aqueous hydrochloric
acid, extracted with dichloromethane, dried over sodium sulfate,
filtered through magnesium sulfate, and concentrated under vacuum
to give 421 mg (83%) of an orange solid. The product was
1
recrystallized from ether:petroleum ether: mp 105-106 °C; H
NMR (CDCl3) δ 3.30 (s, 6 H), 7.19 (s, 1 H), 8.97 (s, 1 H), 11.10
(s, 1 H); 13C NMR (CDCl3) δ 40.72, 104.58, 124.16, 126.45, 129.45,
155.43, 158.25; UV (ethanol) λmax (ꢀ) 279 nm (16.3 mM-1 cm-1
)
and 388 nm (9.5 mM-1 cm-1). Anal. Calcd for C8H9N5O7: C,
33.46; H, 3.16; N, 24.39. Found: C, 33.58; H, 3.18; N, 24.21.
2,4-Dinitro-1-fluoro-5-[N-methyl-(p-methoxyphenyl)amino]-
benzene (JS-25-119). To a solution of 1.87 g (0.0092 mol) of 1,5-
difluoro-2,4-dinitrobenzene in 10 mL of tetrahydrofuran was added
1 g (0.0095 mol) of anhydrous sodium carbonate. The slurry was
flushed with nitrogen followed by the dropwise addition of 1.26 g
(0.0092 mol) of N-methylanisidine in 10 mL of tetrahyrofuran and
stirred overnight at room temperature. The reaction mixture was
evaporated to dryness under vacuum and the residue was extracted
with dichloromethane, dried over sodium sulfate, and filtered
through a layer of magnesium sulfate to give 2.8 g of an orange
1
solid that was recrystallized from ethanol: mp 138-140 °C; H
Preparation of Bis-diazeniumdiolate 6b (CB-4-36). Two
equivalents of sodium1-[4-(ethoxycarbonyl)piperazin-1-yl]diazen-
1-ium-1,2-diolate 1a was reacted with 1,5-difluoro-2,6-dinitro-
benzene according to method A. Purification was carried out using
a Flash 40 system utilizing a 10:1 dichloromethane:ethyl acetate
solvent system. Two major fractions were collected; the least polar
fraction contained the mono adduct 6a (see experiment below). The
second fraction contained the bis-adduct 6b and it was further
NMR (CDCl3) δ 3.38 (s, 3 H), 3.82 (s, 3 H), 6.76 (d, 1 H, J )
13.62 Hz), 6.88-6.92 (m, 2 H), 7.04-7.09 (m, 2 H), 8.57 (d, 1 H,
J ) 7.78); 13C NMR (CDCl3) δ 43.54, 55.06, 106.94, 107.20,
115.42, 125.87, 126.61, 138.51, 148.49, 156.66, 158.54, 159.35;
UV (ethanol) λmax (ꢀ) 246 nm (13.5 mM-1 cm-1) and 374 nm (13.7
mM-1 cm-1). Anal. Calcd for C14H12N3O5F: C, 52.34; H, 3.76;
N, 13.08. Found: C, 52.59; H, 3.80; N, 12.98.
1
O2-[2,4-Dinitro-5-(N-methyl-4-methoxyphenylamino)phen-
yl] 1-(N,N-Dimethylamino)diazen-1-ium-1,2-diolate (11c) (JS-
25-147). To a solution of 2.56 g (0.020 mol) of DMA/NO in 20
mL of 5% aqueous sodium bicarbonate was added a partial solution
of 2.63 g (0.008 mol) of 2,4-dinitro-1-fluoro-5-[N-methyl-(p-
methoxyphenyl)amino]benzene in 75 mL of tetrahydrofuran and
75 mL of tert-butyl alcohol. The resulting reaction mixture was
stirred at room temperature for 48 h. The tetrahydrofuran was
removed on a rotary evaporator and replaced with 200 mL of
dichloromethane, and the solution was filtered into a 500-mL
separatory funnel. The organic solution was washed with water,
dried over sodium sulfate, filtered through magnesium sulfate, and
evaporated to give 1.0 g of a red solid. The crude product was
recrystallized from ethanol to give 1.44 g of pure 11c as red-orange
crystals: mp 185-187 °C; 1H NMR (CDCl3) δ 3.17 (s, 6 H), 3.38
(s, 3 H), 3.81 (s, 3 H), 6.87-6.91 (m, 2 H), 6.95 (s, 1 H), 7.05-
7.09 (m, 2 H), 8.59 (s, 1 H); 13C NMR (CDCl3) δ 41.89, 43.21,
55.51, 106.29, 115.23, 125.74, 126.91, 127.86, 133.36, 139.15,
148.10, 153.33, 158.15; UV (ethanol) λmax (ꢀ) 250 (6.8 mM-1 cm-1),
286 (7.5 mM-1 cm-1), 374 (5.9 mM-1 cm-1). Anal. Calcd for
C16H18N6O7: C, 47.29; H, 4.46; N, 20.68. Found: C, 47.57; H,
4.85; N, 20.51.
