Diazeniumdiolated carbamates: A novel class of nitric oxide donors

Article abstract of DOI:10.1016/j.bmc.2012.01.046

We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.

Full text of DOI:10.1016/j.bmc.2012.01.046

Bioorganic & Medicinal Chemistry 20 (2012) 2025–2029
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Diazeniumdiolated carbamates: A novel class of nitric oxide donors
Rahul S. Nandurdikar a, , Anna E. Maciag b, Zhao Cao b, Larry K. Keefer a, Joseph E. Saavedra b,
⇑
⇑
a Drug Design Section, Chemical Biology Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA
b Basic Science Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates.
Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine dia-
zeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activa-
tion release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient
sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 December 2011
Revised 18 January 2012
Accepted 26 January 2012
Available online 4 February 2012
Keywords:
Nitric oxide
Diazeniumdiolate prodrugs
Diazeniumdiolated carbamates
TOM protecting group
Intracellular nitric oxide
1. Introduction
R1,R2
= alkyl
NO
+ R1R2NH2
(a)
Diazeniumdiolate prodrugs1 (1, Fig. 1) are considered as reliable
sources of the potent bioregulatory agent, nitric oxide2 (NO). These
prodrugs (1), on metabolic activation or hydrolysis, form the parent
anion (2), which further decomposes to release up to two moles of
NO and the parent amine (Fig. 1a). The therapeutic applications of
these prodrugs are diverse, and largely depend on the O-2 protect-
ing group (‘R’ in structure 1, Fig. 1) and its mechanism of activation.
For example, vinyl protected prodrug V-PYRRO/NO (3) is activated
by cytochrome P450 to release NO, and shows hepatoprotective
properties against a variety of toxins.3 Glutathione (GSH)-activated
arylated prodrug JS-K (4) is a lead anticancer agent.4 Recently, pri-
mary amine diazeniumdiolate prodrug AcOM-IPA/NO (5)5 was re-
ported to release nitroxyl (HNO), another potent bioeffector
molecule with possible applications in treating heart failure and
alcohol abuse.6
Secondary amine diazeniumdiolate ions are protonated at N-3
(see Fig. 1 for numbering) to release NO,1 whereas, primary amine
diazeniumdiolates release nitroxyl5 (HNO) on protonation at N-2.
Given the difference in product distribution between primary
and secondary amine diazeniumdiolate ions seen on hydrolyzing
them (mixed NO/HNO donors versus pure NO donors, respec-
tively), we speculated that diazeniumdiolated carbamates (6a)
and amides (6b) might hydrolyze with different and potentially
advantageous product distributions and/or kinetics (Fig. 1c). We
hydrolysis/
metabolic
trigger
3
R1
O
R2
R1
O
R2
N
N
N
(2 moles)
1
N
2
N
O
R1= alkyl
2 = H
N
O
1
2
R
HNO
R
O
O
N O
O
H
N
N
N
N
N
Ar
N
N
(b)
O
N
O
N
O
O
JS-K (4)
3
OAc
Ar = 2,4-dinitrophenyl
5
O
R1
O
N
N
R
pH 7.4
(c)
HNO
NO
or
?
N
O
6 (a) R = alkoxy; diazeniumdiolated carbamate
6 (b) R = alkyl; diazeniumdiolated amide
Figure 1. (a) Activation of diazeniumdiolate prodrugs to release NO or HNO (b)
Structures of, V-PYRRO/NO (3), JS-K (4) and AcOM-IPA/NO (5). (c) Hydrolysis of
diazeniumdiolated amides and carbamates (6).
have until now had no success in forming diazeniumdiolate ions
by reacting amides or other acylated amines directly with NO un-
der basic conditions, so we have resorted to an indirect method for
accessing a relevant prototype. Here we describe the synthesis of
prodrugs that can be used to probe the chemistry of anionic
⇑
Corresponding authors. Tel.: +1 301 846 1602; fax: +1 301 846 5946.
