Synthesis, nitric oxide release, and anti-leukemic activity of glutathione-activated nitric oxide prodrugs: Structural analogues of PABA/NO, an anti-cancer lead compound

Article abstract of DOI:10.1016/j.bmc.2007.11.035

Diazeniumdiolate anions and their prodrug forms are reliable sources of nitric oxide (NO) that have generated interest as promising therapeutic agents. A number of structural analogues of O²-(2,4-dinitro-5-(4-(N-methylamino)benzoyloxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), an anti-cancer lead compound that is designed to release NO upon activation by glutathione, were prepared. The nitric oxide release patterns of these O²-(2,4-dinitrophenyl) diazeniumdiolates in the presence of glutathione were tested and it was found that in the absence of competing pathways, these compounds release nearly quantitative amounts of NO. The ability of PABA/NO and its structural analogues to inhibit human leukemia cell proliferation was determined and it was found that compounds releasing elevated amounts of NO displayed superior cytotoxic effects.

Full text of DOI:10.1016/j.bmc.2007.11.035

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2657–2664
Synthesis, nitric oxide release, and anti-leukemic activity
of glutathione-activated nitric oxide prodrugs: Structural
analogues of PABA/NO, an anti-cancer lead compound
Harinath Chakrapani,a,* Thomas C. Wilde,a Michael L. Citro,b Michael M. Goodblatt,a
Larry K. Keefera and Joseph E. Saavedrab,*
aChemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD 21702, USA
bBasic Research Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702, USA
Received 16 October 2007; revised 9 November 2007; accepted 13 November 2007
Available online 17 November 2007
Abstract—Diazeniumdiolate anions and their prodrug forms are reliable sources of nitric oxide (NO) that have generated interest as
promising therapeutic agents. A number of structural analogues of O2-(2,4-dinitro-5-(4-(N-methylamino)benzoyloxy)phenyl)
1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), an anti-cancer lead compound that is designed to release NO upon
activation by glutathione, were prepared. The nitric oxide release patterns of these O2-(2,4-dinitrophenyl) diazeniumdiolates in
the presence of glutathione were tested and it was found that in the absence of competing pathways, these compounds release nearly
quantitative amounts of NO. The ability of PABA/NO and its structural analogues to inhibit human leukemia cell proliferation was
determined and it was found that compounds releasing elevated amounts of NO displayed superior cytotoxic effects.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Nitric oxide (NO) prodrugs of the ionic diazeniumdio-
late class (1, Scheme 1) that spontaneously dissociate
at definitive rates to form NO are not only widely used
in biological studies but are also of interest as possible
therapeutic agents in the treatment of several disease
states, notably cancer.1–6 Due to the involvement of
NO in diverse physiological processes, site-directed
delivery of therapeutic nitric oxide is essential to avoid
any undesirable side-effects. Derivatization of the O2-
position of the diazeniumdiolate ion with protective
groups, converting them into substrates for specific en-
Scheme 1. Mechanism of nitric oxide release from O2-arylated
diazeniumdiolates that are metabolized by glutathione S-transferase.
zymes in a metabolic pathway, is one frequently used
strategy.7–11 The inactive prodrug circulates freely until
it encounters the specific protein, which then acts as a
O2-arylated diazeniumdiolates targeting the detoxifica-
tion enzyme glutathione S-transferase (GST), often
trigger, cleaving the protective group, resulting in selec-
tive accumulation of NO in that tissue. A successful
over-expressed in cancer tissue.12–14 The general mecha-
example of such an approach is the development of
nism of action of such prodrugs involves nucleophilic
aromatic substitution15 by glutathione (GSH), generat-
ing the nitric oxide-releasing diazeniumdiolate ion 1
(Scheme 1).16
Keywords: Nitric oxide; Glutathione; GST; Anti-cancer; Prodrugs;
PABA/NO.
*
Corresponding authors. Tel.: +1 301 846 1601; fax: +1 301 846 5946
(H.C.); tel.: +1 301 846 1602 (J.E.S.); e-mail addresses:
chakrah@ncifcrf.gov; saavj@ncifcrf.gov
Of the several isoforms of GST, GSTp is over-expressed
in many tumors, which makes it an attractive molecular
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.035

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H. Chakrapani et al. / Bioorg. Med. Chem. 16 (2008) 2657–2664
Scheme 3. Proposed synthetic intermediates
syntheses.
