Nitroreductase-activated nitric oxide (NO) prodrugs

Article abstract of DOI:10.1016/j.bmcl.2013.08.066

Due to the involvement of nitric oxide (NO) in numerous and diverse physiological processes, site-directed delivery of therapeutic NO in order to minimize unwanted side-effects is necessary. O²-(4-Nitrobenzyl) diazeniumdiolates are designed as

Full text of DOI:10.1016/j.bmcl.2013.08.066

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5964–5967
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Nitroreductase-activated nitric oxide (NO) prodrugs
Kavita Sharma a, Kundan Sengupta b, Harinath Chakrapani a,
⇑
a Department of Chemistry, Indian Institute of Science Education and Research Pune, Pune, Maharashtra, India
b Department of Biology, Indian Institute of Science Education and Research Pune, Pune, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to the involvement of nitric oxide (NO) in numerous and diverse physiological processes, site-direc-
ted delivery of therapeutic NO in order to minimize unwanted side-effects is necessary. O2-(4-Nitroben-
zyl) diazeniumdiolates are designed as substrates for Escherichia coli nitroreductase (NTR), an enzyme
that is frequently used to facilitate directed delivery of cytotoxic species to cancers. O2-(4-Nitrobenzyl)
diazeniumdiolates are found to be stable in aqueous buffer but are metabolized by NTR to produce
NO. A cell viability assay revealed that cytotoxic effects of O2-(4-nitrobenzyl)1-(2-methylpiperidin-1-
yl)diazen-1-ium-1,2-diolate (4b) towards two cancer cell lines is significantly enhanced in the presence
of NTR suggesting the potential for use of this compound in nitric oxide-based directed prodrug therapy.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 4 June 2013
Revised 25 July 2013
Accepted 14 August 2013
Available online 22 August 2013
Keywords:
Nitric oxide
Prodrug
Nitroreductase
Diazeniumdiolate
Directed prodrug therapy
Nitric oxide (NO) mediates numerous physiological processes
including vasodilation, neurotransmission and immune response.1
At elevated concentrations NO can damage biomacromolecules
such as proteins, DNA and lipids and can cause cell death by trig-
gering apoptosis.2 Nitric oxide has also been considered as a poten-
tial cancer therapeutic agent.3–5 However, due to its involvement
in a myriad of biological processes, delivery of NO must be local-
ized at the tumour site.3
Due to the limited utility of NO gas, typically, nitric oxide ‘do-
nors’ are used as surrogates for NO in biological studies.6 NO do-
nors are compounds that are otherwise stable but when exposed
to physiological conditions dissociate to produce NO. Among the
available NO donors, diazeniumdiolates are routinely used in bio-
logical studies as reliable sources of NO (Scheme 1).5,7 These NO
donors are also highly suited for site-directed delivery of NO. Dia-
zeniumdiolate anions can be derivatized using appropriate func-
tional groups into stable compounds.7 The protective group is
chosen so that the stable diazeniumdiolate derivative is cleaved
in the presence of an enzyme to produce a diazeniumdiolate anion,
which dissociates at pH 7.4 to produce NO (Scheme 1). Depending
on the choice of the enzyme and its distribution in tissues, these
protected diazeniumdiolates can be optimized for localized deliv-
ery of NO. Some notable examples of triggers are glutathione/glu-
tathione S-transferases, glycosidases, esterases, DT-diaphorase8
and cytochromes P450.7,9–11 Together, these compounds form a co-
hort of metabolically-activated NO prodrugs with potential for
application as therapeutic agents against numerous diseases
including cancer.4,5,10
Escherichia coli nitroreductase (NTR) is an enzyme that is
frequently used as a metabolic trigger for ‘directed’ prodrugs
including gene-directed enzyme prodrug therapy (GDEPT) and
antibody-directed enzyme prodrug therapy (ADEPT).12–14 As NTR
is not usually found in human cells, this enzyme is introduced
either by transfection methodologies (for GDEPT)15 or by the use
of tumour-specific antigens conjugated to the enzyme (for ADEPT).