E-mail addresses: nandurdikarr@mail.nih.gov (R.S. Nandurdikar), saavedjo@
mail.nih.gov (J.E. Saavedra).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.046

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R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 2025–2029
presence of a 4-fold excess of 2-bromo-l- [((trifluoromethane)sul-
O
t
H
fonyl)oxy]ethane (10) and triethylamine (TEA) to obtain the re-
quired bromide (Scheme 2). This crude bromide on treatment
with Verkade’s superbase (11) gave the vinyl carbamate prodrug
12 in 34% yield (2 steps). Removal of silyl protection in the pres-
ence of bromomethyl acetate (13) and TEA gave esterase-labile
acetoxymethylated diazeniumdiolate prodrug 14. Similar depro-
tection in the presence of 2,4-dinitrofluorobenzene (15) gave ary-
lated prodrug 16 in 38% yield. To the best of our knowledge,
these are the first examples of diazeniumdiolates with a carbamate
functional group at N-3 (for atom numbering system, see structure
1, Fig. 1).
LiO Bu, Boc2O
H
Ref. [7]
Na
N
N
N
N
N
N
O
hexane, 0 °C,
71%
O
N
O
O
N
O
O
N
O
CH2OSi(iPr)3
7
8
CH2OSi(iPr)3
9
(IPA/NO)
Scheme 1. Synthesis of carbamate diazeniumdiolate 9.
i. TfOCH2CH2Br (10),
TBAF, TEA,
O
P
N
N
N
N
N
O
CH2Cl2, 0 °C
2.2. NO-release
9
9
O
N
O
ii. 11, CH3CN, rt,
N
11
It was important to identify and quantitate the NO and/or HNO
released on activation of these prodrugs. Chemiluminescence assay
was used to determine NO. After NO evolution had ceased, the
residual solution was further used for Griess’s assay in the pres-
ence of air (oxygen). The Griess’s assay was used to determine
amount of nitrite (NOÀ2 ) present in the solution. Nitrite is the oxi-
dation product of NO in aerobic aqueous solution. Quantitation
of HNO was carried out using quantitation of nitrous oxide
(N2O), a dimerization product of HNO, by gas chromatography.
The results are summarized in Table 1. Compound 14 on activation
by porcine liver esterase under physiological conditions for 5 days
gave 1.25 mol (63%) of NO, 0.06 mol (3%) of nitrite and an unde-
tectable amount of N2O. The decreased amount of NO measured
on hydrolysis of 14 may be attributed to one or more effects. Nitro-
sative inactivation of the enzyme could occur, ceasing hydrolysis
and resulting in protein nitrosation products not detectable by
Griess assay. Formation of N-nitrosocarbamate might also be ex-
pected. This product could further rearrange to the O-isopropyl
carbamate with expulsion of N2, a difficult product to measure.
Similarly, compound 16 on activation by GSH in PBS under physi-
ological conditions gave about 1.35 mol of NO, 0.32 mol of nitrite
and trace amount of N2O. Prodrug 16 also shows less than the cal-
culated amount of NO, probably due to similar reasons as specu-
lated for compound 14. Thus, these N-diazeniumdiolated
carbamates release NO via a pathway similar to that of their sec-
ondary amine counterparts as shown in Figure 2.
60 min, 34% (2 steps)
Verkade's base
12
O
TBAF, TEA,
N
O
N
BrCH2OAc (13),
O
N
O
CH2Cl2, 2 h, 0 °C,
O
14
31%
NO2
O
O
F
N
O
NO2
15
N
9
O
N
O
NO2
TBAF, CH2Cl2,
2 h, 0 °C, 38%
16
NO2
Scheme 2. Synthesis of carbamate diazeniumdiolate prodrugs 12, 14 and 16.
diazeniumdiolated carbamates as well as to generate analogues
that can be used for studies of their biological properties.
