Structural modifications modulate stability of glutathione-activated arylated diazeniumdiolate prodrugs

Article abstract of DOI:10.1016/j.bmc.2012.02.045

JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical development as an anti-cancer drug candidate. This prodrug was designed to be activated by glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH activation and the drugs' biological activity. We show that nitro-to-cyano substitution increases the half-life of the prodrug in the presence of GSH without compromising the compound's in vivo antitumor activity.

Full text of DOI:10.1016/j.bmc.2012.02.045

Bioorganic & Medicinal Chemistry 20 (2012) 3094–3099
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural modifications modulate stability of glutathione-activated arylated
diazeniumdiolate prodrugs
Rahul S. Nandurdikar a, , Anna E. Maciag b, Ryan J. Holland a, Zhao Cao b, Paul J. Shami c,
⇑
Lucy M. Anderson a, 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
c Division of Hematology and Hematologic Malignancies, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
JS-K, a diazeniumdiolate-based nitric oxide (NO)-releasing prodrug, is currently in late pre-clinical
development as an anti-cancer drug candidate. This prodrug was designed to be activated by
glutathione (GSH) to release NO. To increase the potency of JS-K, we are investigating the effect of
slowing the reaction of the prodrugs with GSH. Herein, we report the effect of replacement of nitro
group(s) by other electron-withdrawing group(s) in JS-K and its homo-piperazine analogues on GSH
activation and the drugs’ biological activity. We show that nitro-to-cyano substitution increases the
half-life of the prodrug in the presence of GSH without compromising the compound’s in vivo anti-
tumor activity.
Received 23 December 2011
Revised 13 February 2012
Accepted 20 February 2012
Available online 9 March 2012
Keywords:
Nitric oxide
Diazeniumdiolate prodrugs
JS-K
Ó 2012 Elsevier Ltd. All rights reserved.
Glutathione
Anti-cancer agents
1. Introduction
2. Results and discussion
JS-K (1, Fig. 1), an arylated diazeniumdiolate prodrug, is cur-
rently in late pre-clinical development as an anti-cancer drug can-
didate.1–5 JS-K was designed to be activated by glutathione (GSH)
to release nitric oxide (NO), a potent bioregulatory agent. Struc-
ture–activity relation (SAR) studies have shown that a homo-piper-
azine analogue of JS-K, compound 2 (Fig. 1), exhibits almost
identical in vitro activity and better in vivo efficacy.5 Recently,
we have shown that JS-K and 2 are potent cytotoxic agents against
human non-small cell lung cancer (NSCLC) cell lines both in vitro
and as xenografts in mice.4,5
To improve on these lead compounds, we are currently seeking
to design JS-K analogues with diminished uncatalyzed reactivity
towards GSH, which may subsequently improve their anti-cancer
properties. The aryl group ‘caging’ the NO-generating diazeniumdi-
olate moiety is a key structural feature that controls this reaction.
Herein, we report synthesis and evaluation of arylated dia-
zeniumdiolate prodrugs with several possible combinations of
electron-withdrawing groups.
2.1. Synthesis
Arylated diazeniumdiolate prodrugs JS-K and 2 are activated
by reaction with GSH to form NO. Earlier, we have reported that
compound 2 exhibits anti-proliferative properties comparable to
those of JS-K. However, it shows an extended half-life in
the presence of GSH.5,6 We hypothesized that slowing the reac-
tion time with GSH might translate to a prolonged lifetime in
the blood stream. This may lead to selective accumulation of
the drug in the tumor, thus leading to better anti-tumor
efficacy.
To pursue such delayed reaction with GSH, we planned to
change the substituents in the aromatic ring of JS-K. SAR studies
of JS-K show that both the electron-withdrawing groups in the
aromatic ring are important for the activation by GSH and subse-
quent anti-proliferative activity.2,6,7 Recently, we reported that
adding electron-donating groups like methyl and methoxy in
the aromatic ring slows down the reaction with GSH, but their
in vivo efficacy was compromised.5 Thus, the new analogues re-
quired two electron-withdrawing groups. We decided to replace
one or both nitro group(s) of JS-K with one or two other elec-
tron-withdrawing functional group(s) like methoxycarbonyl, flu-
oro and cyano groups.
