INDQ/NO, a bioreductively activated nitric oxide prodrug

Article abstract of DOI:10.1021/ol400884v

The design, synthesis, and development of INDQ/NO, a novel nitric oxide (NO) prodrug targeted by a bioreductive trigger, are described. INDQ/NO, an indolequinone-diazeniumdiolate is found to be metabolized to produce NO by DT-diaphorase, a bioreductive enzyme that is overexpressed in certain cancers and hypoxic tumors. Cell-based assays revealed that INDQ/NO induces DNA damage and is a potent inhibitor of cancer cell proliferation.

Full text of DOI:10.1021/ol400884v

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
INDQ/NO, a Bioreductively Activated Nitric
Oxide Prodrug
000–000
†
Kavita Sharma, Aishwarya Iyer, Kundan Sengupta, and Harinath Chakrapani*
‡
‡
,†
Departments of Chemistry and Biology, Indian Institute of Science Education and
Research Pune, Pune 411 008, Maharashtra, India
harinath@iiserpune.ac.in
Received March 31, 2013
ABSTRACT
The design, synthesis, and development of INDQ/NO, a novel nitric oxide (NO) prodrug targeted by a bioreductive trigger, are described. INDQ/NO,
an indolequinone-diazeniumdiolate is found to be metabolized to produce NO by DT-diaphorase, a bioreductive enzyme that is overexpressed in certain
cancers and hypoxic tumors. Cell-based assays revealed that INDQ/NO induces DNA damage and is a potent inhibitor of cancer cell proliferation.
Numerous studies demonstrate the efficacy of nitric
1
oxide (NO) as a potent tumoristatic agent. However,
are highly suited for site-directed delivery of therapeutic
3
NO. These NO donors can be derivatized into stable
due to its multifarious biological effects, controlled and
localized generation of therapeutic NO using prodrugs is
2
necessary. Among various available methods for generation
of NO under physiological conditions, diazeniumdiolates
protected forms; activation of these prodrugs by a suit-
able metabolic trigger, typically an enzyme that is over-
expressed in certain tissues, produces NO in a localized
manner.
NAD(P)H:quinone oxidoreductase (DT-diaphorase;
DT-D) has received considerable attention for prodrug
development, as this enzyme is overexpressed in numerous
cancers as compared with the paired normal tissue as well
as hypoxic (low oxygen tension) regions of solid tumors.
Although prodrug methodologies for numerous cytotoxic
5
species are known, a candidate for DT-diaphorase-activated
†
Department of Chemistry.
‡
Department of Biology.
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1
0.1021/ol400884v r XXXX American Chemical Society