Upon exposure to the exogenous enzyme, the inactive prodrug,
which is a substrate for the enzyme is metabolized to produce
the cytotoxic species either intracellularly or in the proximity of
tumours. As normal cells do not express this enzyme, potential del-
eterious side-effects can be minimized.16 NTR-activated cancer
drugs and cytotoxic species including DNA alkylating agents are
previously reported.17 However,
a NTR-activated nitric oxide
prodrug is yet unavailable. Here, we report our results of design,
synthesis and evaluation of NTR-activated NO donor candidates
which have potential for applications in directed or targeted
prodrug therapy.
A typical NTR-activated prodrug consists of a 4-nitrobenzyl
group attached to a leaving group such as a phosphoramide or a
Cleavage
R
N
R
N
N
N
O
N
O
R
NO
2
O
N
O
R
pH 7.4
⇑
Corresponding author. Tel.: +91 20 2590 8090; fax: +91 20 2589 9790.
E-mail address: harinath@iiserpune.ac.in (H. Chakrapani).
Scheme 1. Diazeniumdiolate-based prodrugs of nitric oxide that can be used for
enzyme-activated delivery of NO.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.066

K. Sharma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5964–5967
5965
Table 2
R
Cyclic voltammetry analysis and NTR-mediated decomposition and nitric oxide
release profiles
N
N
O
N
O
R
NTR
Entry
Compd
Ered (V)a
Remainingb (%)
NO yieldc
(lM)
HOHN
R
1
2
3
4
5
1b
2b
3b
4b
5b
ꢀ0.09
ꢀ0.11
ꢀ0.11
ꢀ0.11
ꢀ0.14
60
60
28
31
68
13.6
6.8
18.3
19.7
1.1
R
N
N
N
N
O
N
O
R
O
N
O
R
a
One electron reduction potential measured by cyclic voltammetry. Conditions:
Pt disc working electrode; Pt wire auxiliary electrode; Ag/AgNO3 reference elec-
trode; scan rate = 25 mV/s; NBu4PF6 = 100 mM as the background electrolyte in
O2N
H2N
MeCN. Ered for 4-nitrotoluene was ꢀ0.11 V.
b
Decomposition of 1b–4b (50 lM) in the presence of nitroreductase (NTR) in pH
7.0 buffer after 1 h was estimated by HPLC.
Nitric oxide released during decomposition of 1b–5b (50 lM) in the presence of
nitroreductase (NTR) in pH 7.0 buffer after 1 h measured using a chemilumines-
R
N
c
N
N
O
O
R
NO
cence assay.
Scheme 2. Design of O2-(4-nitrobenzyl)diazeniumdiolates as NTR-activated pro-
drugs of nitric oxide.
O
PhCO2H
OH
O
Ph
EDC, DMAP
DCM, RT, 3 h
Table 1
O2N
O2N
Reported half-lives of nitric oxide release of 1a–4a and synthesis of O2-(4-nitroben-
zyl) diazeniumdiolates 1b–4b19
5b, 33%
Scheme 3. Synthesis of 5b. EDC: N-(3-Dimethylaminopropyl)-N0-ethylcarbodiim-
ide hydrochloride; DMAP: 4-Dimethylaminopyridine.