HL-60 Screen: Cell Culture, Viability Assay, and IC50
Determination. Human myeloid leukemia HL-60 cells (ATCC,
Manassas, VA) were cultured at a density of 1.5 × 105 mL-1 in
RPMI-1640 supplemented with 10% fetal bovine serum and
penicillin/streptomycin. Cells were cultured at 37 °C in a humidified
5% CO2 atmosphere. Cultures were initiated in 96-well plates in a
total volume of 100 µL. Five millimolar stock solutions of each
compound were made in DMSO and serially diluted in PBS before
addition to the cultures. We established dose-response cultures
with concentrations ranging from 0 to 100 µM. The final concentra-
tion of DMSO added to the cultures was 0.1% or less. For each
experiment, compounds were added to cells in logarithmic phase
purified by recrystallization from ethanol: mp 125-126 °C; H
NMR (CDCl3) δ 1.29 (t, 6 H, J ) 7.1 Hz), 3.58-3.62 (m, 8 H),
3.63-3.75 (m, 8 H), 4.19 (q, 4 H, J ) 7.1 Hz), 7.53 (s, 1 H), 8.81
(s, 1 H); 13C NMR (CDCl3) δ 14.59, 42.11, 50.55, 62.09, 106.32,
125.39, 132.46, 153.63, 154.97; UV (methanol) λmax (ꢀ) 288 nm
(24.8 mM-1 cm-1). Anal. Calcd for C20H28N10O12: C, 40.00; H,
4.70; N, 23.33. Found: C, 40.24; H, 4.54; N, 23.04.
Isolation of Monodiazeniumdiolate 6a (CB-4-39). The first
fraction obtained from the experiment above was concentrated under
vacuum and the residue was recrystallized from 50% ethanol in
water: mp 102-104 °C; 1H NMR (CDCl3) δ 1.29 (t, 3 H, J ) 7.1
Hz), 3.64-3.67 (m, 4 H), 3.72-3.75 (m, 4 H), 4.19 (q, 2 H, J )
7.1 Hz), 7.43 (d, 1 H, J ) 11.5 Hz), 8.92 (d, 1 H, J ) 7.5 Hz); 13
C
NMR (CDCl3) δ 14.59, 42.14, 50.48, 62.11, 106.60, 106.87, 125.50,
132.86, 154.95, 157.19, 159.93; UV (ethanol) λmax (ꢀ) 296 nm (17.5
mM-1 cm-1). Anal. Calcd for C13H15FN6O8: C, 38.81; H, 3.76;
N, 20.89. Found: C, 39.08; H, 3.88; N, 20.74.