Scheme 1. Design of a DT-Diaphorase (DT-D)-Activated Nitric Oxide (NO) Prodrug
7
delivery of NO is not yet available. Here, we proposed to
develop a new nitric oxide prodrug that is selective toward
activation by DT-D to produce NO.
NO (Figure 1b). During incubation of 1a (50 μM) in the
presence of DT-D, a rapid increase in NO within 5 min was
8
observed and the yield of NO was nearly 30 μM for 3 h.
DT-D is an obligate two-electron reductase, and bio-
reductively activated prodrug candidates by placing a
suitable leaving group on an appropriate indolequinone
scaffold are in development. It was envisaged that a
diazeniumdiolate placed in the 10-position of a 4,7-diox-
oindole might be well suited for nitric oxide delivery
In the absence of DT-D, negligible NO was produced
during this time period (Figure 1b). Taken together, our
data are indicative of 1a undergoing selective decomposi-
tion under bioreductive conditions in the presence of
DT-D to produce NO.
5
DT-D is overexpressed in several colon cancers includ-
9
ing DLD-1 human adenocarcinoma cells. In order to test
(
Scheme 1). Bioreduction of this indolequinone-based
diazeniumdiolate by DT-D produces a 4,7-dihydroxyin-
dole intermediate (I, Scheme 1). As a consequence of this
transformation, the nitrogen which was previously under the
influence of an electron-withdrawing quinone group is now
conjugated with two electron-donating hydroxy groups.
Thus, the lone pair on the indole nitrogen becomes available
for a rearrangement to release a diazeniumdiolate anion,
which is expected to dissociate in physiological pH to form
whether 1a was a source of NO upon incubation with
DLD-1 cells, extracellular nitrite produced during incuba-
tion of DLD-1 cells with 1a during 5 h was evaluated. We
found that, in the absence of 1a, negligible amounts of
nitrite were produced during this time period (Figure 1c),
but upon incubation of DLD-1 cells with 1a (50 μM),
we found a significant increase in the amount of
nitrite produced, implying that NO was liberated
5
NO and II, which is capable of alkylating DNA (Scheme 1).
In order to test this hypothesis, INDQ/NO, 1a, was
(Figure 1c).
Reactive nitrogen species including NO are known to
synthesized in eight steps starting from 5-methoxyindole
a (Scheme 2). Briefly, formylation of 2a produced 3a in
damage biomacromolecules such as DNA leading to dou-
ble strand breaks; the inability of the cell to completely
12
repair the damage is partially responsible for cell death.
2
quantitative yield; methylation of 3a using methyl iodide in
the presence of sodium hydride gave 4a in 100% yield.
Nitration of 4a using conc. HNO in acetic acid gave 5a
An early specific cellular response to double strand breaks
includes phosphorylation of the histone protein H2AX at
3
in 90% yield. Reduction of the nitro group of 5a using
Sn/HCl produced the amine 6a followed by oxidation
using Fremy’s salt gave 7a, which was subsequently re-
1f
Ser139 (γ-H2AX). The formation of foci is an essential
step for signaling the recruitment of appropriate repair
enzymes. We next examined whether INDQ/NO treat-
ment induced DNA double strand breaks, as detected by
γ-H2AX foci formation in HeLa cervical cancer cells.
Immunofluorescence analysis (Figure 2a) of HeLa cells
treated with 1a during 1.5 h showed that INDQ/NO
induces significant and dose-dependent γ-H2AX foci
formation as early as 1.5 h after INDQ/NO exposure
duced using NaBH to afford 8a in 55% yield. In order to
4
install a diazeniumdiolate at the 10-position, we converted
thealcohol8aintothebromide9ausing PBr inDCM. The
3
bromide 9a (crude) was reacted soon after preparation
with sodium N,N-(diethylamino)diazen-1-ium-1,2-diolate
(DEA/NO) in THF in the presence of 15-crown-5 to give
INDQ/NO, 1a, in 10% yield.
(Figure 2b). These observations are consistent with a
Next, HPLC analysis was used to monitor the decom-
position of 1a in the presence of DT-D, NADPH in pH 7.4
buffer. We found that, within 5 min, a significant amount
of 1a was decomposed in the presence of DT-D and, in
previous report on the nitric oxide prodrug JS-K, which
induced significant H2AX phosphorylation at early time
1f
points.
1
50 min, nearly 80% of the compound was consumed
Figure 1a). During the same time period, in the absence of
(
7) (a) Chakrapani, H.; Showalter, B. M.; Kong, L.; Keefer, L. K.;
(
DT-D, 1a remained stable in pH 7.4 buffer.
Nitric oxide produced during incubation of 1a in pH 7.4
was assayed using a chemiluminescence-based assay for
Saavedra, J. E. Org. Lett. 2007, 9, 3409. (b) Chakrapani, H.; Maciag,
A. E.; Citro, M. L.; Keefer, L. K.; Saavedra, J. E. Org. Lett. 2008, 10,
155. (c) Chakrapani, H.; Bartberger, M. D.; Toone, E. J. J. Org. Chem.
009, 74, 1450.
(8) Under the assay conditions any nitrite that is produced during
6
5
2
decomposition is also measured.
9) (a) Collard, J.; Matthew, A. M.; Double, J. A.; Bibby, M. C. Br. J.
(
(6) The decomposition pattern of 1a in pH 7.4 was nearly identical in the
presence of gluathione suggesting that 1a was not highly reactive with thiols.
Cancer 1995, 71, 1199. (b) Kelland, L. R.; Sharp, S. Y.; Rogers, P. M.;
Myers, T. G.; Workman, P. J. Natl. Cancer Inst. 1999, 91, 1940.
B
Org. Lett., Vol. XX, No. XX, XXXX

Scheme 2. Synthesis of Nitric Oxide Prodrug Candidates 1a and 1b
Figure 1. (a) Decomposition profiles of 1a and 1b in pH 7.4 in
the presence and absence of DT-D were determined by HPLC
analysis of reaction mixtures. (b) Nitric oxide release profiles of
1
a and 1b in pH 7.4 in the presence and absence of DT-D were
1
0
analyzed by chemiluminescence-based assay for NO. (c) Nitric
oxide released during independent incubation of 1a and 1b
with cultured DLD-1 human adenocarcinoma cells for 5 h was
estimated by analysis of an aliquot of extracellular medium
Figure 2. (a) Representative images of Hela cells labeled with anti-
γ-H2AX antibody in immunofluorescence assays after treatement
with 1a for 1.5 h. Nuclei were counterstained with DAPI. Arrow
indicates γ-H2AX foci (Scale bar: ∼5 μm). (b) Average foci per
HeLa nucleus upon treatment with increasing concentrations
of 1a.
1
1
using a chemiluminescence-based assay.
The efficacy of 1a to inhibit proliferation of DLD-1 cells
was evaluated using a standard cell viability assay after
incubation for 72h. We found 1ato be a potent inhibitor of
proliferation of DLD-1 cells (Figure 3a), and its IC50 was
found as 0.25 μM (Figure 3b). A similar potent antiproli-
ferative activity of INDQ/NO was found when tested against
human urinary bladder carcinoma T-24 (IC50 = 0.56 μM)
and HeLa human cervical cancer (IC = 0.96 μM) cells
both known to overexpress DT-D.
Next, in order to study the effect of introduction of a
methyl group on the stability of 4,7-dioxoindole-based
5
0
1
3
(10) The yield of NO was 30% (assuming DEA/NO produces
mol NO/mol) suggesting that NO was trapped before it could be
(11) An aliquot of the reaction mixture is treated with an aqueous
solution of NaI and acetic acid, which converts nitrite to NO (which is
quantified by a chemiluminescence detector).
2
detected.
Org. Lett., Vol. XX, No. XX, XXXX
C