R1
N
R1
N
N
4-NO2PhCH2Br
THF, 15-crown-5
N
R2
N
O
O
Na
O
N
O
R2
O2N
the cyclic derivatives 3b and 4b, 28 and 31% of compound
remained during 1 h (Table 2, entries 1–4). It appears that the
cyclic compounds 3b and 4b were better substrates for NTR in
comparison with 1b and 2b. During this time period, no significant
decomposition of 1b–4b was observed in the absence of NTR sug-
gesting that these compounds were not candidates for hydrolysis
in pH 7.0 buffer and the decomposition must be due to metabolism
by NTR.22
Nitric oxide produced during decomposition of 1b–4b was
measured using a chemiluminescence-based assay for NO (Table 2,
entries 1–4).21 Compounds 1b–4b were reacted with NTR in the
presence of NADPH as a co-factor in pH 7.0 buffer. An aliquot
was taken after 1 h for nitric oxide analysis. Among O2-(4-nitro-
benzyl) diazeniumdiolates tested, 3b and 4b were found to be
R1NR2
Diazeniumdiolate
t1/2
Product
Yieldb
a
Me2N
Et2N
Pyrrolidine
2-Me-piperidine
DMA/NO, 1a
DEA/NO, 2a
PYRRO/NO, 3a
4a
24 s
2 min
2 s
1b
2b
3b
4b
9
15
16
25
8.3 min
a
Reported half-lives of nitric oxide release upon decomposition of the anion in
pH 7.4 buffer (See Ref. 19).
b
Isolated yield in %.
carboxylate. As diazeniumdiolate anions resemble carboxylate ions
in their leaving group ability, we proposed O2-(4-nitrobenzyl) dia-
zeniumdiolates as candidates for NTR activation (Scheme 2).18
Reduction of the nitro group by NTR produces a hydroxylamine/
amine; this transformation converts an electron-withdrawing nitro
group into an electron donating group. Rearrangement of electrons
would be expected to produce a NO-releasing diazeniumdiolate
anion.
the best sources of NO with nearly 20 lM generated in 1 h fol-
lowed by 1b and then 2b (Table 2). In the absence of NTR, we found
negligible amounts of NO, again, indicating the selectivity of O2-(4-
nitrobenzyl) diazeniumdiolates towards activation by NTR.
Next, in order to study if the nitro group on the aryl group was a
source of NO, 4-nitrobenzyl benzoate (5b)23 was prepared by reac-
tion of 4-nitrobenzyl alcohol with benzoic acid (Scheme 3). This
compound is expected to undergo decomposition in the presence
of NTR but without generating NO. Indeed, 5b was metabolized
by NTR with 68% remaining after 1 h and analysis of reaction mix-
tures of 5b in the presence and absence of NTR showed negligible
NO formation during decomposition of this compound suggesting
that the nitro group on the aromatic ring itself was not was not a
significant source of NO (Table 2, entry 5).24,25
O2-(4-Nitrobenzyl) diazeniumdiolates 1b–4b were prepared by
treatment of the corresponding diazeniumdiolate anion 1a–4a19
with 4-nitrobenzyl bromide (Table 1).20 The NO donors 1a–4a
were chosen in part due to a good range in half-lives of NO release
from 2 s to 8.3 min (Table 1).
In order to assess the suitability of the nitro group for reduction,
cyclic voltammetric (CV) analysis of 1b–4b was conducted to
determine reduction potentials. 4-Nitrotoluene was used as a ref-
erence for nitro-group reduction and a 1ꢀeꢀ reduction potential
(Ered) of ꢀ0.11 V was recorded (Table 2). Ered of 1b–4b was found
to be nearly identical suggesting that the diazeniumdiolate group
did not significantly affect the propensity for the nitro group to
undergo reduction (Table 2). Next, in order to test whether
O2-(4-nitrobenzyl) diazeniumdiolates were substrates for NTR,
1b–4b were independently treated with NTR in the presence of
NADPH and decomposition was studied using HPLC.21 During 1 h,
we found nearly 60% of 1b and 2b remained while in the cases of
Thus, amongst O2-(4-nitrobenzyl)diazeniumdiolates synthe-
sized in this study, 3b and 4b were the best sources of NO (Table 2,
entries 3–4). This observation is also consistent with HPLC data on
decomposition of 3b and 4b that these compounds were also the
best substrates for NTR amongst 1b–4b (Table 2, entries 1–4). Next,
a cell viability assay was conducted to study the selectivity of 1b–
4b in inhibiting proliferation of DLD-1 human colon adenocarci-
noma cells. Our initial assessment was conducted at an elevated
concentration of 75 lM and we found moderate inhibitory activity

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K. Sharma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5964–5967
Figure 3. Nitric oxide released during decomposition of 4b (50 lM) in the presence
of NTR in pH 7.4 phosphate buffered saline.