O2-(2,4-Dinitro-5-fluorophenyl) 1-(N,N-Dimethylamino)di-
azen-1-ium-1,2-diolate (11a) (JS-41-117). A partial solution of
4.03 g (0.0197 mol) of 1,5-difluoro-2,4-dinitrobenzene in 50 mL
of tert-butyl alcohol was placed in a 250-mL round-bottom flask
and stirred at room temperature. A solution of 2.92 g (0.023 mol)
of sodium 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (DMA/
NO) in 50 mL of 5% sodium bicarbonate was added dropwise at
a rapid rate. A thick yellow solid formed as the diazeniumdiolate
was added. Upon completion of the addition, the slurry was stirred
for 15 min at room temperature. Water was added, and the product
was collected by filtration and recrystallized from ethanol to give
1
2.3 g (40%) of 11a: mp 129-130 °C; H NMR (CDCl3) δ 3.32
(s, 6 H), 7.43 (d, 1 H, J ) 7.4 Hz), 8.92 (d, 1 H, J ) 7.6 Hz); 13
C
NMR (CDCl3) δ 41.94, 106.58, 106.86, 125.74, 155.48, 157.58,
160.30; UV (ethanol) λmax (ꢀ) 300 nm (22.1 mM-1 cm-1). Anal.
Calcd for C8H8FN5O6‚1/3H2O: C, 32.55; H, 2.96; N, 23.73.
Found: C, 32.90; H, 2.84; N, 23.36.

Antitumor ActiVity of JS-K in Vitro and in ViVo
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4365
growth. Three days after addition of the compounds, cell viability
was measured using the CellTiter 96 assay from Promega (Madison,
WI) according to the manufacturer’s protocol. For each compound
screened, there were at least three replicates. The IC50 of each
compound was determined from growth curves generated by
expressing the absorbance of each variable as a percentage of that
of untreated controls.
Supporting Information Available: Results from elemental
analysis. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) 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.
(2) 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. Leukemia Res. 2006, E
Published.
(3) Shami, P. J.; Kaur, G.; Thillainathan, J.; Jia, L.; Saavedra, J. E.;
Keefer, L. K. JS-K, a novel nitric oxide (NO) generator, shows potent
anti-angiogenic activity. Blood 2004, 104 (Suppl. 1), 931a.
(4) Ren, Z.; Kar, S.; Wang, Z.; Wang, M.; Saavedra, J. E.; Carr, B. I.
JS-K, a novel nonionic 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.
(5) 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.
(6) Townsend, D. M.; Tew, K. D. The role of glutathione-S-transferase
in anti-cancer drug resistance. Oncogene 2003, 22, 7369-7375.
(7) Shami, P. J.; Sauls, D. L.; Weinberg, J. B. Schedule and concentra-
tion-dependent induction of apoptosis in leukemia cells by nitric
oxide. Leukemia 1998, 12, 1461-1466.
Anticancer Screen of O2-Aryl Diazeniumdiolate Analogues
against the NCI 51 Human Cancer Cell Lines. The anticancer
screening test was conducted as previously described.10,32 Cells from
nine cancer subpanels of 51 human lines were grown in RPMI 1640
culture medium supplemented with 5% fetal bovine serum and 2
mM L-glutamine for 24 h at 37 °C to allow stabilization prior to
addition of the O2-aryl diazeniumdiolates. The nine human cancer
subpanels are leukemia, non-small-cell lung, colon, melanoma,
ovarian, renal, prostate, breast, and central nervous system cancer.
Stock solutions of the analogues in DMSO were serially diluted
with the RPMI 1640 medium and added immediately to the
microtiter plates to produce five concentrations of the analogues,
i.e., 10-4, 10-5, 10-6, 10-7, and 10-8 M. Each analogue was
incubated with cells for 48 h. At the end of incubation, the cells
were fixed in situ with 10% trichloroacetic acid, washed five times
with water, and dried. Sulforhodamine B (0.4% in 1% acetic acid)
was added to each well. After incubation for 10 min at room
temperature, unbound sulforhodamine B was removed by washing
five times with acetic acid. Then the plates were air-dried. Bound
stain was solubilized with Tris buffer, and the optical densities were
read at 515 nm. The optical densities generated from sulforhodamine
B staining are a function of cell mass and growth rate. The dose-
response curve (not shown) was created by plotting the percent
growth against the log of concentrations of the corresponding
analogue for each cell line and grouped by disease subpanels. The
molar concentrations that caused 50% growth inhibition (GI50), total
growth inhibition (TGI), and 50% cell killing (LC50) of the 51 cell
lines were determined.
(8) Ju, C.; McCoy, J. P.; Chung, C. J.; Graf, M. L. M.; Pohl, L. R.