⇑
Corresponding authors. Tel.: +1 301 846 7145; 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.
http://dx.doi.org/10.1016/j.bmc.2012.02.045

R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 3094–3099
3095
O
O
N
O
O
NO2
NO2
4 mM GSH in 0.1 M phosphate buffer pH 7.4 was monitored over
time (Table 2). Replacing nitro groups of the parent compound
(JS-K) with cyano groups significantly increased the half-life of
the prodrugs in GSH-containing buffer. The half-life for JS-K under
identical conditions was 2.8 min; modifications described here led
to a decrease in reactivity with GSH by approximately an order of
magnitude, and indeed much more for dicyano compound 11.
Modest to moderate increases to the compound’s stability resulted
in improved in vitro efficacy, as indicated by the IC50 values for
reduction in cell numbers for the NSCLC line H1703. However,
extending the half-life to over 1 h resulted in a decrease in efficacy,
suggesting that the rate of reaction with GSH is important for
biological activity.
N
N
N
N
O
O
NO2
NO2
N
N
N
O
O
JS-K (1)
2
Figure 1. Structures of JS-K and its homo-piperazine analogue 2.
Table 1
Synthesis of arylated diazeniumdiolate prodrugs 9–13
F
R1
The prodrugs which were stabilized toward GSH-induced
decomposition while maintaining in vitro efficacy, that is, com-
pounds 12, 13, 15 and 16, were evaluated for their NO-releasing po-
tential. GSH-activated nitric oxide yields were measured for these
compounds using a chemiluminescence assay. All four compounds
were found to release essentially quantitative amounts of NO on
reaction with GSH (Table 2).
R2
4-8
Na
O
O
O
N
O
O
O
N
O
O
R1
N
N
N
N N
N
DMSO, THF, rt
3
9-13
R2
Electrophile
R1
R2
Product
Yield (%)
4
5
6
7
8
COOMe
F
CN
CN
NO2
NO2
NO2
CN
NO2
CN
9
63
58
85
88
79
2.4. In vitro metabolism
10
11
12
13
As noted, the replacement of a nitro group of JS-K with a cyano
group significantly retarded the reaction of each prodrug with
GSH, vide supra. It was hypothesized that this would result in a
slowing of metabolic clearance of the compound. As a proof of
concept, the NSCLC cell line H1703 was treated with compound
12, and the metabolism was followed at various time points via
LC/MS. The extracted ion chromatograms in Figure 2 illustrate
the decomposition of compound 12, retention time 10.9 min, over
1 h concomitant with the formation of arylated GSH, retention
time 5.8 min, as observed in the GSH reaction kinetics experi-
ments. It is important to note that similar experiments in which
cells were treated with JS-K demonstrate a near complete meta-
bolic turnover of JS-K in as few as ten minutes, thus demonstrat-
ing the stabilizing effect of these structural modifications on the
rate of cellular metabolism.
Diazeniumdiolate sodium salt 32 was treated with substituted
fluorobenzene derivatives 4–8 in DMSO/THF (Table 1). The reaction
proceeded smoothly to afford new arylated diazeniumdiolate pro-
drugs 9–13 in good yields.
The homo-piperazine analogues of cyano-substituted prodrugs
were synthesized by reacting diazeniumdiolate salt 142 with fluo-
robenzene derivatives 7 and 8 to obtain 15 and 16, respectively, in
good yields (Scheme 1).