that introduction of a substituent at the 2-position of the
,7-dioxoindole scaffold resulted in a slower DT-D meta-
4
1
4
bolism rate. A cell viability assay showed that 1b was a
good inhibitor of proliferation of DLD-1 cells with an IC50
of 3.0 μM, but this was 10-fold higher than the IC50 for 1a
(0.25 μM, Figure 3b). When tested against T-24 renal
carcinoma cells, the IC50 of 1b was 9.4 μM, again, dimin-
ished in comparison with 1a. Together, these results sug-
gest that the propensity to undergo metabolism by DT-D
to produce NO played a role in the observed inhibitory
potency of 1a. Further evidence for NO-mediated cytotoxic
effects was obtained when indolequinones 8a and 8b, which
were not sources of NO, were found to have diminished
inhibitory potency against DLD-1 cells (see Supporting
15
Information, Table S4).
DT-D forms the basis for targeted delivery of a number
of bioreductive prodrugs of cytotoxic species, including
DNA alkylating agents, which are in development for use
4
in targeting hypoxic tumors. Approximately one-third of
human tumors contain areas with hypoxia in comparison
Figure 3. (a) Cancer cell proliferation inhibitory activity of 1a
was determined using a standard cell viability assay against
DLD-1 human adenocarcinoma, T-24 human renal carcinoma,
and HeLa human cervical carcinoma cells. (b) IC50 of 1a against
4
with normal cells. As oxygen is a potent radiosensitizer,
hypoxic tumors are often less responsive to radiation
2
DLD-1 cells was obtained by curve fitting (R = 0.996,
16
treatment. Due to a diminished and chaotic blood sup-
P < 0.0001) as 0.25 μM. (c) Nitric oxide release profile of 1a
in pH 6.5 in the presence and absence of DT-D was analyzed by a
chemiluminescence-based assay for NO.
ply, hypoxic areas are poorly accessible to cancer drugs
and hence chemotherapeutics also have diminished effi-
4
e
cacy in shrinking hypoxic tumors. Together, these effects
lead to higher levels of treatment failures in tumors with
significant areas of hypoxia. NO is also a chemical radio-
sensitizer and has been shown to synergize with radiation
diazeniumdiolate, we synthesized 1b from 2b (Scheme 2).
In comparison with 1a, the analogue 1b was a poor
substrate for metabolism by DT-D (Figure 1a); did not
produce significant amounts of NO in the presence of
DT-D (Figure 1b); and generated diminished amounts of
nitrite during incubation with DLD-1 cells (Figure 1c).
Diminished decomposition of 1b by DT-D in comparison
with 1a is consistent with previous reports which indicate
1
7
to promote apoptosis in hypoxic cells. Furthermore, due
to its highly diffusible nature, NO can penetrate cells
1
8
leading to enhancement in cytotoxic effects. As hypoxic
regions are acidic in nature, we evaluated the ability of 1a
to produce NO in pH 6.5. We found that 1a was capable of
producing NO in the presence of DT-D in pH 6.5 suggest-
ing that INDQ/NO may have potential in targeting hy-
poxic tumors as well (Figure 3c).
(
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Acknowledgment. This work was supported in part by
IISER Pune. H.C. acknowledges financial support from
the Department of Biotechnology (DBT, Grant No.
BT/05/IYBA/2011). K. Sengupta acknowledges an Inter-
mediate Fellowship from the Wellcome Trust-DBT
2001, 166, 2500.
(
14) (a) Naylor, M. A.; Jaffar, M.; Nolan, J.; Stephens, M. A.; Butler,
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research fellowship from the Council for Scientific and
Industrial Research (CSIR). A.I. acknowledges the fellow-
ship from WT-DBT India Alliance. The authors thank
Mr. K. R. Vignesh, IISER Pune for help with scale-up of
certain synthetic steps. DEA/NO was a gift from Dr. Larry
Keefer (National Cancer Institute at Frederick) and
Dr. Joseph Saavedra (SAIC-Frederick).
(
15) 3-(Acetoxymethyl)-5-methoxy-1,2-dimethyl-1H-indole-4,7-dione
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produced upon production of NO, might contribute to the cytotoxicity of
1
a. See ref 5g.
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Supporting Information Available. Preparative proce-
dures, spectral characterization data, and assay condi-
tions. This material is available free of charge via the
Internet at http://pubs.acs.org.
(
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The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX