Hydroxyl radical generation via photoreduction of a simple pyridine N-oxide by an NADH analogue

Article abstract of DOI:10.1039/b509447j

Photoreduction of pyridine N-oxide, which has a key structure of antitumor agents for hypoxic solid tumors, by 1-benzyl-1,4-dihydronicotinamide in deaerated aprotic media resulted in generation of hydroxyl radical, leading to the oxidation of salicylic acid to 2,3- and 2,5-dihydroxybenzoic acids, and catechol. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b509447j

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C O M M U N I C A T I O N
Hydroxyl radical generation via photoreduction of a simple pyridine
N-oxide by an NADH analogue†
a,b
a,c
b
d
c
Ikuo Nakanishi,* Chiho Nishizawa, Kei Ohkubo, Keizo Takeshita, Kazuo T. Suzuki,
a
e
f
f
g
Toshihiko Ozawa, Sidney M. Hecht, Masayuki Tanno, Shoko Sueyoshi, Naoki Miyata,
f
b
a
f
Haruhiro Okuda, Shunichi Fukuzumi, Nobuo Ikota* and Kiyoshi Fukuhara*
a
Redox Regulation Research Group, Research Center for Radiation Safety, National Institute
of Radiological Sciences (NIRS), Inage-ku, Chiba, 263-8555, Japan.
E-mail: nakanis@nirs.go.jp; Fax: +81 43 255 6819; Tel: +81 43 206 3131
b
c
Department of Material and Life Science, Graduate School of Engineering, Osaka University,
SORST, Japan Science and Technology Agency (JST), Suita, Osaka, 565-0871, Japan.
E-mail: fukuzumi@chem.eng.osaka-u.ac.jp; Fax: +81 6 6879 7370; Tel: +81 6 6879 7368
Department of Toxicology and Environmental Health, Graduate School of Pharmaceutical
Science, Chiba University, Chuo-ku, Chiba, 260-8675, Japan
d
e
Faculty of Pharmaceutical Sciences, Sojo University, Ikeda, Kumamoto, 860-0082, Japan
Department of Chemistry and Biology, University of Virginia, Charlottesville, Virginia,
29901, USA
f
Division of Organic Chemistry, National Institute of Health Sciences (NIHS), Setagaya-ku,
Tokyo, 158-8501, Japan. E-mail: fukuhara@nihs.go.jp; Fax: +81 3 3707 6950;
Tel: +81 3 3700 1141
g
Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya,
Aichi, 467-8603, Japan
Received 5th July 2005, Accepted 22nd July 2005
First published as an Advance Article on the web 2nd August 2005
Photoreduction of pyridine N-oxide, which has a key struc-
ture of antitumor agents for hypoxic solid tumors, by 1-
benzyl-1,4-dihydronicotinamide in deaerated aprotic media
resulted in generation of hydroxyl radical, leading to the
oxidation of salicylic acid to 2,3- and 2,5-dihydroxybenzoic
acids, and catechol.
of dihydronicotinamide adenine dinucleotide (NADH) in DMF
under anaerobic conditions. The effects of the substituent at
the C-4 position of pyridine N-oxides on the mechanism of
photoinduced electron transfer from BNAH to pyridine N-
oxides as well as the reactivity of the corresponding radical
anions are clarified based on the spectral and electrochemical
data together with the calculated molecular structures by the
density functional method, providing a valuable insight into the
development of antitumor agents for hypoxic cells.
Recently, considerable effort has been made to develop effective
drugs against solid tumors, which exist under hypoxic (oxygen-
•
1,2
poor) conditions in inefficient vascular systems. Tirapazamine
3-amino-1,2,4-benzotriazine 1,4-di-N-oxide), which has a hete-
Salicylic acid (SA) was employed to detect OH generated in
(
the photoreaction of pyridine N-oxides with BNAH in deaerated
•
rocyclic N-oxide structure, is a clinically promising antitumor
DMF. SA reacts with OH to form 2,3-dihydroxybenzoic acid
3
agent against hypoxic cells. The DNA damage induced by
(2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA) as
8–13
tirapazamine is proposed to result from the generation of
major products and catechol as a minor product.
These
•
hydroxyl radical ( OH) or the direct oxidation of the deoxyribose
oxidized products of SA are stable and are readily isolated
and quantified by a reverse-phase HPLC equipped with an
electrochemical detector (HPLC-ECD). SA does not react with
backbone of DNA after one-electron reduction of the N-oxide
4–6
to form an activated intermediate. However, the actual DNA-
damaging species has yet to be clarified. On the other hand,
photosensitizers available for photodynamic therapy are advan-
tageous to localize the toxicity to a selected site (tumor cells),
•
−
•
O
2
at an appreciable rate as compared to OH. Although SA
1
also reacts with singlet oxygen ( O
2
), only 2,5-DHBA is formed
exclusively, instead of the mixture of 2,3-DHBA, 2,5-DHBA,
7
14
thus avoiding toxicity to normal cells. However, since most
and catechol.
photosensitizers require O
2
to produce reactive oxygen species,
Aprotic solvents, such as DMF and acetonitrile (MeCN) were
used because of the poor solubility of pyridine N-oxides toward
water, although the reactivity in aqueous media is important
for an in vivo situation. When a deaerated DMF solution of
they are not effective toward anaerobic solid tumors. Thus,
photoactivated compounds, which generate reactive oxygen
species under anaerobic conditions, are certainly required for
the development of drugs effective against solid tumors without
affecting normal cells.
−
3
−3
−3
−3
PyO (5.0 × 10 mol dm ) and BNAH (5.0 × 10 mol dm )
was irradiated with UV light (k > 290 nm) in the presence of
•
−2
−3
We report herein OH generation from a simple unsubstituted
SA (3.2 × 10 mol dm ), 2,3-DHBA, 2,5-DHBA, and catechol
were detected by the HPLC-EC analysis as shown in Fig. 1a. The
ratio of the yields of 2,5-DHBA, 2,3-DHBA, and catechol are
48:35:16. Irradiation of PyO or BNAH alone in the presence of
SA resulted in no formation of oxidized products of SA (Fig. 1c
and d). Under dark conditions, neither DHBA product nor
catechol was formed even in the presence of all the components,
i.e., PyO, BNAH, and SA (Fig. 1e). These results demonstrate
pyridine N-oxide (PyO), which has a largely negative reduction
potential, via one-electron reduction by photoexcited 1-benzyl-
1
,4-dihydronicotinamide (BNAH) used as a model compound
†
Electronic supplementary information (ESI) available: Transient ab-
sorption spectra of the photoreaction between BNAH and PyO (S1),
•
−
EPR spectrum of NO
between BNAH and NO
See http://dx.doi.org/10.1039/b509447j
2
PyO (S2), spectral change in the photoreaction
•
PyO (S3), and DFT minimized structures (S4).
that OH is generated in the photoreaction of PyO with BNAH
2
in deaerated DMF as shown in Scheme 1. In fact, addition of
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 6 3 – 3 2 6 5
3 2 6 3

