PABA/NO as an anticancer lead: Analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity

Article abstract of DOI:10.1021/jm050700k

PABA/NO is a diazeniumdiolate of structure Me₂NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling th

Full text of DOI:10.1021/jm050700k

J. Med. Chem. 2006, 49, 1157-1164
1157
PABA/NO as an Anticancer Lead: Analogue Synthesis, Structure Revision, Solution Chemistry,
Reactivity toward Glutathione, and in Vitro Activity
†
‡
†
§
‡
‡
Joseph E. Saavedra, Aloka Srinivasan, Gregory S. Buzard, Keith M. Davies, David J. Waterhouse, Keiko Inami,
‡
†
‡
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Thomas C. Wilde, Michael L. Citro, Matthew Cuellar, Jeffrey R. Deschamps, Damon Parrish, Paul J. Shami,
&
&
&
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∞
Victoria J. Findlay, Danyelle M. Townsend, Kenneth D. Tew, Shivendra Singh, Lee Jia, Xinhua Ji, and
Larry K. Keefer*,
‡
Basic Research Program, SAIC Frederick, Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, and Biomolecular Structure Section,
Macromolecular Crystallography Laboratory, National Cancer Institute at Frederick, Frederick, Maryland 21702; Department of Chemistry,
George Mason UniVersity, Fairfax, Virginia 22030; Laboratory for the Structure of Matter, NaVal Research Laboratory, Washington,
District of Columbia 20375; DiVision of Medical Oncology, Department of Internal Medicine, UniVersity of Utah, Salt Lake City, Utah 84112;
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical UniVersity of South Carolina, Charleston,
South Carolina 29425; Department of Pharmacology, UniVersity of Pittsburgh Cancer Institute, Pittsburgh, PennsylVania 15213; and
DeVelopmental Therapeutics Program, National Cancer Institute, Bethesda, Maryland 20892
ReceiVed July 21, 2005
2
PABA/NO is a diazeniumdiolate of structure Me NN(O)dNOAr (where Ar is a 5-substituted-2,4-dinitrophenyl
ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian
cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water.
Here we report structure-based optimization efforts resulting in three analogues with improved solubility
and stability in aqueous solution. We sought to explain PABA/NO’s physicochemical uniqueness among
these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence
of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that
PABA/NO’s N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl
oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the
previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric
oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO’s potent suppression of A2780
human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell
line.
Introduction
enhanced resistance to the drug. PABA/NO also increased levels
of phosphorylated and thus activated forms of the stress-
2
PABA/NO is an O -arylated diazeniumdiolate that has shown
activated protein kinases c-Jun NH -terminal kinase (JNK)
2
tumoristatic activity against A2780 human ovarian cancer
xenografts in female SCID mice with a potency comparable to
that of cisplatin. It was designed using a structure-based
and p38, events that have been linked to drug-induced
apoptosis.
1
1
On the basis of its in vivo activity and the relevant
mechanistic findings summarized above, we consider PABA/
NO to be a promising anticancer lead deserving further
preclinical characterization. In the present work, we compare it
with three close structural analogues with regard to structure,
solubility, hydrolytic stability, reactivity with GSH, and in vitro
anticancer activity.
modeling approach to generate potentially cytolytic nitric oxide
(
(
NO) through mediation of π-class glutathione S-transferase
GST), which is overexpressed in many tumors. Consistent with
the structure-based design considerations, PABA/NO was
metabolized to NO more efficiently by GSTπ than by
1
GSTR, which is the predominant isoform in the liver. Its
cytotoxicity was shown to increase with increases in the
intracellular expression levels of GSTπ. Conversely, elevated
levels of multidrug resistance-associated protein 1 (MRP1), a
key component of the cellular efflux pump system, conveyed
Results
Synthesis and Solubility. A milestone in our structure-based
drug design effort was the synthesis of 4, which satisfied all
the criteria for a good fit in the active site of GSTπ but was
predicted to be poorly accommodated in that of GSTR. It was
synthesized as in Scheme 1 and confirmed by X-ray crystal-
*
Author for correspondence. Phone: 301-846-1467. Fax: 301-846-5946.
E-mail: keefer@ncifcrf.gov.
†
Basic Research Program, National Cancer Institute at Frederick.
Laboratory of Comparative Carcinogenesis, National Cancer Institute
‡
2
lography to have the structure shown, but it proved too
at Frederick.
insoluble to test. Reasoning that a carboxyl group should
overcome that barrier, we substituted N-methylated PABA (p-
aminobenzoic acid) 5a for N-methylaniline in the reaction of
Scheme 1 (see also eq 1) and isolated PABA/NO (6a).
Encouraged by the promising initial testing results described
above but concerned about its low solubility (<2 µM), we
repeated the reaction of Scheme 1 using 5a analogues 5b, 5c,
§
George Mason University.
Naval Research Laboratory.
University of Utah.
Medical University of South Carolina.
University of Pittsburgh Cancer Institute.
Developmental Therapeutics Program.
Macromolecular Crystallography Laboratory, National Cancer Institute
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∞
at Frederick.
1
0.1021/jm050700k CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/10/2006

1
158 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
SaaVedra et al.