7 and 8 and their
Scheme 2. Original synthesis of PABA/NO (3).
derivatives (9–14) differing in the substitution pattern
in the aroate ester functionality were synthesized in
moderate to good yields from 7 or 8 (Table 1).
target for site-directed delivery of tumoristatic agents.17–20
Molecular modeling studies of the Meisenheimer com-
plex (Scheme 1) were used as a guide in the design of
GSTp-selective 2,4-dinitroaryl derivatives of DMA/NO
(structure 2, Scheme 2).12–14
Nitric oxide donors coupled with non-steroidal anti-
inflammatory drugs (NSAIDs) are assuming importance
as versatile drug candidates.21–23 These compounds have
shown to minimize the deleterious effects of traditional
NSAIDs, while retaining the potent anti-inflammatory
activity of the parent NSAIDs.21–23 PABA/NO ana-
logues (15–17) of non-sterodial anti-inflammatory drugs
(NSAIDs) such as aspirin, ibuprofen, and naproxen
were prepared by treating 8 with the corresponding acid
chloride (Scheme 4).
One such compound, PABA/NO (structure 3, Scheme
2), has shown tumoristatic activity against A2780 hu-
man ovarian cancer xenografts in female SCID mice
with a potency comparable to that of cisplatin.14 This
and other in vitro and in vivo activities established
PABA/NO to be a promising anti-cancer lead com-
pound.13 In an effort to identify drug candidates with
superior anti-cancer activity, we attempted to synthesize
and study structural analogues of PABA/NO.
In addition, several structurally similar compounds with
varying functional groups connecting the two aromatic
rings (18–22) were synthesized starting from 4, 7 or 8
(Scheme 5).
Initially, PABA/NO was prepared in 38% overall yield
by the reaction of 1,5-difluoro-2,4-dinitrobenzene (4)
with (N-methyl)-4-aminobenzoic acid (5) to form the
fluoride 6, which was then reacted with DMA/NO
(Scheme 2).13 The second step of this synthesis produced
a mixture of byproducts that rendered isolation of
PABA/NO cumbersome. Hence, devising an efficient
synthetic route to PABA/NO was desired.
The ambident nucleophilic character of 4-aminobenzoic
acid is noteworthy. In acetone, it was reported that 4-
aminobenzoic acid reacted through the amino group
Table 1. Synthesis of PABA/NO and its structural analogues
2. Results and discussion
In order to prepare a variety of PABA/NO analogues,
two synthetic intermediates, structures 7 and 8, of diver-
gent reactivity were envisaged (Scheme 3).
The fluoride 7, which was prepared by treatment of 4
with DMA/NO, is a candidate for reaction with nucleo-
philes (Scheme 3).12 The phenol 8, which can react with
electrophiles, was obtained by hydrolysis of 7 in 83%
yield (Scheme 3).13 This design exploits the opposing or-
ganic reactivity of compounds 7 and 8 and should per-
mit facile access to diverse PABA/NO analogues.
Reactant
Product no
W
Z
Yielda
7
7
7
7
8
8
8
3
9
NHMe
NMe2
NH2
NHAc
H
CH
CH
CH
CH
CH
N
79%b
66%b
64%b
75%b
34%c
44%c
71%c
10
11
12
13
14
H
NO2
CH
a Isolated yield.
b Prepared using the corresponding benzoic acid.
c Prepared using the corresponding aroyl chloride.
As predicted, the two compounds 7 and 8 served as con-
venient divergence points in the synthesis of numerous
PABA/NO analogues. PABA/NO and several ester

H. Chakrapani et al. / Bioorg. Med. Chem. 16 (2008) 2657–2664
2659
lone pair as the most nucleophilic species. However, in
the presence of a weak base such as triethylamine, it is
expected that ionization to the anionic form of 4-amino-
benzoic acid occurs. In such a situation, the carboxylate
ion is the dominant nucleophile, rationalizing the ob-
served outcome of these reactions of 4-aminobenzoic
acid.
PABA/NO and its analogues were independently treated
with GSH in 0.1 M, pH 7.4, phosphate buffer and NO
release was measured (Table 2). The yields of NO from
the ester analogues 9–17 were lower than that of PABA/
NO (Table 2). All other analogues, 18–22, were found to
release higher amounts of NO (P93%).
Under similar conditions but in the absence of GSH,
negligible amounts of NO were detected from 9–22. In
an earlier study, we reported that upon treatment with
Scheme 4. Synthesis of NSAID-based PABA/NO analogues.
a
range of nucleophiles, 2,4-dinitroarylated dia-
zeniumdiolates released NO, suggesting that arylated
diazeniumdiolates are susceptible to activation with
other nucleophiles to release nitric oxide.15
The sub-stoichiometric yield of NO suggests that a path-
way other than just nucleophilic aromatic substitution
by GSH to release DMA/NO ion is operational. In addi-
Scheme 6. Reactivity of 4-aminobenzoic acid under different reaction
conditions with aryl fluorides.
Table 2. Nitric oxide release and anti-leukemic activity for PABA/NO
and its structural analogues
Compound
% NO released
IC50 (lM)
PABA/NO, 3
9
73
30
63
20
15
4
3.9
>25
13
10
11
12
13
14
15
16
17
18
19
20
21
22
8
22
>25
>25
>25
>25
>25
>25
6.8
Scheme 5. Synthesis of some PABA/NO structural analogues.