Figure 1. Cell viability assay to assess anti-proliferative activity of 1b–4b at 75 lM
in the presence and absence of NTR conducted with DLD-1 human adenocarcinoma
cells. Blank is untreated cells.
an enhanced bystander effect is predicted as a freely diffusible
reactive gas NO is produced.2,3 However, for this to occur, NO must
be released rapidly in the proximity of the tumour. A time course of
for all compounds in the absence of NTR (Fig. 1).26 However, in the
presence of NTR, we found significant inhibition of proliferation in
the case of 4b with only 10% of cells being viable with respect to
control. Although 3b produced similar amounts of NO in compari-
son with 4b, the enhancement in cytotoxicity in the presence of
NTR was higher in the case of 4b (Fig. 1). The origin of this differ-
ence is unclear. However, a recent study on NTR-activated DNA
alkylating agents indicated that the anti-proliferative activity of
the NTR substrate did not always correlate with propensity for
its metabolism by NTR.17f Based on these results, 4b was identified
as a NTR-activated nitric oxide donor with enhanced cytotoxicity
in the presence of exogenously added NTR and was chosen for fur-
ther evaluation.
NO release from 4b (50
lM) in the presence of NTR showed an in-
crease in levels of NO (17
lM in 5 min) suggesting that a burst of
NO would be generated upon exposure to NTR (Fig. 3). Previous
studies have indicated that NO by itself and in conjunction with
radiation inhibits cancer cell growth under hypoxic conditions.28–31
As hypoxic tumours are known to be acidic, we recorded NO
release from 4b in pH 6.5. NO (6 lM) was produced in 5 min in
pH 6.5 (Fig. S2, Supporting Information) suggesting that 4b might
be useful for NO delivery to hypoxic tumours.32 The sensitivity of
Mycobacterium tuberculosis to reactive nitrogen species has been
demonstrated and the use of such NTR-activated NO prodrugs to
target this bacterium might be possible.25
In conclusion, we report 1-(2-methylpiperidin-1-yl)diazen-1-
ium-1,2-diolate (4b) as a nitroreductase activated nitric oxide pro-
drug with enhanced cytotoxicity towards two cancer cell lines in
the presence of NTR suggesting potential applications for this com-
pound in nitric oxide-based directed prodrug therapy.
A concentration-dependent cell viability profile of 4b with
DLD-1 cells showed that the potency of 4b was enhanced in the
presence of NTR at concentrations below 25 lM as well (Fig. 2).
Compound 5b also did not show significant inhibitory activity
against DLD-1 cells even at elevated concentrations tested suggest-
ing that the metabolites formed during decomposition of 4b may
not have significant cytotoxicity (Table S4, Supporting Informa-
tion). A similar cell viability assay was conducted with 4b using
HeLa human cervical cancer cells and again, a dose-dependent
enhancement in toxicity in the presence of NTR was observed
(See Supporting Information, Fig. S1).
Taken together, our data indicates that the nitric oxide prodrug
4b is a potential candidate for nitroreductase-based directed pro-
drug therapy. The use of directed prodrug therapy using NTR has
been reported to be effective in inhibiting tumour growth.27–29
Some examples of NTR-mediated therapeutics include generation
of alkylating agents, ene-diynes, pyrazolidines, and 2-fluoroade-
nines.17 However, the efficiency of gene transfer or antibody
conjugation determines the efficiency of generation and transfer
of toxic metabolites. Hence, the success of such targeted therapy
is in part dependent on the ability of the toxic species to diffuse
and kill non-transfected neighboring cells as well. Also known as
bystander effect, this collateral killing of neighboring cells aids in
tumour regression.16 By the use of nitric oxide-based therapeutics,
Acknowledgments
This work was supported in part by IISER Pune. HC acknowl-
edges financial support from the Department of Biotechnology
(DBT, Grant number BT/05/IYBA/2011). K. Sengupta acknowledges
an Intermediate Fellowship from the Wellcome Trust-DBT India
Alliance. K. Sharma acknowledges a research fellowship from
Council for Scientific and Industrial Research (CSIR). The authors
are grateful to Dr. Larry Keefer (National Cancer Institute at
Frederick) and Dr. Joseph Saavedra (SAIC-Frederick) for a gift of
diazeniumdiolate salts.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.08.