Table 1. Physicochemical Parameters for PABA/NO and Three
Scheme 1. Synthesis of 42
Analogues
solubilitya
(mM)
khydrolysis
b
kGSH
-1
c
νCdO
-
1
(M s-1
-1
compound
(s
)
)
(cm )
(4.9 ( 2.4) × 10-
4 d
0.31 ( 0.02d
1730
6
a
<0.002
(PABA/NO)
6
6
6
b
c
d
>3
>2
3
∼0
0.043 ( 0.003 1685
0.056 ( 0.003 1684
∼0
∼0
0.13 ( 0.004
1685
a
b
Maximum solubility in 0.1 M phosphate buffer, 37 °C. First-order
c
rate constants for hydrolysis at pH 7.4 and, except as noted, 37 °C. Second-
order rate constants for reaction with glutathione at pH 7.4 and, except as
noted, 37 °C. d Rate data for PABA/NO were obtained at room temperature.
Scheme 2. Structures of PABA/NO and Three Analogues
Investigated Here
and 5d in place of N-methylated PABA and isolated three new
compounds, 6b-6d. All were 3 orders of magnitude more
soluble in pH 7.4 buffer (Table 1).
the only one of the four that was not soluble in sodium
bicarbonate solution. Moreover, its infrared carbonyl stretching
-
1
frequency was the only one to appear at 1730 cm , consistent
with an ester linkage, with the other three in the free carboxylate
-
1
range, 1684-1685 cm . These physical data strongly suggest
that PABA/NO is the only one of the four without a free
carboxyl group, the PABA moiety being bound to the remainder
of the molecule through an ester linkage rather than via the
anilino nitrogen. This was confirmed by X-ray crystallography,
which provided an unambiguous view of the connectivity
(Figure 1).
Structure Assignments, Including Revisions. The signifi-
cant difference in solubility between PABA/NO and its three
analogues suggested that PABA/NO is fundamentally different
from the other three, despite the fact that all four compounds
were prepared by the same two-step (difluoride 1 plus substituted
aniline 5, then product plus diazeniumdiolate ion 3) reaction
sequence as 4, whose structure was confirmed as shown in
Scheme 1 by X-ray crystallography.
On the basis of these physicochemical data, summarized in
Table 1 and the Supporting Information, we conclude that the
structures of these compounds are as shown in Scheme 2. Thus,
PABA/NO has structure 6a, a revision of the one reported
1
previously. This implies that the structure of its immediate
PABA/NO differed from its three analogues not only in its
solubility at physiological pH but also in the fact that it was
precursor, i.e., the one formed by reaction of 1 with 5a as shown
in eq 1, was also incorrect as originally assigned and must
1

PABA/NO as an Anticancer Lead
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1159
Figure 1. Structure of 6a showing the labeling of the non-hydrogen atoms. Thermal ellipsoids are shown at the 50% probability level.
be revised. The correct connectivity is as shown in eq 2,
structure 7.
While the infrared and acid-base behavior of 6b, 6c, and
d strongly support the structural assignments listed in Scheme
6
2
, we sought confirmation through further crystallographic
Figure 2. Structure of 6c showing the labeling of the non-hydrogen
atoms. Thermal ellipsoids are shown at the 30% probability level. Only
one of the four molecules in the asymmetric unit is shown. Molecules
studies. Thus far, only 6c has yielded a suitable crystal (Figure
), which crystallized with four molecules in the asymmetric
2
2
-4 as well as disordered positions for N3, C3′, and C3′′ are omitted
unit, three of which were disordered. In these three molecules,
the N3 atom and the two attached carbons of each dimethyl-
amino group were disordered over two positions, with ratios of
for clarity.
the reactivity of 6a was characterized by plotting the decrease
in its HPLC peak area as a function of time. Because its
hydrolysis and its reaction with glutathione were too fast to
follow reliably by this method at 37 °C, its rate constants were
determined at room temperature. The results are given in Table
1. Despite the lower temperature (by approximately 15 °C),
PABA/NO’s kGSH was 2-7 times as great as those of its
analogues. Chemiluminescence traces confirming the generation
of NO in the reactions of 6a-6d with GSH are shown in Figures
S2 and S3 of Supporting Information.
4
8:52, 69:31, and 48:52, respectively. This contributed to the
poor scattering exhibited by the crystal and resulted in slightly
higher R1 and wR2 values than typically expected. Structurally,
the four unique molecules varied significantly only in the
orientation of the benzoate ring (Figure S1 of Supporting
Information). The torsion angles about the N7-C8 bonds of
molecules A and D were -35.5° and -37.0°, respectively, while
molecules B and C have torsion angles of 44.5° and 36.2°,
respectively.
Hydrolysis and Reaction with GSH. PABA/NO also dis-
tinguished itself from 6b-6d in its reactivity. Its reaction rates
could not be followed easily by the standard ultraviolet method
used for the other three compounds, partly because of the
complexity of the reaction and partly because the products’
spectra were closely similar to that of PABA/NO. Consequently,
Hydrolysis of PABA/NO (6a) occurred at both the ester and
diazeniumdiolate groups (Scheme 3), so the observed khydrolysis
rate reflects a combination of two parallel processes. Integrating
the HPLC peaks for products 8 and 9 showed that hydrolysis
of the ester was at least an order of magnitude faster than
cleavage of the diazeniumdiolate group. Once hydrolysis

1
160 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
SaaVedra et al.
Scheme 4. Products Observed in the Reaction of PABA/NO
Scheme 3. PABA/NO Hydrolysis Products Determined by
HPLC, LC/MS, and Chemiluminescence Analysis
(6a) with Glutathione (GSH) under Catalysis by Glutathione
S-Transferase µ (GSTµ) in 0.1 M Phosphate Buffer (pH 7.4)
a
occurred at one position, though, further hydrolysis became
extremely slow. No significant hydrolysis of analogues 6b-6d
was observed under these conditions.
To further probe PABA/NO’s reactivity with GSH, we
conducted the reaction in the presence of GSTµ: to catalyze
the initial glutathionylation. GSH adduct 10 was formed very
rapidly, dominating the HPLC-UV trace within 35 min of
mixing (Figure 3); NO was seen as a byproduct by chemi-
luminescence. Cleavage of the ester function of 6a also occurred,
giving rise to 8. On further incubation at 37 °C, the ester group
of 10 was cleaved to generate hydrolysis products 5a and 11.
The products formed on prolonged incubation under these
conditions are shown in Scheme 4.
a
*
Organic product identities were confirmed by mass spectrometry or
HPLC, with the exception** of 3 and dimethylamine. Production of NO
was confirmed by chemiluminescence.
Table 2. Cytotoxic Concentrations (µM) of 6a and 6c against the Most
Sensitive Human Cancer Cell Lines in the NCI 51-Cell-Line Screen
a
PABA/NO (6a)
analogue 6c
cell line
GI50
TGI
LC50
GI50
TGI
LC50
Biological Results. Initial in vitro screening was performed
with HL-60 human leukemia cell cultures. Results were disap-
pointing, giving IC50 values that were at least an order of
colon HCT-15
2.0
1.9
2.6
1.8
5.3
4.4
6.9
4.0
19
10
2.6
8.9
20
19
3.9
2.2
36
3.4
30
68
60
58
melanoma LOXIMVI
ovarian OVCAR-3
renal CAKI-1
51
>100
2
magnitude higher than that of JS-K, an O -arylated diazenium-
a
Compound 6b inhibited the ovarian OVCAR-3 cells with GI50 ) 5.5
µM, TGI ) 9 µM, and LC50 ) 100 µM, and the other three cell lines with
GI50, TGI, and LC50 > 100 µM. Analogue 6d was inactive at a concentration
of 100 µM.
diolate that has consistently shown sub-micromolar potency in
this screen and has proven active against human leukemia and
3
prostate cancer xenografts in NOD-SCID mice. Similar results
were seen with NIH3T3 mouse skin fibroblasts, a cell line used
in the mechanistic studies with PABA/NO described earlier.1
IC50 values for compounds 6a-6d in these two cell lines are
summarized in the Supporting Information.
The compounds were next subjected to the NCI 51-cell-line
screen. The dose-response curves revealed that the overall
inhibitory potency of PABA/NO and its three analogues ranked
in the order 6a > 6b > 6c > 6d. PABA/NO exhibited the
greatest growth inhibition against all the tested cell lines with
a mean GI50 value of 9.8 µM, a mean TGI value of 33 µM, and
a mean LC50 value of 69 µM. Analogue 6b showed the next
greatest potency in inhibiting the 51 cell lines with a mean GI50
value of 23 µM, a mean TGI value of 52 µM, and mean LC50
value of 85 µM. Compound 6c inhibited the 51 cell lines with
a mean GI50 value of 68 µM, a mean TGI value of 96 µM, and
a mean LC50 value of more than 100 µM. Compound 6d showed
the least inhibitory potency with mean GI50, TGI, and LC50
values of more than 100 µM.
Among the 51 cell lines, colon cancer HCT-15, melanoma
LOXIMVI, ovarian OVCAR-3, and renal CAKI-1 showed the
greatest sensitivity to the compounds. Their proliferation was
most inhibited by PABA/NO and next by 6c (Table 2). The
Figure 3. Chromatograms of reaction mixtures containing 5 µM
PABA/NO (6a), 0.1 mM GSH, and 2.9 nM GSTµ (molecular weight
2
5.7 kDa) in 0.1 M phosphate at pH 7.4 and 37 °C at (A) 2 min and
(B) 35 min after mixing.

PABA/NO as an Anticancer Lead
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1161
Scheme 5. Results of Molecular Modeling Experiments1
Leading to the Design of PABA/NO (6a) as a Potential
GSTπ-Selective Improvement over JS-K [12, O -(2,4-
Dinitrophenyl) 1-[(4-Ethoxycarbonyl)piperazin-l-yl]diazen-
l-ium-1,2-diolate]
slightly so, and showed promising in vivo activity against a
human ovarian cancer xenograft in SCID mice. As may have
1
2
been predicted, in vitro experiments implicated proteins involved
in clearing electrophilic species from the cell (GSTπ, MRP1,
and γCS) in PABA/NO’s mechanism of action. The drug was
also shown to activate the MAPK suite of protein kinases (ERK,
JNK, and p38). Unfortunately, PABA/NO was not very soluble,
and its spontaneous hydrolysis and its uncatalyzed reaction with
glutathione were more rapid than might be desired, given the
goal of systemically administering it for selective metabolism
only in GSTπ-overexpressing cells without collaterally exposing
other NO-sensitive compartments.
In view of PABA/NO’s promising activity against human
ovarian cancer xenografts in mice, however, we began a lead
optimization effort by preparing analogues 6b-6d and compared
their chemistry with that of PABA/NO. All were orders of
magnitude more soluble than PABA/NO. They were also
significantly less reactive with GSH in the absence of GST.
Unfortunately, their in vitro anticancer activity was not improved
over that of PABA/NO.
With the view that a full physicochemical characterization
of a medicinally promising agent is a crucial prerequisite to
systematic lead optimization, we report here on the fundamental
properties of PABA/NO. Not only have we provided new details
concerning its reactivity, but a comparison with its analogues
has revealed the need for a revision in its structure. PABA/NO
is an ester, as diagrammed in Schemes 2 and 4, rather than the
1
diarylamine derivative that was originally reported.
It appears that resonance interaction with the electron-
withdrawing p-carboxyl group of 5a diminishes the nucleo-
philicity of the methylamino nitrogen, allowing a carboxyl
oxygen to displace a fluoride ion of 1 by default, as shown in
eq 2. It is understandable that this resonance effect would not
be operative when the carboxyl group is meta to the amino
nitrogen, leading to the diarylamine connectivity shown in
Scheme 2 for 6c and 6d, but it is surprising that 5b did not
displace a fluoride of 1 via its oxygen as 5a did. Apparently,
the N-methyl group of 5a that distinguishes it from 5b, whose
presence might have been expected to increase the inherent
nucleophilicity of the nitrogen, offers enough steric interference
at the nitrogen center to force attack on 1 by the oxygen of 5a.
low GI50 along with low LC50 values indicate that PABA/NO
has both strong cytostatic and cytotoxic effects on these four
cell lines, while 6c has cytotoxic effects less potent than PABA/
NO’s.
Discussion
This work was initiated in an effort to improve on structure
2 (JS-K, Scheme 5) as an anticancer lead. JS-K was active
We have been unable thus far to confirm the structural
assignment of 6b by X-ray crystallography, but the structure
shown in Scheme 2 is consistent with the data at hand and seems
1
against both HL-60 human leukemia and PPC-1 human prostate
cancer xenografts in NOD-SCID mice, but it was metabolized
to the putative cytostatic/cytotoxic intermediate nitric oxide
almost 100-fold more efficiently by the R isoform of GST than
-1
secure. Unlike PABA/NO (νCdO ) 1730 cm ), but like 6c and
-1
6d, 6b has a carbonyl stretching frequency (1685 cm ) typical
of a free carboxylic acid instead of an ester grouping (Table 1).
The greater solubility of 6b as well as 6c and 6d (but not 6a)
in mild alkali than in acid also supports the presence of a free
carboxylate function in 6b-6d.
3
by the enzyme’s π isoform; π-susceptibility should allow for
greater concentration of this active metabolite in tumor tissue,
given that GSTπ expression is upregulated in many malignant
cell types. Also, given the importance of the R isoform to
cellular well-being, we sought to decrease the R-susceptibility
so as to spare normal, R-expressing tissue from potentially
deleterious collateral exposure to NO.
The significantly greater overall activity of 6a relative to its
three analogues in the 51-cell-line screen appears to correlate
with its greater reactivity and unique structure, possibly also
with its greater lipophilicity (hence membrane permeability).
Future work will be aimed at preparing differently substituted
3
To these ends, an earlier structure-based drug design exercise
2
2
involving transition state modeling of O -arylated diazenium-
O -arylated diazeniumdiolates patterned after the core PABA/
diolate structures in the active sites of three GST isoforms was
conducted. The results (Scheme 5) suggested that decreasing
the bulk on the amine side of the N2O2 group should make the
substrate more π-compatible, while adding a sterically demand-
ing substituent at the 5-position of the aryl ring should decrease
its accommodation in the R active site. These considerations
led to the synthesis of PABA/NO (6a), which was indeed
metabolized more efficiently by GSTπ than GSTR, if only
NO structure in an effort to increase its solubility without
lowering its cytostatic/cytotoxic activity against tumor cells, as
well as to decrease its reactivity toward GSH in the absence of
GST. With all three of the in vivo tests that have to our
2
knowledge been conducted with O -arylated diazeniumdiolates
in anticancer screens thus far having been positive, we are
hopeful that continued lead optimization work will generate
further improvements still.

1
162 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
SaaVedra et al.
Experimental Section
836 mg (2.6 mmol) of 3-[N-(2,4-dinitro-5-fluorophenyl)amino]-
benzoic acid in 15 mL of dimethyl sulfoxide (DMSO) was
introduced slowly through a syringe and gradually allowed to warm
to room temperature. After 24 h the solution was poured over ice-
water containing hydrochloric acid and the product was extracted
with ethyl acetate. The ethyl acetate solution was extracted with
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-d
sulfoxide-d . Chemical shifts (δ) are reported in parts per million
ppm) downfield from tetramethylsilane. Quantification of NO by
6
, or dimethyl
6
5% sodium bicarbonate solution. The aqueous layer was separated
(
and washed with dichloromethane to remove the DMSO and then
acidified with dilute hydrochloric acid, and the product was
extracted with ethyl acetate. The organic layer was dried over
sodium sulfate, filtered through magnesium sulfate, and concen-
trated on a rotary evaporator to give 501 mg of product that was
recrystallized from acetone: mp 175-176 °C; UV (in 5% sodium
chemiluminescence was done with a Thermal Energy Analyzer
Model 502A instrument (Thermedics, Inc., Woburn, MA), as
previously described.4 Elemental analyses were performed by
Atlantic Microlab, Inc. (Norcross, GA) or Midwest Analytic
(
Indianapolis, IN). Compounds 6a and 7 were prepared as previ-
1
ously described (though see structure revisions reported above).
-1
-1
1
bicarbonate solution) λmax 354 nm (ꢀ ) 20.5 mM cm ); H NMR
CDCl ) δ 3.02 (s, 6H), 6.98 (s, 1H), 7.63-7.67 (m, 2H), 7.88-
2
The sodium salt of 3 was prepared as described earlier.
(
3
4
-[N-(2,4-Dinitro-5-fluorophenyl)amino]benzoic Acid. A solu-
tion of 3.84 g (0.0187 mol) of 1,5-difluoro-2,4-dinitrobenzene in
0 mL of acetone was placed in a 500-mL flask equipped with a
13
7
1
1
0
3
.98 (m, 2H), 8.93 (s, 1H), 10.23 (s, 1H); C NMR (CDCl
00.36 ppm, 126.03, 126.59, 126.96, 127.19, 127.39, 129.97,
14 6 8
32.16, 138.10, 146.67, 154.14, 166.52. Anal. Calcd for C15H N O ‚
.2 acetone: C, 44.83; H, 3.67; N, 20.11. Found: C, 44.83; H,
.55; N, 19.82.
3
) δ
5
stirring bar and a pressure-equalizing addition funnel. To the stirring
solution was added 2.56 g (0.0187 mol) of 4-aminobenzoic acid
5b in 50 mL of acetone. The resulting bright yellow solution was
3
-Methylaminobenzoic Acid (5d). 3-Aminobenzoic acid (42.5
g; 0.31 mol) was dissolved in 600 mL of water containing 25 g
0.62 mol) of sodium hydroxide. To the basic solution was added
9 mL (0.31 mol) of dimethyl sulfate, and the resulting mixture
stirred at room temperature and the progress of the reaction was
monitored by TLC on silica gel plates using 10:1 dichloromethane:
methanol as the developer. After stirring for 24 h at room
temperature, the reaction mixture was heated at reflux for 8 h.
Filtration and evaporation left a residue that was recrystallized from
(
2
was stirred at room temperature overnight. The aqueous solution
was washed with dichloromethane to remove any neutral organic
material, acidified with 10% hydrochloric acid, and extracted with
ethyl acetate. The solution was dried over sodium sulfate and filtered
through magnesium sulfate. The solvent was removed on a rotary
evaporator to give 13 g of a solid that contained the desired product
along with starting material and 3-(N,N-dimethylamino)benzoic
acid. The mixture was adsorbed on 20 g of silica gel and deposited
on a 1.1- × 15-cm precolumn reservoir connected to a prepacked
silica gel column (2.7 × 29 cm) that had been conditioned with
5:1 dichloromethane:acetonitrile and eluted with the same solvent
system to give fractions containing 3.03 g of 3-methylaminobenzoic
ethanol:water to give 2.6 g of red-orange plates: mp 238-239 °C;
1
H NMR (DMSO-d ) δ 7.10 (d, 2H, J ) 14 Hz), 7.49-7.54 (m,
6
2
1
1
H), 8.01-8.05 (m, 2H), 8.92 (d, 1H, J ) 7.96 Hz), 10.24 (b, 1H),
2.99 (b, 1H). Anal. Calcd for C13 F: C, 48.61; H, 2.51; N,
8 3 6
H N O
3.08. Found: C, 48.65; H, 2.60; N, 12.97.
2
O -[2,4-Dinitro-5-(4-carboxylatophenyl)amino]phenyl 1-(N,N-
Dimethylamino)diazen-1-ium-1,2-diolate (JS-38-12) (6b). A solu-
tion of 731 mg (2.28 mmol) of 4-[N-(2,4-dinitro-5-fluorophenyl)-
amino]benzoic acid in DMSO was added to a slurry of 508 mg (4
mmol) of 3 (sodium salt) in tetrahydrofuran and cooled to 0 °C
under nitrogen. After gradually warming to room temperature and
then stirring for another day, the reaction mixture was poured into
ice-cold hydrochloric acid. The product was extracted with ethyl
acetate, and the organic layer was extracted with 5% sodium
bicarbonate. The aqueous layer was washed with dichloromethane,
acidified, and extracted with ethyl acetate. The organic extract was
dried and evaporated to give 414 mg of crude product. Purification
5
1
acid (5d): mp 115-116 °C (lit.
6
mp 127 °C); H NMR (acetone-
d ) δ 2.82 (s, 2H), 6.81-6.86 (m, 1H), 7.25-7.27 (m, 3H). Anal.
Calcd for C H NO : C, 63.56; H, 6.00; N, 9.27. Found: C, 63.54;
8
9
2
H, 5.94; N 9.19.
3-[N-(2,4-Dinitro-5-fluoro)-N-methylamino]benzoic Acid. A
solution of 1.10 g (0.0073 mol) of 3-methylaminobenzoic acid (5d)
in 50 mL of 5% sodium bicarbonate solution was cooled in an
ice-bath. A solution of 1.50 g (0.0073 mol) of 1,5-difluoro-2,4-
dinitrobenzene in 15 mL of tetrahydrofuran and 25 mL of tert-
butyl alcohol was added. The resulting orange reaction mixture was
stirred for 4 h, concentrated under vacuum, extracted with water,
and washed with ethyl acetate. The aqueous solution was acidified
with 10% hydrochloric acid, extracted with ethyl acetate, dried over
sodium sulfate, filtered, and concentrated under vacuum to give
538 mg of an orange glass. The crude product was flash chromato-
graphed on silica gel using a glass column and eluted with 5:1
dichloromethane:ethyl acetate. The fractions containing the desired
product were pooled and evaporated to give 342 mg of a yellow
of the product required two subsequent recrystallizations from
1
ethanol: mp 163-165 °C; H NMR (DMSO-d
6
) δ 3.07 (s, 6H),
7
.19 (s, 1H), 7.55 (d, 2H, J ) 8.6 Hz), 8.02 (d, 2H, J ) 8.4 Hz),
.92 (s, 1H), 10.19 (s, 1H), 13.0 (b, 1H). Anal. Calcd for
‚0.5 ethanol: C, 44.76; H, 3.47; N, 19.57. Found: C,
4.90; H, 3.55; N 19.41.
-[N-(2,4-Dinitro-5-fluorophenyl)amino]benzoic Acid. A solu-
tion of 6.42 g (0.0313 mol) of 1,5-difluoro-2,4-dinitrobenzene in
00 mL of acetone was placed in a 500-mL flask equipped with a
8
15 14 6 8
C H N O
4
3
1
stirring bar and a pressure-equalizing addition funnel. To the stirred
solution was added 4.29 g (0.0313 mol) of 3-aminobenzoic acid
1
5c in 100 mL of acetone. The resulting bright yellow solution was
powder: mp 95-96 °C; H NMR (CDCl ) δ 3.50 (s, 3H), 7.00 (d,
3
stirred at room temperature and the progress of the reaction was
monitored by TLC on silica gel plates using 10:1 dichloromethane:
methanol as the eluant. After 24 h the reaction mixture was filtered
to remove any acetone-insoluble particles, and the yellow filtrate
was evaporated under vacuum to give a yellow solid. The product
was triturated with acetone:ether, collected by filtration, and dried
under vacuum to give 7.12 g (71%) of product: mp 239-240 °C;
1H, J ) 12.9 Hz), 7.45-7.60 (m, 2H), 7.74-7.97 (m, 2H), 8.62
1
(d, 1H, J ) 7.8 Hz). Anal. Calcd for C H N O FC‚ / H O: C,
14
10
3
6
3
2
49.27; H, 3.15; N, 12.31. Found: C, 49.10; H, 3.21; N, 12.11.
2
O -{2,4-Dinitro-5-[N-(3-carboxylatophenyl)-N-methyl]amino}-
phenyl 1-(N,N-Dimethylamino)diazen-1-ium-1,2-diolate (JS-39-
47) (6d). A slurry of 239 mg (1.88 mmol) of 3 (as the sodium salt)
in 2.5 mL of tetrahydrofuran under nitrogen was cooled to 0 °C. A
solution of 324 mg (0.91 mmol) of 3-[N-(2,4-dinitro-5-fluoro)-N-
methylamino]benzoic acid in 2.5 mL of tetrahydrofuran was added
to the cold slurry, which was gradually allowed to warm to room
temperature with stirring overnight. Ethyl acetate was added and
the mixture was poured over dilute hydrochloric acid. The aqueous
portion was discarded and the organic layer was extracted with
5% sodium bicarbonate. The aqueous solution was separated,
acidified with hydrochloric acid, and extracted with ethyl acetate.
The solution was dried over sodium sulfate, filtered, and concen-
-
1
-1
1
UV (methanol) λmax 342 nm (ꢀ ) 8.9 mM cm ); H NMR
(
7
1
CDCl
3
) δ 6.86 (d, 1H, J ) 14 Hz), 7.18-7.32 (m, 1H), 7.63-
.66 (m, 2H), 7.92 (s, 2H), 8.93 (d, 1H, J ) 8.0 Hz), 10.27 (s,
H). Anal. Calcd for C13 F: C, 48.61; H, 2.51; N, 13.08.
8 3 6
H N O
Found: C, 48.21; H, 2.64; N, 12.73.
2
O -[2,4-Dinitro-5-(3-carboxylatophenyl)amino]phenyl 1-(N,N-
Dimethylamino)diazen-1-ium-1,2-diolate (JS-36-120) (6c). A
slurry of 800 mg (6.3 mmol) of 3 (sodium salt) in 25 mL of
tetrahydrofuran was cooled to 0 °C under nitrogen. A solution of

PABA/NO as an Anticancer Lead
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1163
trated under vacuum to give 196 mg of a brown glass. Flash
chromatography of the material was performed on silica gel using
a 1.3- × 13.5-cm glass column and elution with 9:1 dichloro-
methane:methanol. A second round of chromatography was neces-
inhibition (TGI), and 50% cell killing (LC50) of the 51 cell lines
were determined, respectively.
Rate Constant Determinations. The reaction of 5 µM PABA/
NO with GSH was monitored by HPLC analysis of PABA/NO
concentrations, obtained from peak areas with reference to a 5 µM
PABA/NO standard as a function of time, in 0.10 M phosphate
buffer at pH 7.4. The greater solubility and simpler decomposition
behavior of the three PABA/NO analogues permitted their reactions
to be monitored directly by ultraviolet spectrophotometry using a
Hewlett-Packard 8452A diode-array spectrophotometer. The wave-
lengths followed were 341, 335, and 346 nm for 6b, 6c, and 6d,
respectively, and their starting concentrations were 20, 10, and 50
µM, respectively. The decreases in substrate concentration displayed
excellent first-order behavior over several half-lives. Pseudo-first-
order rate constants were obtained with GSH in large excess of
substrate. Measured values showed a linear dependence on the
concentration of GSH. The slopes and y-intercepts of the plots
yielded rate constants for the reactions with GSH and water,
respectively.
sary for better purification, giving 12 mg of product: mp 150-
1
1
7
53 °C; H NMR (DMSO-d
6
) δ 3.21 (s, 6H), 3.49 (s, 3H), 7.21-
.34 (m, 2H), 7.37 (s, 1H), 7.57-7.69 (m, 2H), 8.56 (s, 1H). Anal.
Calcd for C16
Found: C, 44.58; H, 3.86; N, 19.34.
H N
16 6
O
8
·0.5H
2
O: C, 44.76; H, 3.99; N, 19.57.
Characterization of GSH Adduct 10 Isolated from the GSTµ-
Catalyzed Reaction of PABA/NO with GSH. A solution of
PABA/NO (50 µM final concentration), GSH (2 mM final
concentration), and GSTµ (3 fµ/mL final concentration) in 100 mM
phosphate containing 1% DMSO, pH 7.4, was incubated at 37 °C
for 5 min, whereupon the entire 1-mL volume was injected into a
1
2
-mL loop for isolation by HPLC. The mobile phase consisted of
5:75 acetonitrile:water with 0.1% formic acid for 5 min followed
by a linear gradient to 70:30 acetonitrile:water over the next 10
min at a flow rate of 1 mL/min. The separation was performed on
a Phenomenex 5-µm C18 Luna (2) column (250 × 4.6 mm) with
Single-Crystal X-ray Diffraction Studies. For 6a, a clear,
3
orange needle of dimensions 0.08 × 0.09 × 0.43 mm was mounted
315-nm detection.
on glass fiber using a small amount of Cargille immersion oil. Data
were collected on a Bruker four-circle diffractometer equipped with
a SMART 1000 CCD detector. The crystals were irradiated using
graphite-monochromated Mo KR radiation (λ ) 0.71073). An MSC
X-Stream low-temperature device was used to keep the crystals at
a constant -170 °C during data collection. Data collection was
The product was isolated by collecting the fraction at 13.8 min,
which had a λmax of 320 nm. The solution was frozen and
lyophilized. The resulting off-white solid was stored in the
refrigerator overnight. For LC-MS, the residue was dissolved in
100 mM, pH 7.4 sodium phosphate buffer and sampled periodically
over 3 h. Initially, there was a very large peak in the total ion
performed and the unit cell was initially refined using SMART
+
chromatogram with an (M + H) value of 623 m/z, corresponding
8
(
v5.625). The crystals were monoclinic, P2
1
/n, with a ) 14.153(4)
to 10. On continued incubation, the intensity of this peak began to
Å, b ) 7.881(2) Å, c ) 18.163(5) Å, â ) 112.336(5)°, V )
+
decrease, giving way to two other peaks having (M + H) values
3
3
-1
1
873.8(8) Å , Z ) 4, Fcalc ) 1.490 Mg/m , µ ) 0.122 mm , and
of 490 and 152 m/z, corresponding to 11 and 5a, respectively.
F(000) ) 872. Data reduction was performed using SAINT
Hydrolysis of PABA/NO at Physiological pH. PABA/NO (final
concentration 5 µM) in 1 mL of 0.1 M, pH 7.4 phosphate buffer
containing 0.1% DMSO was analyzed for hydrolytic decomposition
by HPLC. The HPLC conditions utilized consisted of acetonitrile:
water with 0.1% formic acid added, and the separation was
performed using a Phenomenex 5-µm C18 Luna (2) column (250
v6.36A)9 and XPREP (v6.12). Corrections were applied for
10
(
Lorentz, polarization, and absorption effects using SADABS
11
(
v2.03). The structure was solved and refined with the aid of the
12
programs in the SHELXTL-plus (v6.10) system of programs. The
full-matrix least-squares refinement on F included atomic coor-
2
dinates and anisotropic thermal parameters for all non-H atoms.
The H atoms were included using a riding model. The final R value
×
4.6 mm) at a flow rate of 1 mL/min with 315-nm detection. The
gradient consisted of 25% acetonitrile for 5 min, followed by a
linear gradient to 70% acetonitrile over the next 10 min. Upon
adding the PABA/NO to the buffer, the solution was immediately
analyzed using the above HPLC conditions. A second analysis was
performed after the solution had been incubated at 37 °C for 35
min.
(
R1) was 0.0629 for 1930 observed (I > 2σI) reflections and 0.1240
for all 3419 reflections using 271 parameters, and goodness-of-fit
was 1.020.
3
For 6c, a yellow needle of dimensions 0.04 × 0.06 × 0.36 mm
was mounted on glass fiber using a small amount of epoxy. Data
were collected on a Bruker three-circle diffractometer equipped with
a SMART 6000 CCD detector. The crystals were irradiated using
a rotating anode Cu KR source (λ ) 1.54178) with incident beam
G o¨ bel mirrors. Data collection was performed and the unit cell was
Anticancer Screen of PABA/NO Analogues against the NCI
5
1 Human Cancer Cell Lines. The anticancer screening test was
6,7
conducted as previously described. 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 PABA/NO analogues. 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
8
initially refined using SMART (v5.625). The crystals were triclinic,
P 1h , with a ) 11.2367(11) Å, b ) 12.8743(10) Å, c ) 25.143(2)
Å, R ) 89.451(5)°, â ) 88.172(6)°, γ ) 87.516(5)°, V ) 3631.9(6)
3
3
-1
Å , Z ) 8, Fcalc ) 1.483 Mg/m , µ ) 1.063 mm , and F(000) )
9
1672. Data reduction was performed using SAINT (v6.36A) and
XPREP (v6.12).10 Corrections were applied for Lorentz, polariza-
11
tion, and absorption effects using SADABS (v2.03). The structure
was solved and refined with the aid of the programs in the
SHELXTL-plus (v6.10) system of programs. The full-matrix least-
1
640 medium and added immediately to the microtiter plates to
-4
-5
-6
12
produce five concentrations of the analogues, i.e., 10 , 10 , 10
1
,
-
7
-8
2
0
, and 10 M. Each analogue was incubated with cells for 48
squares refinement on F included atomic coordinates and aniso-
h. At the end of the incubation, the cells were fixed in situ by 10%
trichloroacetic acid, washed five times with water, and dried.
Sulforhodamine B (0.4% in 1% acetic acid) was added to each
well and incubated 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
tropic thermal parameters for most non-H atoms. The H atoms were
included using a riding model. The final R value (R1) was 0.0916
for 5783 observed (I > 2σI) reflections and 0.2693 for all 10 961
reflections using 1130 parameters, and goodness-of-fit was 1.033.
Acknowledgment. This project was funded in part by the
Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research, by the National Cancer
Institute under Contract No. NO1-CO-12400, and by the
National Institute on Drug Abuse under Contract No. Y1-DA-
(
not shown) was created by plotting the percent growth against the
1
002, as well as NIH Grant R01 CA84496 and a Translational
log of the corresponding PABA/NO analogue’s concentrations for
each cell line and grouped by disease subpanels. The molar analogue
concentrations that cause 50% growth inhibition (GI50), total growth
Research Award to P.J.S. from the Leukemia and Lymphoma
Society.

1
164 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
SaaVedra et al.
Supporting Information Available: Complete crystal structure
of the Diazeniumdiolate Class with Potent Antineoplastic Activity.
Mol. Cancer Ther. 2003, 2, 409-417.
reports for 6a and 6c, as well as a figure showing the superposition
of distinct conformational orientations in crystalline 6c; IC50 data
for compounds 6a-d in the HL-60 and NIH3T3 cell line screens;
kinetic data used to derive the rate constants of Table 1; chemi-
luminescence traces confirming that NO was generated on reaction
of 6a-d with GSH. This material is available free of charge via
the Internet at http://pubs.acs.org.
(
4) 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.
(5) Houben, J.; Brassert, W. U¨ ber die Einwirkung von alkoholischem
Chlorwasserstoff auf m-Methylnitrosamin-benzoes a¨ ure. Chem. Ber.
1910, 43, 206-213.
(
6) 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
Cultured Human Tumor Cell Lines. J. Natl. Cancer Inst. 1991, 83,
References
(
1) 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.
757-766.
(
7) Jia, L.; Tomaszewski, J.; Jothianandan, D.; Furchgott, R. F.; Keefer,
L.; Shami, P. EDRF-Like Activity and Anticancer Spectrum of JS-
K, a Novel Diazeniumdiolate Nitric Oxide Donor. Proc. Am. Assoc.
Cancer Res. 2003, 44, 1059.
(
2) 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
(
(
8) SMART, v5.625; Bruker AXS Inc., Madison, WI, 2001.
9) SAINT, v6.36A; Bruker AXS Inc., Madison, WI, 2002.
10) XPREP, v6.12; Bruker AXS Inc., Madison, WI, 2001.
11) SADABS, v2.03; Bruker AXS Inc., Madison, WI, 2000.
12) SHELXTL, v6.10; Bruker AXS Inc., Madison, WI, 2000.
-
Ion R2N[N(O)NO] as Nucleophile and Leaving Group in SNAr
Reactions. J. Org. Chem. 2001, 66, 3090-3098.
(
(
(
(
3) 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
JM050700K