0
7
to form 23 (tC@O = 1685 cmꢀ1), in two steps from 4
(Scheme 6).13 However, in the presence of triethylamine,
the major product of the reaction of 7 and 4-aminoben-
zoic acid (Scheme 6) was through the carboxylic acid
functionality to form the ester 10 (tC@O = 1742 cmꢀ1).
0
5
100
99
100
96
93
0
1.6
1.4
3.9
2.3
In a neutral organic solvent such as acetone, presumably
the carboxylic acid does not ionize, leaving the nitrogen
>25

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H. Chakrapani et al. / Bioorg. Med. Chem. 16 (2008) 2657–2664
active or inactive in the cell proliferation bioassay. Ta-
ken together, these observations indicate a role for nitric
oxide in the cytotoxic activity of these compounds.
In conclusion, we report the synthesis of a number of
O2-(2,4-dinitrophenyl) diazeniumdiolates that are de-
signed to be activated by glutathione to release nitric
oxide using several approaches. The nitric oxide release
patterns of these prodrugs in the presence of glutathione
were determined; in the absence of competing pathways,
nearly quantitative amounts of NO release were ob-
served. Their in vitro anti-leukemic activity indicates
that nitric oxide is an important component of cytotox-
icity. Finally, this study reveals structural patterns that
may assist in future design of improved nitric oxide-
based anti-cancer agents.
Scheme 7. Mechanism of nitric oxide release from PABA/NO.
tion to nitric oxide release, PABA/NO, in the presence
of GSH, is reported to undergo ester hydrolysis to form
8 as a byproduct (Scheme 7).13 Such a competitive path-
way appears to be pronounced in 9–17, leading to lower
yields of NO from these compounds in comparison with
that of PABA/NO.
3. Experimental
3.1. General
The compound 8 itself does not release appreciable
amounts of nitric oxide (Table 2), probably due to equil-
ibration to its phenolate ion 8a (Scheme 7) that is ex-
pected to resist reaction with nucleophilic species such
as GSH (pKa of 2,4-dinitrophenol is 4.09).24
NO was purchased from Matheson Gas Products
(Montgomeryville, PA). Starting materials were pur-
chased from Aldrich Chemical Co. (Milwaukee, WI) un-
less otherwise indicated. NMR spectra were obtained in
chloroform-d, DMSO-d6, or acetone-d6 on a Varian
UNITY INOVA spectrometer; chemical shifts (d) are
reported in parts per million (ppm) downfield from tet-
ramethylsilane. NMR spectra for publication were pre-
pared using the software MestRe Nova (Mestrelab
Research, Spain). Quantification of NO after treatment
of compounds with glutathione (GSH) was carried out
by chemiluminescence with a Thermal Energy Analyzer
Model 502A instrument (Thermedics, Inc., Woburn,
MA) or a Sievers 280i Nitric Oxide Analyzer (Boulder,
CO).12 Ultraviolet (UV) spectra were recorded on an
Agilent Model 8453 or a Hewlett-Packard model
8451A diode array spectrophotometer. Infrared spectra
were recorded on M500 IR spectrometer (Buck Scien-
tific, East Norwalk, CT). Elemental analyses were per-
formed by Midwest Microlab (Indianapolis, IN).
Sodium 1-(N,N-dimethylamino)diazen-1-ium-1,2-dio-
late (DMA/NO, 2) was prepared as described earlier.4
O2-(2,4-Dinitro-5-fluorophenyl) 1-(N,N-dimethylamino)-
From the higher amounts of NO released from 18–22, it
is inferred that an amine, amide, sulfonate, or ether lin-
ker is less labile to cleavage under these conditions. Fu-
ture work will focus on preparing analogues with such
linkages that are resistant to hydrolysis in the presence
of GSH.
Next, PABA/NO and its analogues were tested for their
ability to inhibit leukemia cell proliferation and IC50
values were determined (Table 2). It was found that in
addition to PABA/NO, the compounds 18–22 were po-
tent inhibitors of cell proliferation. When the % NO re-
leased upon treatment with GSH and IC50 values of
PABA/NO and its analogues were plotted, a noteworthy
trend emerged (Fig. 1). Compounds that released ele-
vated amounts of nitric oxide (>70%) were found to dis-
play potent anti-proliferative activity (<10 lM).
Furthermore, all compounds in this study that released
negligible amounts of NO were found to be moderately
diazen-1-ium-1,2-diolate
7
and O2-(2,4-dinitro-5-
hydroxyphenyl) 1-(N,N-dimethylamino)diazen-1-ium-
1,2-diolate 8 were prepared by reported methods.12
3.2. General synthetic procedures
Unless otherwise specified, these were the general meth-
ods used for the synthesis of PABA/NO analogues.