2.2. In vitro screening
The new arylated prodrugs 9–13 and 15–16 were screened for
in vitro anti-proliferative activity against the H1703 human NSCLC
cell line. With the exception of compound 11, cyano-substituted
prodrugs showed good anti-proliferative activity, with IC50 values
in the low micromolar level, thus comparable to JS-K and com-
pound 2 (Table 2). The rest of the compounds displayed IC50
2.5. In vivo studies
NSCLC cell line H1703, with established in vivo sensitivity to JS-
K,4,5 was chosen for assessment of activity of compound 12 in vivo
against xenografted cells in athymic mice. Compound 12 formu-
>10 lM. Hence, only the prodrugs 12, 13, 15 and 16 were consid-
ered for further investigation.
lated in Pluronic P1234,5 was administered at 20
lmol/kg via intra-
peritoneal (ip) injections, five times a week for a four-week period.
Treatment with 12 significantly reduced growth of H1703 cells in
vivo, when compared with cells in control animals treated with
vehicle only (Fig. 3).
2.3. NO release and GSH reaction kinetics
The GSH reaction kinetics for each of the shortlisted prodrugs
were studied to determine the effect of modulating the electron-
withdrawing groups on GSH reactivity. The decomposition of the
chromophoric diazeniumdiolate functional group in presence of
2.6. Stress signaling and apoptosis
Treatment with compound 12 activated the stress-activated
protein kinese/c-jun terminal kinase (SAPK/JNK) stress signaling
pathway. SAPK/JNK phosphorylation was observed after 4 h with
the drug (Fig. 4), as well as phosphorylation of its downstream
effector c-jun. Activating transcription factor 3 (ATF3) was also
upregulated. We have shown previously that JNK/ATF3 pathway
activation is necessary for triggering apoptosis in the cells treated
with diazeniumdiolate-based NO-releasing compounds.5 In the
current experiment apoptosis was activated within 4 h of incuba-
tion with the drug, as evidenced by cleavage of PARP and caspases
3 and 7 (Fig. 4).
F
R1
R1
O Na
O
N
N
O
O
N
N
N
N
N
N
7-8
R2
O
O
O
O
DMSO, THF, rt
14
R2
R1 = CN, R2 = NO2; 15 (79%)
R1 = NO2, R2 = CN; 16 (82%)
Scheme 1. Synthesis of prodrugs 15 and 16.

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R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 3094–3099
Table 2
GSH-induced NO-release rates for JS-K and its structural analogues
a
Compound
NO yield (mol)/mol of compound mean (SE)
kmax Monitored (nm)
kobs (sÀ1) mean (SE)
T
½
(min) mean (SE)
IC50 (lM) mean (SE)
JS-K
2
1.96 (0.03)
2.00 (0.04)
2.02 (0.06)
2.04 (0.01)
1.96 (0.04)
2.01 (0.03)
n.d.
300
312
273
302
286
312
273
4.08 eÀ3 (1.45 eÀ4
3.46 eÀ3 (1.04 eÀ4
5.23 eÀ4 (4.20 eÀ5
)
)
)
2.8 (0.1)
3.3 (0.1)
22.1 (3.1)
33.6 (1.7)
42.6 (0.6)
66.0 (4.0)
1.08 (0.12)
0.95 (0.16)
0.73 (0.10)
0.55 (0.08)
0.68 (0.10)
2.62 (0.18)
13
12
16
15
11
3. eÀ4 (3.72 eÀ5
)
2.71 eÀ4 (3.80 eÀ5
1.75 eÀ4 (1.04 eÀ4
1.26 eÀ5 (2.30 eÀ6
)
)
)
918.0 (69.0)
>20.00
n.d. = not determined.
a
H1703 human NSCLC cell line.
Figure 2. Extracted ion chromatogram showing decomposition of compound 12 in H1703 cells. Cells were treated with 5 lM compound 12 for 5, 30 and 60 min (top, middle
and bottom panel, respectively).
2.7. Summary
3. Experimental
3.1. Synthesis
3.1.1. General
Starting materials were purchased from Aldrich Chemical Co.
(Milwaukee, WI) unless otherwise indicated. NMR spectra were
recorded on a Varian UNITY INOVA spectrometer; chemical shifts
(d) are reported in parts per million (ppm) downfield from tetra-
methylsilane. The NMR spectra of compounds 15 and 16 were re-
corded at 65 °C in DMSO-d6. Ultraviolet (UV) spectra were
recorded on an Agilent Model 8453 or a Hewlett–Packard model
8451A diode array spectrophotometer. Infrared (IR) spectra were
JS-K, an arylated diazeniumdiolate prodrug, has shown promise
as a selective anti-cancer agent. To delay the metabolic clearance of
the compound we sought to modulate the rate of its reaction with
GSH. To this end SAR studies have elucidated the effect of varying
electron-withdrawing groups on drug efficacy. While decreasing
the strength of the electron-withdrawing groups slows the aro-
matic substitution reaction with GSH, over-stabilization results in
a loss of efficacy. We found that switching one nitro group to a cy-
ano group increases the half-life of the prodrug in the presence of
GSH without compromising the compound’s in vitro and in vivo
anti-tumor activity.

R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 3094–3099
3097
3.1.2. General procedure for arylation of diazeniumdiolate salts
To a partial solution of the diazeniumdiolate 3 or 14 (1 equiv) in
DMSO (4 mL/mmol of diazeniumdiolate salt) was added the fluoro
compound (4–8) (1 equiv) in THF (2 mL/mmol of fluoro compound)
at room temperature. The resulting solution was stirred at room
temperature overnight. To this homogeneous solution, water was
added (8 mL/mmol of diazeniumdiolate), producing a yellow pre-
cipitate that was collected by filtration, washed with water, and
dried. The crude product was purified by flash column chromatog-
raphy or by recrystallization.
3.1.3. Compound 9
[JS-59-16] mp 55–56 °C; UV (ethanol) kmax (e 304 nm 14.0 mM
À1 cmÀ1); 1H NMR (CDCl3) d 1.29 (t, J = 7.1 Hz, 3H), 3.60–3.62 (m,
4H), 3.72–3.75 (m, 4H), 3.99 (s, 3H), 4.18 (q, J = 7.1 Hz, 2H), 7.53
(d, J = 9.0 Hz, 1H), 8.38 (dd, J = 9.0, 2.7 Hz, 1H), 8.80 (d, J = 2.7 Hz,
1H); 13C NMR (CDCl3) d 163.4, 159.7, 155.0, 143.0, 128.7, 128.0,
119.7, 116.1, 62.0, 53.0, 50.8, 14.6. Anal. Calcd for C15H19N5O8: C,
45.34; H, 4.82; N, 17.63, found: C, 45.41; H, 4.94; N, 17.66.
3.1.4. Compound 10
[JS-59-165] mp 131–133 °C; UV (ethanol) kmax (e 308 nm
9.36 mMÀ1 cmÀ1); 1H NMR (DMSO-d6) d 1.21 (t, J = 7.1 Hz, 3H),
3.61–3.64 (m, 8H), 4.08 (q, J = 7.1 Hz, 2H), 7.77 (t, J = 8.9 Hz, 1H),
8.14–8.17 (m, 1H), 8.34 (dd, J = 10.9, 2.7 Hz, 1H); 13C NMR
(DMSO-d6) (aromatic region is complex due to 13C–F couplings)
d 155.3, 154.8, 150.5, 149.2, 149.1, 148.0, 143.4, 143.3, 121.7,
121.7, 117.5, 113.6, 113.4, 61.5, 53.0, 50.2, 42.2, 14.9. Anal. Calcd
for C13H16FN5O6: C, 43.70; H, 4.51; N, 19.60: F, 5.32, found: C,
43.50; H, 4.46; N, 19.56; F, 5.01.