2. Results and discussion
2.1. Synthesis
2.3. Intracellular NO-release
We envisaged that the triisopropylsilyloxymethyl (TOM) pro-
tecting group strategy7 developed for O-2 substituted dia-
zeniumdiolate prodrugs might be useful for the synthesis of such
carbamate or amide derivatives. Thus, IPA/NO (7, Scheme 1) was
converted to TOM-ylated IPA/NO (8, Scheme 1) following the re-
ported procedure.7 This compound 8 on treatment with a base
and Boc-anhydride (Boc2O) gave carbamate 9. After experimenta-
tion with several bases and reaction conditions for this transforma-
tion, it was observed that compound 9 is efficiently formed by
using lithium tert-butoxide in hexane at 0 °C.
Next, we studied the intracellular NO release and cell perme-
ability of these new compounds. The intracellular NO was esti-
mated by using the nitric oxide-sensitive and commercially
available dye, 4-amino-5-methylamino-2’,7’-difluorofluorescein
diacetate (DAF-FM diacetate).8 Normal human skin fibroblast BJ-
5ta cells and U937 human leukemia cells were selected, and JS-K
(4) was taken as a positive control for the experiments. Both the
cell lines were pre-loaded with DAF-FM diacetate, followed by
treatment with DMSO solutions of prodrugs 14, 16 and JS-K. The
fluorescence measurements after 90 min provided estimates of
the levels of intracellular NO. It was observed that at equal concen-
trations, prodrug 16 showed significantly higher levels of NO as
With compound 9 in hand, it was important to remove the TOM
protecting group and introduce a biologically significant protecting
group helpful for site-directed delivery of NO. We treated com-
pound 9 with tetra-n-butylammonium fluoride (TBAF) in the
Table 1
Quantitation of NO, NO2À and N2O for prodrugs 14 and 16.
Prodrug
Activation conditions
NO mol/mol of prodruga (% yield)
NOÀ2 mol/mol of prodruga (% yield)
N2O mol/mol of prodrug
14
16
Porcine liver esterase in PBS at 37 °C
GSH (4 mM) in PBS at 37 °C
1.25 (63%) over 5 days
1.35 (68%) over 400 min
0.06 (3%)
0.32 (16%)
N.D.b
0.02
a
2 mol/mol prodrug = 100% yield.
N.D. = not detected.
b

R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 2025–2029
2027
NO by a mechanism similar to their secondary amine counterparts.
We have also demonstrated their ability to release NO inside cells.
The compounds synthesized have led to a new class of NO-donors
with potential biological applications.
O
O
H+
N
H
O
N
N
O
NO
+
O
N
O
18
17
4. Experimental
4.1. Synthesis
Figure 2. Proposed path for activation of diazeniumdiolated carbamate 17 to
release NO, the predominant hydrolysis product.
4.1.1. General
Starting materials were purchased from Aldrich Chemical Co.
(Milwaukee, WI) unless otherwise indicated. NMR spectra were re-
corded on a 400 MHz Varian UNITY INOVA spectrometer; chemical
shifts (d) are reported in parts per million (ppm) downfield from
tetramethylsilane. Ultraviolet (UV) spectra were recorded on an
Agilent Model 8453 or a Hewlett-Packard model 8451A diode array
spectrophotometer. High resolution mass spectra (HRMS) were re-
corded on Agilent 6250 series Accurate-Mass Q-TOF LC/MS by elec-
trospray ionization (ESI). Elemental analyses were performed by
Midwest Microlab (Indianapolis, IN). Chromatography was per-
formed on a Biotage SP1 Flash Purification System. Prepacked silica
gel flash chromatography columns were purchased from Yamazen
Science Inc. (San Bruno, CA). Compounds 87 and 109 were prepared
by using the reported procedures. Diethyl ether was used to dis-
solve and transfer all the liquid compounds from rotary evaporator
flask to smaller containers and sample vials.