Method A: Ester formation. A solution or a partial solu-
tion of 1.5 mmol of the aryl carboxylic acid in dichloro-
methane (65 mL) was treated with triethylamine (2
molar excess) giving a homogeneous solution. To this
was added a solution of 7 (420 mg, 1.45 mmol) in dichlo-
romethane (65 mL) and the mixture was stirred at room
temperature. The progress of the reaction was moni-
tored by TLC and after 72 h, the solution was washed
with 5% sodium bicarbonate, then with 1 M HCl, dried
Figure 1. Variation of observed cytotoxic effects with % NO released
upon activation by GSH by PABA/NO and its analogues.

H. Chakrapani et al. / Bioorg. Med. Chem. 16 (2008) 2657–2664
2661
(sodium sulfate), filtered and evaporated to give a solid
that was recrystallized from ethanol or from dichloro-
methane:ether. Unless otherwise indicated, no chroma-
tography was required.
ing to Method B. The crude product was recrystallized
from ethanol to give a 66% yield of 9: mp 160–161 °C;
UV kmax (e) 229 nm (29.1 mMꢀ1 cmꢀ1), 320 nm
1
(47.5 mMꢀ1 cmꢀ1); H NMR (CDCl3) d 3.11 (s, 6 H),
3.27 (s, 6H), 6.71 (d, 2H, J = 9.2 Hz), 7.55 (s, 1H),
8.03 (d, 2H, J = 9.2 Hz), 8.90 (s, 1H); 13C NMR d
40.1, 41.8, 110.9, 113.4, 124.6, 132.7, 133.4, 135.9,
150.0, 153.9, 154.3, 163.4. Anal. Calcd for
C17H18N6O8Æ0.05 EtOH: C, 47.02; H, 4.23; N, 19.24.
Found: C, 46.59; H, 4.17; N, 18.85.
Method B: Alternate method for ester formation. To a
solution of 7 (151 mg, 0.52 mmol) in acetonitrile
(6 mL), a solution of 0.53 mmol of the aryl carboxylic
acid and a slight molar excess of triethylamine in aceto-
nitrile (4 mL) were added and the resulting solution was
stirred at room temperature. A precipitate formed with-
in 30 min of reaction and the resulting slurry was stirred
overnight. The reaction mixture was diluted with water,
and the yellow precipitate was collected by filtration and
recrystallized from ethanol afforded pure material.
3.2.3. O2-(2,4-Dinitro-5-(4-(amino)benzoyloxy)phenyl) 1-
(N,N-dimethylamino)diazen-1-ium-1,2-diolate (10).
A
solution of 7 in acetonitrile was treated with aminoben-
zoic acid and triethylamine according to Method B. The
crude product was recrystallized from ethanol to give a
Method C: Acylation reaction. To a solution of 8
(96 mg, 0.33 mmol) in dichloromethane (5 mL), triethyl-
amine (56 lL, 0.44 mmol) was added. To this an equi-
molar amount of the acid chloride in dichloromethane
(5 mL) was added and the mixture was stirred at room
temperature for 20 min to 2 h while being monitored
by TLC. The reaction mixture was diluted with dichlo-
romethane (20 mL), then washed with 1 M HCl and
subsequently with 5% sodium bicarbonate. The organic
layer was dried over sodium sulfate, filtered through
magnesium sulfate and evaporated to give either a pow-
der or a glass. The powder was normally recrystallized
from mixtures of ethanol and dichloromethane, while
the glassy products were dissolved in ether and allowed
to crystallize slowly on standing.
64% yield of 10: mp 160–1601 °C; IR (film) 1742 cmꢀ1
;
1H NMR (CDCl3) d 3.27 (s, 6H), 4.30 (broad, 2H),
6.70–6.72 (m, 2H), 7.53 (s, 1H), 7.98–8.00 (m, 2H),
8.91 (s, 1H); 13C NMR d 41.8, 113.4, 113.9, 116.3,
128.6, 133.1, 135.8, 149.8, 152.4, 152.5, 154.0, 163.1.
Anal. Calcd for C15H14N6O8: C, 44.34; H, 3.47; N,
20.68. Found: C, 43.90; H, 3.45; N, 20.97.
3.2.4. O2-(2,4-Dinitro-5-(4-(N-acetylamino)benzoyloxy)-
phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate
(11). This product was obtained in 75% yield from the
reaction of 4-acetamidobenzoyl chloride and 8 as de-
scribed in Method C: mp 151–2 °C; UV (ethanol) kmax
(e) 292 nm (46.1 mMꢀ1 cmꢀ1); 1H NMR (acetone-
d6)
d 2.25 (s, 3H), 3.28 (s, 6H), 7.43 (broad, 1H),
7.56 (s, 1H), 7.73 (d, 2H, J = 8.8 Hz), 8.17–8.19 (m,
2H), 8.96 (s, 1H); 13C NMR d 24.9, 41.8, 113.3, 119.0,
122.7, 124.9, 132.2, 133.9, 135.5, 143.7, 149.3, 154.1,
162.8, 168.5. Anal. Calcd for C17H16N6O9: C, 45.54;
H, 3.60; N, 18.74. Found: C, 45.41; H, 3.58; N, 18.45.