Tolerogenic role of Kupffer cells in allergic reactions. Chem. Res.
Toxicol. 2003, 16, 1514-1519.
(9) 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.
In Vivo Study in Rats. JM-1 rat hepatoma cells, which were
derived from and established to grow in Fischer rats,33 were a gift
(G. Michalopoulos, University of Pittsburgh). Two-month-old male
Fischer rats were obtained from Hilltop Labs (Scottsdale, PA). 2′-
Amino-3′-methoxyflavone was purchased as PD 98059 from
Calbiochem (La Jolla, CA) and PTIO was from Sigma (St. Louis,
MO). JM-1 cells were cultured in minimum essential medium (Life
Technology, Gaithersburg, MD) in a humidified atmosphere of 5%
CO2 and 95% air at 37 °C. The medium contained 10% fetal bovine
serum. Cells were harvested after trypsinization, washed twice with
cold PBS, and suspended at a concentration of 106 cells/mL in PBS.
Five groups (four rats per group) were used for the experiments.
One million cells were injected into each rat’s liver through the
mesenteric vein. A stock solution of JS-K was prepared at a
concentration of 10 mg/mL in DMSO. One week after cell
transplantation in the rat’s liver, JS-K was injected intraperitoneally
in three groups five times, at a dose of 0, 3, or 30 mg/kg of rat
body weight every other day. Control rats received the same volume
of DMSO as those in the other groups. Five milliliters of 75 µM
2′-amino-3′-methoxyflavone or PTIO in 0.37% DMSO was injected
intravenously in the remaining two groups through the mesenteric
vein 1 h before JS-K (30 mg/kg) treatment. Rats were sacrificed 3
weeks after tumor cell injection and 2 weeks after the start of JS-K
treatment. Tumor-bearing livers were removed and the tumors were
excised and weighed. The statistical significance of the difference
between different treatments was determined by the t-test.
(10) Jia, L.; Tomaszewski, J. E.; Furchgott, R. F.; Jothianandan, D.; Keefer,
L. K.; Shami, P. J. EDRF-like activity and anticancer spectrum of
JS-K, a novel diazeniumdiolate nitric oxide donor. Proc. Am. Assoc.
Cancer Res. 2003, 44, 919 (abstract 4619).
(11) Deora, A. A.; Hajjar, D. P.; Lander, H. M. Recruitment and activation
of Raf-1 kinase by nitric oxide-activated Ras. Biochemistry 2000,
39, 9901-9908.
(12) Ingram, A. J.; James, L.; Thai, K.; Ly, H.; Cai, L.; Scholey, J. W.
Nitric oxide modulates mechanical strain-induced activation of p38
MAPK in mesangial cells. Am. J. Physiol. Renal. Physiol. 2000, 279,
F243-F251.
(13) Go, Y.-M.; Patel, R. P.; Maland, M. C.; Park, H.; Beckman, J. S.;
Darley-Usmar, V. M.; Jo, H. Evidence for peroxynitrite as a signaling
molecule in flow-dependent activation of c-Jun NH2-terminal kinase.
Am. J. Physiol. 1999, 277, H1647-H1653.
(14) Go, Y.-M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard,
K. A., Jr.; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H. Protein
kinase B/Akt activates c-Jun NH2-terminal kinase by increasing NO
production in response to shear stress. J. Appl. Physiol. 2001, 91,
1574-1581.
(15) Caselli, A.; Camici, G.; Manao, G.; Moneti, G.; Pazzagli, L.; Cappugi,
G.; Ramponi, G. Nitric oxide causes inactivation of the low molecular
weight phosphotyrosine protein phosphatase. J. Biol. Chem. 1994,
269, 24878-24882.
(16) Callsen, D.; Sandau, K. B.; Bru¨ne, B. Nitric oxide and superoxide
inhibit platelet-derived growth factor receptor phosphotyrosine phos-
phatases. Free Radical Biol. Med. 1999, 26, 1544-1553.