066.
References and notes
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Figure 2. Cell viability assay to assess anti-proliferative activity of 4b in the
presence and absence of NTR was conducted with DLD-1 human colon adenocar-
cinoma cells.

K. Sharma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5964–5967
5967
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were stirred at 37 °C and after 1 h an aliquot was analyzed for decomposition
and nitric oxide generation. Decomposition: HPLC (Eclipse Plus C-18, 5 m,
4.6 ꢁ 250 mM; flow rate: 1 mL/min; eluant 70% MeCN:H2O) analysis was used
to determine amount of compound remaining. For quantifying amount of NO
release from enzymatic decomposition of 1b–5b, the reaction mixtures were
l
prepared in pH 6.5, pH 7.0, and 7.4. 10 lL of the reaction mixture and blank
solution were injected at the specified time points into a Sievers Nitric Oxide
Analyzer (NOA 280i) using argon as the carrier gas. The reservoir contained NaI
and acetic acid, which converts nitrite to NO. A calibration curve was generated
using NaNO2 standards, which under the assay conditions would get converted
to NO. The so formed NO is quantified using the chemiluminescence detector.
Under the enzymatic assay conditions any nitrite that is produced during
decomposition is also measured.
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23. To an ice cold solution of 4-nitrobenzyl alcohol (0.652 mmol), benzoic acid
(0.979 mmol) and 4-dimethylaminopyridine (DMAP, 1.30 mmol) in dry DCM
(10 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC.HCl,
14. Xu, G.; McLeod, H. L. Clin. Cancer Res. 2001, 7, 3314.
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1.30 mmol) was added. The reaction was stirred at room temperature for
3 h.The reaction mixture was diluted with water (25 mL) and the resulting
aqueous mixture was extracted with DCM (3 ꢁ 10 mL). The combined organic
layer was washed with brine, dried over anhydrous Na2SO4, filtered and the
filtrate was evaporated to give crude product. Silica gel chromatography (10%
ethyl acetate: pet ether) of the resulting crude gave 5b (33%) as a white solid.
Analytical data for 5b: 1H NMR (CDCl3, 400 MHz) d: 8.23 (d, J = 8.7 Hz, 2H), 8.07
(d, J = 8.6 Hz, 2H), 7.57–7.60 (m, 3H), 7.45 (t, J = 7.4 Hz, 2H), 5.44 (s, 2H); 13C
NMR (CDCl3, 100 MHz) d: 166.0, 147.6, 143.3, 133.4, 129.7, 128.5, 128.3, 123.8,
65.1. See: Ohno, O.; Ye, M.; Koyama, T.; Yazawa, K.; Mura, E.; Matsumoto, H.;
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24. Some possible mechanisms of NO generation from nitroaromatic compounds
are discussed in Ref. 25. The levels of NO produced by 5b is ꢂ1% and hence
might be a minor component of NO generating pathways from 1b–4b. The
precise mechanism for NO generation from 5b is not clear at this time and
requires further investigations.
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20. General procedure for synthesis of O2-(4-nitrobenzyl) diazeniumdiolates. To an ice
cold solution of the diazeniumdiolate salt (1.041 mmol) in THF (10 mL), 15-
crown-5 (0.025 mL) was added. After 15 min of stirring at 0 °C, 4-nitrobenzyl
bromide (0.694 mmol) was added. The reaction mixture was stirred at room
temperature for 3–5 h. Reaction mixture was concentrated under reduced
pressure. The resulting crude was chromatographed using a silica gel support
to isolate pure material.