3.1.5. Compound 11
[JS-59-145] mp 170–171 °C; UV (ethanol) kmax (e 275 nm
9.5 mMÀ1 cmÀ1); IR (film) 3139 cmÀ1, 3040, 2396, 1720, 1661,
1572, 1431; 1H NMR (DMSO-d6) d 1.20 (t, J = 7.4 Hz, 3H), 3.60–
3.61 (m, 4H), 3.64–3.66 (m, 4H), 4.07 (q, J = 7.4 Hz, 2H), 7.77 (d,
J = 8.6 Hz, 1H), 8.24 (dd, J = 8.6, 2.0 Hz, 1H), 8.56 (d, J = 2.0 Hz,
1H); 13C NMR (DMSO-d6) d 160.2, 154.8, 139.7, 139.0, 117.4,
116.9, 107.6, 100.8, 61.6, 50.1, 42.2, 14.9. Anal. Calcd for
Figure 3. Compound 12 significantly reduced growth of H1703 cells in vivo. The
compound was administered ip at 20 lmol/kg five times a week for four weeks, and
tumors were measured with a caliper. Values are means (SE); ⁄⁄P <0.01; ⁄P <0.05 by
t-test with the Welch correction (top panel). Treatment did not affect body weights
(bottom panel).
C
15H16N6O4Á0.25H2O: C, 51.43; H, 4.80; N, 24.41, found: C, 51.41;
H, 4.51; N, 24.18.
Treatment time (hours)
0 .4 .2 1
6 12 24
Treatment time (hours)
0 .4 .2 1
6 12 24
2
2
4
4
3.1.6. Compound 12
[JS-59-4] mp 149–151 °C; UV (ethanol) kmax (e 302 nm 13.6 mM
À1 cmÀ1); IR (film) 3135 cmÀ1, 3032, 2397, 1715, 1668, 1594, 1358;
1H NMR (CDCl3) d 1.29 (t, J = 7.0 Hz, 3H), 3.64–3.67 (m, 4H), 3.73–
3.75 (m, 4H), 4.18 (q, J = 7.0 Hz, 2H), 7.51 (d, J = 9.0 Hz, 1H), 8.24
(dd, J = 9.0, 2.3 Hz, 1H), 8.55 (d, J = 2.3 Hz, 1H); 13C NMR (CDCl3)
d 161.4, 155.0, 143.1, 129.9, 129.6, 116.1, 113.1, 101.6, 62.1, 50.5,
42.2, 14.6. Anal. Calcd for C14H16N6O6: C, 46.16; H, 4.43; N, 23.07,
found: C, 46.05; H, 4.62; N, 23.08.
P-SAPK/JNK
P-c-jun
PARP
cleaved
caspase 3
cleaved
caspase 7
ATF3
Figure 4. Treatment of H1703 cells with
1 lM compound 12 induced stress
3.1.7. Compound 13
signaling and apoptosis in lung adenocarcinoma H1703 cells. Phosphorylation of
SAPK/JNK and its downstream effectors c-jun and ATF3 and PARP cleavage/effector
caspases 3 and 7 activation are shown by Western blot. The star indicates full length
PARP protein, while the arrow indicates the 89-kDa cleaved fragment.
[JS-59-131] mp 146–147 °C; UV (ethanol) kmax (e 275 nm
16.0 mMÀ1 cmÀ1); IR (film) 3139 cmÀ1, 3039, 2397, 1715, 1571,
1485, 1264; 1H NMR (DMSO-d6) d 1.20 (t, J = 7.1 Hz, 3H), 3.60 (br,
4H), 3.62–3.63 (m, 4H), 4.07 (q, J = 7.1 Hz, 2H), 7.89 (d, J = 8.9 Hz,
1H), 8.24 (dd, J = 8.9, 2.0 Hz, 1H), 8.67 (d, J = 2.0 Hz, 1H); 13C
NMR (DMSO-d6) d 154.8, 151.9, 139.2, 138.0, 130.6, 118.9, 117.2,
107.1, 61.6, 50.1, 42.19, 14.9. Anal. Calcd for C14H16N6O6: C,
46.16; H, 4.43; N, 23.07, found: C, 46.00; H, 4.71; N, 23.11.
measured on a Buck Scientific Infrared Spectrophotometer Model
500. Elemental analyses were performed by Midwest Microlab
(Indianapolis, IN). Chromatography was performed on a Biotage
SP1 Flash Purification System. Prepacked silica gel flash chroma-
tography columns were purchased from Silicycle (Quebec City,
Canada). Compounds 12 (JS-K), 2,2 32 and 142 were prepared
using reported methods.