4.1.2. Procedures and analytical data
Compound 9. Under N2, a 1 M solution of LiOtBu in hexane
(3.8 mL, 3.84 mmol) was added to an ice-cold solution of com-
pound 8 (1.06 g, 3.49 mmol) in hexane (10 mL). After 20 min, a
solution of Boc2O in hexane was added to this ice-cold reaction
mixture. After 20 min of stirring, the reaction was diluted with dis-
tilled water (10 mL) and hexane (10 mL). The organic layer was
washed with brine, dried over anhydrous Na2SO4 and evaporated.
The crude product was purified by flash column chromatography
(9:1 hexane/ethyl acetate) to give compound 9 as an oil (1.0 g,
71%). UV (ethanol) kmax (e
) 235 nm (5.9 mMÀ1 cmÀ1); 1H NMR
(CDCl3, 400 MHz) d 1.05–1.17 (m, 3H), 1.08 (d, J = 6.1 Hz, 18H),
1.32 (d, J = 6.7 Hz, 6H), 1.49 (s, 9H), 4.36 (septet, J = 6.7 Hz, 1H),
5.53 (s, 2H); 13C NMR (CDCl3, 100 MHz) d 151.42, 91.58, 83.53,
51.44, 27.96, 19.86, 17.64, 11.81. HRMS (ESI) m/z calculated for
C
18H43N4O5Si [M+NH4]+ 423.2997, found 423.2993. Anal. Calcd
for C18H39N3O5SiÁ0.5Et2O: C, 54.27; H, 10.02; N, 9.49, Found: C,
Figure 3. Levels of intracellular NO formation upon treatment of (a) BJ-5ta and (b)
U-937 cells with compounds (10 lM final concentration) and DMSO (control) as
54.36; H, 9.79; N, 9.66.
determined by DAF-FM diacetate fluorescence study.
Compound 12. To a solution of 9 (140 mg, 0.35 mmol), triethyl-
amine (TEA) (0.2 mL, 1.4 mmol) and 2-bromo-l-[((trifluorome-
thane)sulfonyl)oxy]ethane (10) (360 mg, 1.4 mmol) in CH2Cl2
(3 mL) was added tetrabutylammonium fluoride trihydrate (TBAF)
(133 mg, 0.42 mmol) in CH2Cl2 (2 mL) at 0 °C. Reaction was al-
lowed to attain rt over 2 h, then the reaction was diluted with 5%
sodium bicarbonate, and extracted with CH2Cl2. The combined or-
ganic layer was washed with brine, dried over anhydrous Na2SO4
and evaporated. The crude product was used for the next step
without further purification. This crude product was dissolved in
acetonitrile (2 mL) and a solution of Verkade’s superbase 11
(151 mg, 1.4 mmol) in acetonitrile (1 mL) was added at rt. After
60 min at rt, volatiles were evaporated and the crude product
was purified by flash column chromatography (9:1 hexane/ethyl
acetate) to give compound 12 as a colorless oil (29 mg, 34% for 2
compared to JS-K, while 14 showed a comparable fluorescence le-
vel as that of JS-K for BJ-5ta and U937 cells (Fig. 3). A reviewer has
pointed out that the DAF-FM assay is subject to false positive read-
ings under certain experimental settings. Nevertheless, we con-
clude that the present results are consistent with the view that
the prodrugs 14 and 16 are taken up by the cells and are activated
therein to release NO.