25. Gurumurthy, M.; Mukherjee, T.; Dowd, C. S.; Singh, R.; Niyomrattanakit, P.;
Tay, J. A.; Nayyar, A.; Lee, Y. S.; Cherian, J.; Boshoff, H. I.; Dick, T.; Barry, C. E.;
Manjunatha, U. H. FEBS J. 2012, 279, 113.
26. DLD-1 human adenocarcinoma cells were seeded at 3 ꢁ 10ꢀ4 cells/well
overnight in
a 96-well plate in complete RPMI 1640 media. E. Coli
nitroreductase (lyophilized powder, Sigma) was used to prepare
a stock
Analytical Data: O2-(4-Nitrobenzyl) 1-(N,N-dimethyl)diazen-1-ium-1,2-diolate
solution containing 6 mg in 2 mL of Dulbecco’s phosphate-buffered saline
(DPBS). NADPH stock solution (0.5 mM) was prepared in DPBS. Cells were
exposed to varying concentrations of the test compound prepared as a DMSO
stock solution so that the final concentration of DMS was 1%. The cells were
incubated for 72 h at 37 °C. A stock solution of 3-(4,5-dimethylthiazol-2-yl)-
(1b). Pale yellow solid (15 mg, 9 %); FT-IR (m
max, cmꢀ1): 2933, 1605, 1518, 1495,
1383, 1349, 1271, 1071; 1H NMR (400 MHz, CDCl3) d: 8.21 (d, J = 8.7 Hz, 2H),
7.53 (d, J = 8.8 Hz, 2 H), 5.28 (s, 2H), 2.98 (s, 6H); 13C NMR (100 MHz, CDCl3) d:
147.9, 143.2, 128.7, 123.8, 73.7, 42.3; HRMS (ESI) for [C9H12N4O4+Na]+: Calcd,
263.0756. Found: 263.0779. O2-(4-Nitrobenzyl) 1-(N,N-diethyl)diazen-1-ium-
2,5-diphenyl tetrazolium bromide (MTT) was prepared 3.5 mg in 700
This stock was diluted with 6.3 mL DPBS and 100 L of the resulting solution
was added to each well after aspiration of media. After 4 h incubation, the MTT
solution was removed carefully and 100 of DMSO was added.
lL DPBS.
1,2-diolate (2b). Light brown solid (27 mg, 15 %); FT-IR (
m
max, cmꢀ1): 2359,
l
2341, 1606, 1520, 1384, 1355, 1018; 1H NMR (400 MHz, CDCl3) d: 8.20 (d,
J = 8.7 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 5.34 (s, 2H), 3.08 (q, J = 7.0 Hz, 4H), 1.00
(t, J = 7.1 Hz, 6H); 13C NMR (100 MHz, CDCl3) d: 147.9, 143.1, 128.6, 123.8, 73.9,
48.7, 11.5; HRMS (ESI) for [C11H16N4O4+Na]+: Calcd, 291.1069. Found:
291.1069. O2-(4-Nitrobenzyl) 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate
lL
Spectrophotometric analysis of each well using a microplate reader (Thermo
Scientific Varioscan) at 570 nm was carried out to estimate cell viability. A
similar assay was conducted in the presence of NTR (5
lL stock) and NADPH
(3b). Pale yellow solid (15 mg, 16 %); FT-IR (
m
max, cmꢀ1): 2360, 1652, 1540,
(10 L stock) to analyze the effect of addition of NTR on cell viability.