3.1.8. Compound 15
[RN-3-46] UV (ethanol) kmax (e
310 nm 13.0 mMÀ1 cmÀ1); IR
(film) 3134 cmÀ1, 3006, 2400, 1709, 1630, 1540, 1485, 1358,
1279; 1H NMR (DMSO-d6) d 1.14 (t, J = 7.1 Hz, 3H), 1.90–1.95 (m,

3098
R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 3094–3099
2H), 3.43–3.46 (m, 2H), 3.67–3.70 (m, 2H), 3.94–3.96 (m, 2H),
4.00–4.05 (m, 4H), 7.66 (d, J = 9.3 Hz, 1H), 8.51 (dd, J = 9.3, 2.7 Hz,
1H), 8.74 (d, J = 2.7 Hz, 1H); 13C NMR (DMSO-d6) d 161.9, 155.3,
142.9, 130.8, 115.8, 113.8, 99.9, 61.2, 50.7, 50.2, 45.5, 44.3, 25.2,
14.9. Anal. Calcd for C15H18N6O6: C, 47.62; H, 4.80; N, 22.21, found:
C, 47.69; H, 4.79; N, 22.01.
for 24 h. Compounds were prepared as 10 mM stock solution in
DMSO. Increasing drug concentrations in 10 L of PBS were added
to 100 L of the culture medium for 48 h. MTT assay (Promega,
Madison, WI) was performed according to the manufacturer’s pro-
tocol. Each concentration was represented in eight repeats, and the
screening was performed as two independent experiments. IC50
values were calculated by using Sigma Plot software (Systat Soft-
ware, Inc., San Jose, CA).
l
l
3.1.9. Compound 16
[RN-3-101] UV (ethanol) kmax (e
286 nm 16.2 mMÀ1 cmÀ1); IR
(film) 3147 cmÀ1, 3002, 2401, 1733, 1589, 1554, 1485,1360; 1H
NMR (DMSO-d6) d 1.12 (t, J = 6.9 Hz, 3H), 1.87–1.92 (m, 2H),
3.41–3.44 (m, 2H), 3.65–3.68 (m, 2H), 3.90–3.93 (m, 2H), 3.93–
3.99 (m, 4H), 7.74 (d, J = 8.8 Hz, 1H), 8.15 (dd, J = 8.8, 2.0 Hz, 1H),
8.57 (d, J = 2.0 Hz, 1H); 13C NMR (DMSO-d6) d 155.4, 152.4, 138.9,
130.3, 118.5, 117.1, 106.6, 61.2, 50.9, 50.2, 45.7, 44.3, 25.4, 14.8.
Anal. Calcd for C15H18N6O6: C, 47.62; H, 4.80; N, 22.21, found: C,
47.49; H, 4.86; N, 22.19.
3.5.2. In vivo treatments
All animals used in this project were cared for and used hu-
manely according to the following policies: the U.S. Public
Health Service Policy on Humane Care and Use of Animals
(1996), the National Institutes of Health’s Guide for the Care
and Use of Laboratory Animals (1996), and the U.S. Government
Principles for Utilization and Care of Vertebrate Animals Used in
Testing, Research and Training (1985). All NCI-Frederick animal
facilities and the animal program are accredited by the Associa-
tion for Assessment and Accreditation of Laboratory Animal Care
International. H1703 cells were injected at 5 Â 106 s.c. into a
flank of 7-week-old female athymic NCr-nu/nu mice (Charles
River Laboratories, Inc. Wilmington, MA). The drug injections
were initiated when the tumors reached 2 Â 2 Â 2 mm3 (typi-
cally four weeks). Compound 12 was formulated in Pluronic
P123 (BASF, Florham Park, NJ) as micelles.4,5 Animals were trea-
ted five times a week for four weeks with ip injections of either
3.2. NO-release by chemiluminescence
Chemiluminescence detection and quantification of NO evolv-
ing from the reactions of each prodrug were conducted using a Sie-
vers 280i Nitric Oxide Analyzer (NOA). A solution of pH 7.4
phosphate buffer containing 4 mM GSH at 37 °C was sparged with
inert gas until a steady detector response was established. The NO
release profile was followed over time after injecting each prodrug
to start the reaction. The resulting curve was integrated to quantify
the amount of NO released/mol of compound.
vehicle (2.25% Pluronic P123 in PBS) or compound 12 (20 lmol/
kg in vehicle). Tumors were measured with a caliper twice a
week, and the tumor volumes were calculated using a formula
3.3. GSH reaction kinetics
for ellipsoid volume,
p/6  length  width  height. The non-
parametric Mann–Whitney test was used for statistical compar-
ison of tumors volume at each time points. Body weights were
taken three times a week.
Kinetic experiments were performed at 37 °C on a standard UV–
visible spectrophotometer. Reactions were initiated by addition of
substrate after temperature equilibration. Typical substrate con-
centrations were ꢀ15
l
M with 4 mM GSH in 0.1 M phosphate buf-
fer, pH 7.4, containing 50
lM diethylenetriaminepentaacetic acid
3.5.3. Immonoblotting
Western blot analysis was performed as described previously.4
Primary antibodies to caspase 3, caspase 7, PARP, phosphorylated
SAPK/JNK, phosphorylated c-jun (Cell Signaling Technology, Dan-
vers, MA) and ATF3 (Santa Cruz Biotechnology, Santa Cruz, CA)
were used.
(DTPA). For prodrugs 12, 13, 15 and 16 the rate was derived by fit-
ting the data to an exponential curve typical for first order pro-
cesses. Prodrug 11 was evaluated with initial rate calculations.
3.4. In vitro metabolism
The H1703 cells were plated in 75-cm2 flasks and incubated
3.5.4. Statistical analysis
overnight at 37 °C. The cells were treated with 5
12 and incubated for 5, 30 and 60 min. At each time point the cells
were lysed via scraping in 800 L of 10 mM HCl followed by suc-
cessive rounds of freezing and thawing. To the lysate was added
200 L of a 5% 5-sulfosalicylic acid solution. The precipitate was re-
lM of compound
Statistical tests were carried out by using Instat version 3.00
(GraphPad Software Inc., San Diego, CA). Pairwise comparisons in-
cluded the t test, with the Welch correction or Mann–Whitney test
as appropriate.
l
l
moved by centrifugation at 8000Âg for 10 min, and the superna-
tant was analyzed by LC/MS.
Acknowledgments
The system used for analysis is a Thermoquest Surveyor HPLC
coupled with a Finnigan LCQ deca mass spectrometer. Positive
ions were generated with an atmospheric pressure chemical ion-
ization (APCI) source with a capillary voltage of 15 V and a corona
This project has been funded with Federal funds from the Na-
tional Cancer Institute, National Institutes of Health, under contract
HHSN261200800001E and by the Intramural Research 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 spectrometry studies. We
thank Mr. Ken Kosak of the University of Utah for providing the Plur-
onics P123 formulation.
discharge of 4
lA. Separations were performed on an Agilent
Eclipse XDB-C18 5-
1 mL/min under H2O/acetonitrile/0.1% formic acid gradient
l
m 4.6 Â 150 mm column at a flow rate of
conditions.
3.5. Biological evaluation
3.5.1. Cell culture and proliferation assay
Supplementary data
Human NSCLC cell line H1703 was obtained from the American
Type Culture Collection (ATCC, Manassas, VA) and cultured accord-
ing to the supplier’s protocol. For proliferation assay cells were
seeded at 1 Â 104 per well in 96-well plates and allowed to adhere
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.02.045.

R. S. Nandurdikar et al. / Bioorg. Med. Chem. 20 (2012) 3094–3099
3099
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