(17) Xian, M.; Wang, K.; Chen, X.; Hou, Y.; McGill, A.; Chen, X.; Zhang,
Z.-Y.; Cheng, J.-P.; Wang, P. G. Inhibition of protein tyrosine phos-
phatases by low-molecular-weight S-nitrosothiols and S-nitrosylated
human serum albumin. Biochem. Biophys. Res. Commun. 2000, 268,
310-314.
(18) Traverse, S.; Gomez, N.; Paterson, H.; Marshall, C.; Cohen, P.
Sustained activation of the mitogen-activated protein (MAP) kinase
cascade may be required for differentiation of PC12 cells. Comparison
of the effects of nerve growth factor and epidermal growth factor.
Biochem. J. 1992, 288 (Pt 2), 351-355.
Acknowledgment. This research was supported in part by
the Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research and by NCI Contract NO1-
CO12400 with SAIC-Frederick, Inc. The work was also
supported by NIH Grant R01 CA84496 and by a Translational
Research Award from the Leukemia and Lymphoma Society
(P.J.S.). We thank John Klose for providing the NMR data.
(19) Osada, S.; Osada, K.; Carr, B. I. Tumor cell growth inhibition and
extracellular signal-regulated kinase (ERK) phosphorylation by novel
K vitamins. J. Mol. Biol. 2001, 314, 765-772.

4366 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Shami et al.
(20) Mendes, A. F.; Carvalho, A. P.; Caramona, M. M.; Lopes, M. C.
Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS
II expression in articular chondrocytes. Inflamm. Res. 2002, 51, 369-
375.
(21) Ishii, Y.; Ogura, T.; Tatemichi, M.; Fujisawa, H.; Otsuka, F.; Esumi,
H. Induction of matrix metalloproteinase gene transcription by nitric
oxide and mechanisms of MMP-1 gene induction in human melanoma
cell lines. Int. J. Cancer 2003, 103, 161-168.
(22) Taimor, G.; Rakow, A.; Piper, H. M. Transcription activator protein
1 (AP-1) mediates NO-induced apoptosis of adult cardiomyocytes.
FASEB J. 2001, 15, 2518-2520.
(23) Fan, M.; Goodwin, M. E.; Birrer, M. J.; Chambers, T. C. The
c-Jun NH2-terminal protein kinase/AP-1 pathway is required for
efficient apoptosis induced by vinblastine. Cancer Res. 2001, 61,
4450-4458.
(24) Henry, Y.; Lepoivre, M.; Drapier, J.-C.; Ducrocq, C.; Boucher, J.-
L.; Guissani, A. EPR characterization of molecular targets for
NO in mammalian cells and organelles. FASEB J. 1993, 7, 1124-
1134.
(25) 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.
(26) Drago, R. S.; Paulik, F. E. The reaction of nitrogen(II) oxide with
diethylamine. J. Am. Chem. Soc. 1960, 82, 96-98.
(28) 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.
(29) Stewart, H. W.; Turner, R. J.; Denton, J. J.; Kushner, S.; Brancone,
L. M.; McEwen, W. L.; Hewitt, R. I.; Subbarow, Y. Experimental
chemotherapy of filariasis. IV. The preparation of derivatives of
piperazine. J. Org. Chem. 1948, 13, 134-143.
(30) Toldy, L.; Solyon, S.; Kocka, I.; Toth, G.; Toth, I. Thiourea
derivatives with tuberculostatic action. II. Acylthiocarbamides. Acta
Chim. Acad. Sci. Hungaricae 1971, 69, 221-227.
(31) Angier, R. B.; Murdock, K. C.; Curran, W. V.; Sollenberger, P. Y.;
Casey, J. P. Antiviral agents. I. Analogues and derivatives of
2-diethylaminoethyl 4-methylpiperazine-1-carboxylate. J. Med. Chem.
1968, 11, 720-729.
(32) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. Feasibility
of a high-flux anticancer drug screen using a diverse panel of cul-
tured human tumor cell lines. J. Natl. Cancer Inst. 1991, 83, 757-
766.
(33) Novicki, D. L.; Jirtle, R. L.; Michalopoulos, G. Establishment of
two rat hepatoma cell strains produced by a carcinogen initiation,
phenobarbital promotion protocol. In Vitro 1983, 19, 191-202.
(27) 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.
JM060022H