l
1521, 1507, 1343; 1H NMR (CDCl3, 400 MHz) d: 8.20 (d, J = 8.8 Hz, 2H), 7.52 (d,
J = 8.7 Hz, 2H), 5.24 (s, 2H), 3.50–3.47 (m, 4H), 1.92–1.89 (m, 4H), 1.65–1.79
(m, 4H); 13C NMR (CDCl3, 100 MHz) d: 147.8, 143.6, 128.6, 123.8, 73.5, 50.9,
22.9; HRMS (ESI) for [C11H14N4O4+Na]+: Calcd, 289.0912. Found: 289.0922. O2-
(4-Nitrobenzyl) 1-(2-methylpiperidin-1-yl)diazen-1-ium-1,2-diolate (4b). Pale
27. Mitchell, J. B.; Wink, D. A.; DeGraff, W.; Gamson, J.; Keefer, L. K.; Krishna, M. C.
Cancer Res. 1993, 53, 5845.
28. (a) Ahn, G. O.; Brown, M. Frontiers Biosci. 2007, 12, 3483; (b) Brown, J. M.;
Wilson, W. R. Nat. Rev. Cancer 2004, 4, 437.
29. Zhang, N.; Tan, C.; Cai, P.; Zhang, P.; Zhao, Y.; Jiang, Y. Chem. Commun. 2009,
3216.
30. (a) Prosser, G. A.; Copp, J. N.; Syddall, S. P.; Williams, E. M.; Smaill, J. B.; Wilson,
W. R.; Patterson, A. V.; Ackerley, D. F. Biochem. Pharmacol. 2010, 79, 678; (b)
Sunters, A.; Baer, J.; Bagshawe, K. D. Biochem. Pharmacol. 1991, 41, 1293.
31. (a) Maciag, A. E.; Saavedra, J. E.; Chakrapani, H. Anticancer Agents Med. Chem.
2009, 9, 798; (b) Maciag, A. E.; Nandurdikar, R. S.; Hong, S. Y.; Chakrapani, H.;
Morris, N. L.; Diwan, B.; Shami, P. J.; Shiao, Y.-H.; Anderson, L. M.; Keefer, L. K.;
Saavedra, J. E. J. Med. Chem. 2011, 54, 7751; (c) Nandurdikar, R. S.; Maciag, A. E.;
Citro, M. L.; Shami, P. J.; Keefer, L. K.; Saavedra, J. E.; Chakrapani, H. Bioorg. Med.
Chem. Lett. 2009, 19, 2760.
yellow solid (50 mg, 25 %); FT-IR (m
max, cmꢀ1): 2934, 2857, 1521, 1505, 1383,
1344, 1020; 1H NMR (CDCl3, 400 MHz) d: 8.19 (d, J = 8.6 Hz, 2H), 7.52 (d,
J = 8.6 Hz, 2H), 5.34 (s, 2H), 3.13–3.23 (m, 3H), 1.65–1.79 (m, 4H), 1.31–1.42
(m, 2H), 0.91 (d, J = 6.1 Hz, 3 H); 13C NMR (CDCl3, 100 MHz) d: 147.9, 143.0,
128.5, 123.8, 56.7, 54.1, 25.1, 23.1, 18.2; HRMS (ESI) for [C13H18N4O4+Na]+:
Calcd, 317.1225. Found: 317.1245.
21. Enzyme stock solution was prepared by dissolving 1.5 mg of nitroreductase
(NTR) in phosphate buffer pH 7.0 (100
compound was prepared in DMSO. 50 L of the freshly prepared enzyme stock
was added to a solution consisting of 10 L of the test compound (10 mM stock
in DMSO) and 400 L NADPH (1 mM stock in phosphate buffer pH 7.0) and
1540 L of phosphate buffer pH 7.0. The blank consisted of 10 L of the test
compound (10 mM stock in DMSO) and 1990 L of phosphate buffer pH 7.0.
Data reported are averages of two independent runs. These reaction mixtures
lL). A 10 mM stock solution of the
l
l
l
32. Sogawa, K.; Numayama-Tsuruta, K.; Ema, M.; Abe, M.; Hisaku, A.; Fujii-
Kuriyama, Y. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7368.
l
l
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