Method D: Arylether formation. To a partial solution of
7 (50 mg, 0.17 mmol) in tert-butanol (2 mL), an equiva-
lent of the phenol in 0.1 M NaOH (2 mL) was added.
The slurry was stirred at room temperature overnight
and diluted with water; the resulting yellow precipitate
was collected by filtration and recrystallized from
ethanol.
3.2.5. O2-(2,4-Dinitro-5-(benzoyloxy)phenyl) 1-(N,N-
dimethylamino)diazen-1-ium-1,2-diolate (12). A solution
of 8 in dichloromethane was treated with benzoyl chlo-
ride as described in Method C to give a 34% yield of 12:
mp 130–1 °C; UV (ethanol) kmax (e) 232 nm
(16.9 mMꢀ1 cmꢀ1), 293 nm (8.2 mMꢀ1 cmꢀ1); 1H
NMR (CDCl3) d 3.28 (s, 6H), 7.54 (s, 1H), 7.57–7.59
(m, 2H), 7.70–7.74 (m, 1H), 8.19–8.22 (m, 2H), 8.96 (s,
1H); 13C NMR d 41.8, 113.3, 124.9, 127.6, 128.9,
130.7, 133.9, 134.8, 149.3, 154.1, 163.5. Anal. Calcd
for C15H13N5O8: C, 46.04; H, 3.35; N, 17.90. Found:
C, 45.76; H, 3.34; N, 17.59.
Method E: Amide formation. To a solution of 1.6 g
(11 mmol) of N-substituted benzamide in anhydrous
THF, cooled to ꢀ80 °C, lithium diisopropyl amide
(LDA, 1.5 M in hexane, 8.5 mL, 12.8 mmol) was added.
After 1 h, this solution, by syringe, was added to 7
(2.1 g, 10.1 mmol), also dissolved in anhydrous THF,
and cooled to ꢀ80 °C. After an additional hour, sol-
vents were removed and the remaining solids were ex-
tracted with dichloromethane and washed with dilute
HCl and brine, then dried (Na2SO4 and MgSO4), fil-
tered, and flash chromatographed (1:1 hexanes:ethyl
acetate) to obtain pure material.
3.2.6. O2-(2,4-Dinitro-5-(nicotinoyloxy)phenyl 1-(N,N-
dimethylamino)diazen-1-ium-1,2-diolate (13). A solution
of 8 in dichloromethane was treated with two equiva-
lents of triethylamine followed by acylation with one
equivalent of nicotinoyl chloride hydrochloride as de-
scribed in Method C; recrystallization of the resulting
solid from ether afforded 13 (44% yield): mp 123–
3.2.1. O2-(2,4-Dinitro-5-(4-(N-methylamino)benzoyloxy)-
phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate
(PABA/NO, 3). Prepared using method A in 79% yield.
Its physicochemical properties matched those described
previously.
124 °C;
UV
(ethanol)
kmax
(e)
264 nm
(36.5 mMꢀ1 cmꢀ1), 299 nm (33.7 mMꢀ1 cmꢀ1); 1H
NMR (CDCl3) d 3.30 (s, 6H), 7.54 (s, 1H), 7.53–7.56
(m, 1H), 8.46–8.49 (m, 1H), 8.93–8.94 (m, 1H), 8.99 (s,
1H), 9.41 (s, 1H); 13C NMR d 41.8, 110.6, 121.1,
121.3, 122.4, 131.6, 132.6, 148.9, 151.6, 152.1, 159.7,
3.2.2. O2-(2,4-Dinitro-5-(4-(N,N-dimethylamino)benzoyl-
oxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-dio-
late (9). A solution of 7 in acetonitrile was treated with
4-dimethylaminobenzoic acid and triethylamine accord-

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H. Chakrapani et al. / Bioorg. Med. Chem. 16 (2008) 2657–2664
162.4. Anal. Calcd for C14H12N6O8: C, 42.86; H, 3.08;
N, 21.42. Found: C, 42.64; H, 3.12; N, 21.11.
3.2.11. O2-(2,4-Dinitro-5-(4-(hydroxymethyl)phenyl)ami-
nophenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-dio-
late (18). To a solution of 7 (1.9 g, 6.5 mmol) in 40 mL
of THF/acetone (1:1), 0.81 g (6.6 mmol) of 4-aminoben-
zyl alcohol in acetone (15 mL) was added. The reaction
was stirred at room temperature for 48 h and progress
was monitored by TLC. Solvents were evaporated under
vacuum and the resulting solids were taken up in dichlo-
romethane. This solution was was washed with 5% so-
dium bicarbonate, dried over sodium sulfate, filtered
and evaporated. Recrystallization from ethanol gave a
79% yield of 18: mp 137–140 °C; UV (ethanol) kmax (e)
304 nm (26.1 mMꢀ1 cmꢀ1); 1H NMR (DMSO-d6) d
3.02 (s, 6H), 4.54 (d, 2H, J = 5.7 Hz), 5.28 (t, 1H,
J = 5.7 Hz), 6.87 (s, 1H), 7.36 (d, 2H, J = 8.4 Hz), 7.45
(d, 2H, J = 8.4 Hz), 8.91 (s, 1H), 10.16 (s, 1H); 13C
NMR (DMSO-d6) d 40.9, 62.3, 100.2, 125.7, 126.1,
126.4, 127.3, 127.5, 136.0, 141.7, 147.4, 154.1. Anal.
Calcd for C15H16N6O7: C, 45.92; H, 4.11; 21.42. Found:
C, 45.95; H, 4.05; N, 21.19.
3.2.7. O2-(2,4-Dinitro-5-(4-(nitrobenzoyl)oxy)phenyl) 1-
(N,N-dimethylamino)diazen-1-ium-1,2-diolate (14). This
compound was obtained in 71% yield from 8 and
p-nitrobenzoyl chloride as described in Method C.
The product was recrystallized from ether:dichloro-
methane: mp 133–134 °C; UV (ethanol) kmax (e)
261 nm (35.7 mMꢀ1 cmꢀ1), 299 nm (20.9 mMꢀ1
cmꢀ1); 1H NMR (CDCl3) d 3.29 (s, 6H), 7.55 (s,
1H), 8.40 (m, 4H), 8.99 (s, 1H); 13C NMR d 41.7,
113.1, 124.0, 125.1, 131.9, 132.9, 134.3, 135.0, 148.5,
151.5, 154.3, 161.9. Anal. Calcd for C15H12N6O10:
C, 41.29; H, 2.77; N, 19.26. Found: C, 41.28; H,
2.80; N, 19.09.
3.2.8. O2-(2,4-Dinitro-5-(2-acetoxybenzoyl)oxy)phenyl)
1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (15). A
solution of 8 in dichloromethane was acylated with the
acid chloride of acetylsalicylic acid containing a slight
molar excess of triethylamine according to Method C
to give a 66% yield of 15: mp 128–9 °C; UV (ethanol)
kmax (e) 235 nm (21.8 mMꢀ1 cmꢀ1), 293 nm
3.2.12. O2-(2,4-Dinitro-5-(N-benzoyl-N-methylamino)
phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate
(19). To a slurry of 2 (1.5 g, 12.0 mmol) in anhydrous
THF (30 mL) at 0 °C, 24 (1.3 g, 4.1 mmol) dissolved
in anhydrous THF (25 mL) was added. After 1 h, sol-
vents were removed and solids were extracted with
dichloromethane and brine. The organic layers were
dried and filtered, and solvents were removed. Recrys-
tallization from ethanol gave 19 (640 mg, 1.6 mmol,
39% yield): mp 120–123 °C; UV (ethanol) kmax (e)
1
(11.9 mMꢀ1 cmꢀ1); H NMR (CDCl3) d 2.30 (s, 3H),
3.29 (s, 6H), 7.21–7.24 (m, 1H), 7.42–7.46 (m, 1H),
7.49 (s, 1H), 7.71–7.75 (m, 1H), 8.25–8.26 (dd, 1H,
J = 1.8 Hz, J = 8.0 Hz), 8.95 (s, 1H); 13C NMR d 21.0,
41.7, 113.5, 120.7, 124.2, 124.9, 126.4, 132.4, 134.1,
135.4, 135.8, 148.8, 151.7, 154.2, 160.9, 169.5. Anal.
Calcd for C17H15N5O10: C, 45.44; H, 3.36; N, 15.59.
Found: C, 45.28; H, 3.33; N, 15.29.
1
288 nm (20.8 mMꢀ1 cmꢀ1); H NMR (CDCl3) d 3.26
(s, 6H), 3.46 (s, 3H), 7.28–7.32 (m, 2H), 7.37–7.41 (m,
4H), 8.66 (s, 1H); 13C NMR d 38.6, 41.7, 118.0,
124.6, 128.0, 128.3, 131.1, 138.9, 144.3, 151.2, 170.3.
Anal. Calcd for C16H16N6O7: C, 47.53; H, 3.99; N,
20.78. Found: C, 47.77; H, 4.10; N, 20.41.
3.2.9. O2-(2,4-Dinitro-5-(2-(4-(2-methylpropyl)phenyl)
propionoyloxy)phenyl) 1-(N,N-dimethylamino)diazen-1-
ium-1,2-diolate (16). This adduct was prepared in
85% yield from the reaction of 8 with the acid chlo-
ride of 4-isobutyl-a-methyl phenylacetic acid as de-
scribed in Method C: mp 121–122 °C; UV (ethanol)
3.2.13. O2-(2,4-Dinitro-5-(N-benzoyl-N-ethylamino)phenyl)
1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (20). To a
slurry of 2 (1.1 g, 8.8 mmol) in anhydrous THF
(20 mL) at 0 °C, 25 (0.9 g, 2.8 mmol) dissolved in anhy-
drous THF (15 mL) was added. After 2 h, solvents were
removed and solids were extracted with dichlorometh-
ane and brine. The organic layer was dried and filtered,
and the solvents were removed. Recrystallization from
ethanol gave 20 (670 mg, 1.6 mmol, 57% yield): mp
129–130 °C; UV (ethanol) kmax (e) 288 nm
(20.8 mMꢀ1cmꢀ1); 1H NMR (CDCl3) d 1.28 (t, 3H,
J = 7.1), 3.27 (s, 6H), 3.94 (broad, 2H), 7.29–7.41 (m,
6H), 8.65 (s, 1H); 13C NMR d 13.3, 41.7, 46.4, 118.1,
124.6, 127.8, 128.5, 134.3, 134.4, 139.5, 142.9, 153.0,
170.0. Anal. Calcd for C17H18N6O7: C, 48.81; H, 4.34;
N, 20.09. Found: C, 48.75; H, 4.35; N, 19.94.
1
kmax (e) 297 nm (24.4 mMꢀ1 cmꢀ1); H NMR (CDCl3)
d
0.91 (dd, 6H, J = 0.8, 6.7 Hz), 1.67 (d, 3H,
J = 7.2 Hz), 1.87 (septet, 1H, J = 6.9 Hz), 2.48 (d,
2H, J = 7.2 Hz), 3.24 (s, 6H), 4.06 (q, 1H,
J = 7.2 Hz), 7.15–7.18 (m, 2H), 7.20 (s, 1H), 7.29–
7.31 (m, 2H), 8.87 (s, 1H); 13C NMR d 18.3, 22.4,
30.2, 41.7, 45.0, 45.2, 113.0, 124.8, 127.5, 129.6,
134.1, 135.7, 141.3, 149.0, 154.4, 171.3. Anal. Calcd
for C21H25N5O8: C, 53.05; H, 5.30; N, 14.23. Found:
C, 52.69; H, 5.20; N, 14.37.
3.2.10. O2-(2,4-Dinitro-5-(2-(6-methoxynaphth-2-yl)pro-
pionoyloxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-
1,2-diolate (17). Method C was followed in the synthe-
sis of 17: mp 118–119 °C; UV (ethanol) kmax (e)
299 nm (24.4 mMꢀ1cmꢀ1); 1H NMR (CDCl3) d 1.76
(d, 3H, J = 7.2 Hz), 3.17 (s, 6H), 3.93 (s, 3H), 4.22
(q, 1H, J = 7.2 Hz), 7.15–7.18 (m, 3H), 7.47–7.50 (m,
1H), 7.73–7.77 (m, 3H), 8.87 (s, 1H); 13C NMR
(CDCl3) d 18.2, 41.7, 45.5, 55.4, 77.2, 105.6, 113.0,
119.3, 124.8, 126.2, 126.5, 128.9, 129.3, 133.6, 134.0,
135.4, 149.0, 154.0, 158.0, 171.3; Anal. Calcd for
C22H21N5O9 Æ 0.25H2O: C, 52.43; H, 4.30; N, 13.90.
Found: C, 52.24; H, 4.24; N, 14.06.
3.2.14. O2-(2,4-Dinitro-5-(4-(N-acetylamino)benzenesul-
fonyl)oxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-
1,2-diolate (21). To a solution of acetylsulfanilyl chloride
(145 mg, 0.62 mmol) in acetone (7 mL), a solution of 8
(178 mg, 0.62 mmol) in 0.1 M sodium hydroxide
(7 mL). The resulting homogeneous solution was stirred
at room temperature and gradually became cloudy.
After stirring overnight, the product was collected by fil-

H. Chakrapani et al. / Bioorg. Med. Chem. 16 (2008) 2657–2664
2663
tration, washed with water followed by ether, and al-
lowed to dry to form a yellow powder that was recrystal-
lized from acetone:dichloromethane to afford 21
(193 mg, 0.4 mmol, 64%): mp 138–139 °C; UV (ethanol)
to quantify the amount of NO released/mol of
compound.
3.4. HL-60 cell culture and growth assay
kmax
(e)
280 nm
(28.2 mMꢀ1cmꢀ1),
338 nm
(18.5 mMꢀ1cmꢀ1); 1H NMR (acetone-d6) d 2.17 (s,
3H), 3.26 (s, 6H) , 7.36 (s, 1H), 7.86–7.88 (m, 2H),
7.95–7.97 (m, 2H), 8.82 (s, 1H), 9.79 (broad, 1H); 13C
NMR (acetone-d6) d 24.5, 41.7, 113.7, 119.7, 119.8,
125.5, 127.6, 131.2, 146.6, 146.9, 154.3, 169.9, 170.0.
Anal. Calcd for C16H16N6O10S: C, 39.67; H, 3.33; N,
17.35; S, 6.62. Found: C, 39.42; H, 3.24; N, 16.95; S,
6.97.
Human myeloid leukemia HL-60 cells (American Type
Culture Collection, Manassas, VA) were cultured in
RPMI 1640 with 10% fetal bovine serum at 37 °C in a
5% CO2-humidified atmosphere. Cells were plated in
96-well plates at a concentration of 10,000 cells in
100 lL. Stock DMSO solutions of each compound were
serially diluted in phosphate-buffered saline (PBS) be-
fore addition to cultures with a final DMSO concentra-
tion in the cultures of 0.5%. Agents were added after
24 h of culture initiation. Three days after agent addi-
tion, the number of viable cells was determined using
the MTS assay (CellTiter 96, Promega, Madison, WI)
according to the manufacturer’s protocol. IC50 values
were calculated by nonlinear fitting of the absorbance/
concentration data to the following equation,
3.2.15. O2-(2,4-Dinitro-5-(4-(acetamido)phenoxy)) 1-
(N,N-dimethylamino)diazen-1-ium-1,2-diolate (22).
A
solution of 7 in tert-butanol was reacted with 4-acet-
amidophenol in 0.1 M NaOH according to Method D.
The product was recrystallized from ethanol to give a
75% yield of 22: mp 159–160 °C; UV (acetonitrile) kmax
(e) 224 nm (26.6 mMꢀ1 cmꢀ1), 281 nm (18.9 mMꢀ1
y ¼ Amin þ ðAmax ꢀ AminÞ=ð1 þ 10ðLog IC
Þ
50ꢀxÞꢁHS
1
cmꢀ1); H NMR (acetone-d6) d 2.21 (s, 3H), 3.11 (s,
6H), 7.06 (s, 1H), 7.25–7.28 (m, 2H), 7.81 (d, 2H,
J = 8.7 Hz), 8.86 (s, 1H), 9.35 (broad, 1H); 13C NMR
(acetone-d6) d 23.6, 41.0, 105.7, 121.1, 121.2, 125.6,
131.0, 133.7, 138.3, 149.2, 154.6, 157.2, 168.4. Anal.
Calcd for C16H16N6O8 Æ 0.5 H2O: C, 44.76; H, 3.99;
19.57. Found: C, 44.57; H, 3.81; N, 19.46.
where Amin is the (background) absorbance observed
with no living cells, Amax is the (control) absorbance
with no compound added, Log IC50 is the logarithm
of the IC50, HS is the Hill slope, and x is the concentra-
tion of the compound in the culture medium. Alterna-
tively, the IC50 values were calculated using the fitting
program available in Sigma Plot.
3.2.16. 2,4-Dinitro-1-fluoro-5-(N-benzoyl-N-methylamino)-
benzene (24). N-Methylbenzamide was reacted with 4
according to Method E to afford 24 (43% yield): mp
153–156 °C; 1H NMR (acetone-d6) d 3.54 (s, 3H),
7.34–7.50 (m, 5H), 8.02 (d, 1H, J = 11.5 Hz), 8.72 (d,
1H, J = 7.5 Hz); 13C NMR (acetone-d6) d 38.9, 120.6,
125.0, 128.9, 129.3, 131.6, 135.3, 142.5, 145.5, 157.0,
159.7, 170.5. Anal. Calcd for C14H10FN3O5: C, 52.67;
H, 3.16; 13.16. Found: C, 52.52; H, 3.18; N, 13.01.
Acknowledgments
This research was supported in part by the Intramural
Research Program of the NIH, National Cancer Insti-
tute, Center for Cancer Research, as well as by National
Cancer Institute contract N01-CO-12400 to SAIC. The
authors thank Ms. Janet R. Fields, NCI, for invaluable
support with the biological assay.
3.2.17. 2,4-Dinitro-1-fluoro-5-(N-benzoyl-N-ethylamino)
benzene (25). N-Ethylbenzamide25 was reacted with 4
according to Method E to afford 25 (29% yield): mp
Supplementary data
113–114 °C; 1H NMR (CDCl3)
d 1.29 (t, 3 H,
J = 7.1 Hz), 3.96 (q, 2 H, J = 7.0 Hz), 7.32–7.46 (m,
6H), 8.70 (d, 1H, J = 7.5 Hz); 13C NMR (CDCl3) d
13.5, 46.6, 119.4, 124.5, 127.7, 128.7, 131.2, 134.0,
141.7, 143.6, 156.0, 158.7, 170.2. Anal. Calcd for
C15H12FN3O5: C, 54.06; H, 3.63; 12.61. Found: C,
53.94; H, 3.62; N, 12.51.
NMR spectra for compounds 10–22. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2007.11.035.
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