3. Conclusions
In summary, we have synthesized diazeniumdiolated carba-
mate prodrugs with established, biologically significant protecting
groups starting from TOM-ylated diazeniumdiolate. To the best of
our knowledge, this is the first report of diazeniumdiolated carba-
mate prodrugs. We have studied their activation mechanism and
measured the amount of NO released. These new prodrugs release
steps). UV (ethanol) kmax (e
) 258 nm (5.73 mMÀ1 cmÀ1); 1H NMR
(CDCl3, 400 MHz) d 1.33 (d, J = 6.7 Hz, 6H), 1.50 (s, 9H), 4.37 (sep-
tet, J = 6.7 Hz, 1H), 4.53 (dd, J = 6.6, 2.7 Hz, 1H), 4.97 (dd, J = 14.0,
2.7 Hz, 1H), 6.90 (dd, J = 14.0, 6.6 Hz, 1H); 13C NMR (CDCl3,
100 MHz) d 151.25, 148.27, 94.08, 83.98, 51.66, 27.94, 19.85.

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R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 2025–2029
Anal. Calcd for C10H19N3O4: C, 48.97; H, 7.81; N, 17.13, Found: C,
4.2.2. Griess assay
48.92; H, 7.73; N, 17.16.
Sample volume was measured after the chemiluminescence
Compound 14. To a solution of 9 (400 mg, 0.98 mmol), triethyl-
amine (TEA) (0.6 mL, 3.92 mmol) and bromomethyl acetate (13)
(0.4 mL, 3.92 mmol) in CH2Cl2 (9 mL) was added tetrabutylammo-
nium fluoride trihydrate (TBAF) (372 mg, 1.18 mmol) in CH2Cl2
(4 mL) at 0 °C. Reaction was allowed to attain rt over 2 h, then
the reaction was diluted with 5% sodium bicarbonate and extracted
with CH2Cl2. The combined organic layer was washed with brine,
dried over anhydrous Na2SO4 and evaporated. The crude product
was purified by flash column chromatography (9:1 hexane/ethyl
acetate) to give compound 14 as colorless oil (89 mg, 31%). UV
experiment in the presence of air (oxygen). 100
lL of the Griess re-
agent, 300 L of sample and 2.6 mL of deionized water were added
l
in a spectrophotometer cuvette. The mixture was incubated for
30 min at rt. Photometric reference sample was prepared by mix-
ing 100 lL of the Griess reagent and 2.9 mL of deionized water.
The absorbance of the nitrite-containing sample at 548 nm was
measured relative to the reference sample. Then absorbance read-
ings were converted to the nitrite concentrations according to cal-
ibration curve.
(ethanol) kmax
(e
) 247 nm (5.52 mMÀ1 cmÀ1); 1H NMR (CDCl3,
4.2.3. Gas Chromatography for N2O
400 MHz) d 1.32 (d, J = 6.7 Hz, 6H), 1.50 (s, 9H), 2.12 (s, 3H), 4.35
(septet, J = 6.7 Hz, 1H), 5.86 (s, 2H); 13C NMR (CDCl3, 100 MHz) d
168.99, 151.14, 87.24, 83.86, 51.61, 27.93, 20.64, 19.78. HRMS
(ESI) m/z calculated for C11H22N3O6 [M+H]+ 292.1503, found
292.1500.
Instrument: Shimadzu GC-2014
Detector: ECD (Electron capture detector)
Column: Restek ShinCarbon 80/100 Packed Column
Length = 2 m; ID = 2.0 mm;
Test condition: Injector temp = 250 °C, Detector temp = 250 °C;
Initial column temp = 90 °C; Final temp = 200 °C; Rate = 20 °C/
min; Final temp Hold = 1.1 min; Total program time = 6.7 min;
Carrier: He (UHP) = 30 mL/min.
Compound 16. To a solution of 9 (360 mg, 0.9 mmol) and
2,4-dinitrofluorobenzene (15) (170 mg, 0.9 mmol) in CH2Cl2
(9 mL) was added tetrabutylammonium fluoride trihydrate (TBAF)
(347 mg, 1.10 mmol) in CH2Cl2 (4 mL) at 0 °C. Reaction was al-
lowed to attain rt over 2 h, then the volatiles were evaporated
and the crude product was purified by flash column chromatogra-
phy (5:1 hexane/ethyl acetate) to give compound 16 as a yellow
4.3. Cell culture and intracellular NO-release
BJ-5ta: Human hTERT immortalized BJ-5ta skin fibroblasts were
obtained from the American Type Culture Collection (ATCC,
Manassas, VA) and maintained in Dulbecco’s Modified Eagle
Medium (DMEM) (Gibco, Invitrogen, Carlsbad, CA):199 medium
(Sigma) (4:1) supplemented with 10% fetal calf serum (Gemini
Bio-Products, Sacramento, CA), 100 U/mL penicillin and 2 mM
glutamine. The intracellular level of nitric oxide after diazeniumdi-
olate prodrug treatment was quantified using the NO-sensitive
solid (130 mg, 38%). UV (ethanol) kmax (e
) 248 nm (6.01 mMÀ1
cmÀ1) and 286 nm (7.29 mMÀ1 cmÀ1); 1H NMR (CDCl3, 400 MHz)
d 1.39 (d, J = 6.8 Hz, 6H), 1.53 (s, 9H), 4.43 (septet, J = 6.8 Hz, 1H),
7.73 (d, J = 9.2 Hz, 1H), 8.49 (ddd, J = 9.2, 2.7, 0.6 Hz, 1H), 8.90 (d,
J = 2.7 Hz, 1H); 13C NMR (CDCl3, 100 MHz) d 152.94, 150.94,
143.15, 129.00, 121.98, 118.79, 84.95, 52.50, 27.95, 19.86. HRMS
(ESI) m/z calculated for C14H23N6O8 [M+NH4]+ 403.1572, found
403.1578.
fluorophore
diacetate (DAF-FM diacetate; Invitrogen, Carlsbad, CA). BJ-5ta cells
growing on 96-well plates were loaded with 2.5 M DAF-FM diac-
4-amino-5-methylamino-2’,7’-difluorofluorescein
l
4.2. NO-release
etate in Hanks’ balanced salt solution (HBSS) at 37 °C and 5% CO2.
After 30 min of incubation the cells were rinsed with HBSS to re-
move excess probe, and fresh HBSS was added to the wells. The
4.2.1. Chemiluminescence
Calibration of the Sievers Nitric Oxide Analyzer (NOA), model
280i (Instruments Business Group, Boulder, CO) was performed
by injecting various volumes of known concentrations of NO in he-
lium (50 ppm, 500 ppm and 5%) certified standards into the reac-
tion chamber and recording the peak areas. Samples and reaction
chambers were incubated at 37 °C. The gas was sparged with argon
and swept into the chemiluminescence detector. Data were re-
corded using Agilent Chemstation software and processed using
Microsoft Excel.
compounds were added to the cells at 10 lM final concentration.
After 90-min incubation the fluorescence of the benzotriazole
derivative formed on DAF-FM’s reaction with aerobic NO was ana-
lyzed using a PerSeptive Biosystems CytoFluor 4000 microplate
reader with the excitation source at 485 nm and emission at
525 nm. The mean value of 16 independent experiments is re-
ported (see Table S1 in the Supplementary data).
U937: U937 cell lines were obtained from ATCC, Manassas, VA.
Cells were maintained in RPMI 1640 medium (Gibco, Invitrogen,
Carlsbad, CA) supplemented with 10% fetal calf serum (Gemini
Bio-Products, Sacramento, CA), 100 U/mL penicillin and 2 mM glu-
Esterase-activated: 3.0 mL of pH 7.4 buffer containing porcine li-
ver esterase (ammonium sulfate suspension, Sigma, 5KU) (0.55
lL)
and diethylenetriaminepentaacetic acid (DTPA, 50 M) was placed
l
tamine, at 37 °C and 5% CO2. U937 cells were loaded with 2.5 lM
into the reaction chamber of the NOA and then sparged for several
minutes with argon. A DMSO solution (10 mM) of the prodrug 14
(60 lL) was injected into the reaction chamber and nitric oxide re-
DAF-FM diacetate in HBSS at 37 °C and 5% CO2. After 30 min of
incubation the cells were collected by centrifugation, rinsed with
HBSS to remove excess probe, and resuspended in fresh HBSS.
lease was recorded. Total amount of NO released was determined
by integrating the area under the curve and applying a calibration
curve. The NO release is an average of three independent
experiments.
The compounds were added to the cells at 10 lM final concentra-
tion. After 90-min incubation the fluorescence of the benzotriazole
derivative formed on DAF-FM’s reaction with aerobic NO was ana-
lyzed using a PerSeptive Biosystems CytoFluor 4000 microplate
reader with the excitation source at 485 nm and emission at
525 nm. The mean value of 16 independent experiments is re-
ported (see Table S2 in the Supplementary data).
Glutathione (GSH)-activated: 3.0 mL of pH 7.4 buffer containing
GSH (3.6–3.9 mM) and DTPA (50
chamber of the NOA and then sparged for several minutes with ar-
gon. A DMSO solution (10 mM) of the prodrug 16 (100 L) was in-
lM) was placed into the reaction
l
jected into the reaction chamber and nitric oxide release was
recorded by Sievers NOA. Total amount of NO released was deter-
mined by integrating the area under the curve and applying a cal-
ibration curve. The NO release is an average of three independent
experiments.
Acknowledgments
This project has been funded with Federal funds from the Na-
tional Cancer Institute, National Institutes of Health, under con-
tract HHSN261200800001E and by the Intramural Research

R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 2025–2029
2. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol. Rev. 1991, 43, 109.
2029
Program of the NIH, National Cancer Institute, Center for Cancer
Research. We thank Dr. Sergey Tarasov and Ms. Marzena A. Dyba
of the Biophysics Resource in the Structural Biophysics Laboratory,
NCI-Frederick, for assistance with the high resolution mass spec-
trometry studies.
3. Liu, J.; Saavedra, J. E.; Lu, T.; Song, J.-G.; Clark, J.; Waalkes, M. P.; Keefer, L. K. J.
Pharmacol. Exp. Ther. 2002, 300, 18.
4. Maciag, A. E.; Saavedra, J. E.; Chakrapani, H. Anticancer Agents Med. Chem. 2009,
9, 798.
5. (a) Salmon, D. J.; Torres de Holding, C. L.; Thomas, L.; Peterson, K. V.; Goodman,
G. P.; Saavedra, J. E.; Srinivasan, A.; Davies, K. M.; Keefer, L. K.; Miranda, K. M.
Inorg. Chem. 2011, 50, 3262; (b) Andrei, D.; Salmon, D. J.; Donzelli, S.; Wahab, A.;
Klose, J. R.; Citro, M. L.; Saavedra, J. E.; Wink, D. A.; Miranda, K. M.; Keefer, L. K. J.
Am. Chem. Soc. 2010, 132, 16526.
Supplementary data
6. (a) Nagasawa, H. T.; Kawle, S. P.; Elberling, J. A.; DeMaster, E. G.; Fukuto, J. M. J.
Med. Chem. 1995, 38, 1865; (b) Paolocci, N.; Katori, T.; Champion, H. C.; St. John,
M. E.; Miranda, K. M.; Fukuto, J. M.; Wink, D. A.; Kass, D. A. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 5537.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2012.01.046.
7. Nandurdikar, R. S.; Keefer, L. K.; Saavedra, J. E. Chem. Commun. 2011, 47, 6710.
8. Wardman, F. Free Radical Biol. Med. 2007, 43, 995.
References and notes
9. Chi, D. Y.; Kilbourn, M. R.; Katzenellenbogen, J. A.; Welch, M. J. J. Org. Chem. 1987,
52, 658.
1. Keefer, L. K. ACS Chem. Biol. 2011, 6, 1147.