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When PyO is replaced by 4-nitropyridine N-oxide (NO
2
PyO),
neither DHBA products nor catechol was produced after
irradiation of a deaerated DMF solution containing NO
BNAH, and SA (Fig. 1f).
2
PyO,
This result demonstrates that the radical anion of NO
2
PyO
PyO ) generated in photoinduced electron transfer from
BNAH to NO PyO is relatively stable and does not undergo the
N–O bond cleavage. In fact, an EPR spectrum of NO
which has a g value of 2.0054, was observed after irradiation
of a deaerated MeCN solution of NO PyO and BNAH (Fig.
S2a†). The hyperfine coupling constants (hfc) of the observed
•
−
(
NO
2
2
•
−
2
PyO ,
2
•
−
EPR spectrum of NO
2
PyO were determined by comparison of
the observed spectrum with the computer-simulated spectrum
(
Fig. S2b†) and they were assigned based on the reported hfc
•
−
18
values for NO PyO in DMF (Fig. S2†).
2
The UV-vis spectral titration (Fig. S3†) indicates that BNAH
acts as a two-electron donor to reduce 2 equiv. of NO PyO to
2
•
−
0
NO
2
PyO . Since the E ox value of BNAH* (−2.65 V) is more
Fig. 1 HPLC-ECD chromatograms of products formed during irradia-
0
18
negative than the E red value of NO
duced electron transfer from BNAH* to NO
give a radical ion pair of NO
2
PyO (−0.77 V), photoin-
−
3
−3
−3
−3
−3
tion (1 h) of (a) PyO (5.0 × 10 mol dm ), BNAH (5.0 × 10 mol dm ),
−
2
−3
−3
2
PyO occurs to
and SA (3.2 × 10 mol dm ); (b) PyO (5.0 × 10 mol dm ),
•
•
•
+
−
+
−
3
−3
−2
−3
2
PyO and BNAH . BNAH
undergoes deprotonation to give BNA . The delocalization of
an electron on the NO PyO molecule due to the electron-
BNAH (5.0 × 10 mol dm ), SA (3.2 × 10 mol dm ), and ethanol
•
−
1
−3
−3
−3
(
5.7 × 10 mol dm ); (c) PyO (5.0 × 10 mol dm ) and SA
•
−
−2 −3 −3 −3
(
(
1
3.2 × 10 mol dm ); (d) BNAH (5.0 × 10 mol dm ) and SA
2
−
2
−3
−3
−3
3.2 × 10 mol dm ); (e) PyO (5.0 × 10 mol dm ), BNAH (5.0 ×
withdrawing NO group may preclude the protonation of
2
−
3
−3
−2
−3
•−
0
mol dm ), and SA (3.2 × 10 mol dm ) without irradiation; (f)
−3 −3 −3 −3
2
NO
2
PyO . In fact, no hyperfine structure due to the N–OH
proton was observed in the EPR spectrum of NO
subsequent electron transfer from BNA to NO
NO
(
PyO (5.0 × 10 mol dm ), BNAH (5.0 × 10 mol dm ), and SA
•−
2
PyO . The
3.2 × 10− mol dm ) in deaerated DMF at 298 K.
2
−3
•
2
PyO may also
0
•
occur rapidly, judging from the E ox value of BNA (−1.05 V),
0
18
which is lower than the E red value of NO
2
PyO (−0.77 V). Thus,
once photoinduced electron transfer from BNAH to NO
occurs, 2 equiv of NO PyO are produced.
DFT calculations using B3LYP/6-31G* basis set for PyOH
2
PyO
•
−
2
•
•
and NO
2
PyOH were carried out to investigate the difference in
the reactivity of radical species of pyridine N-oxides depending
on a substituent at the C-4 position (Fig. S4†). The calculated
•
˚
N–O bond length in PyOH (1.50 A) is significantly longer
•
˚
than that in NO
2
PyOH (1.40 A). This suggests that the N–
O bond cleavage in PyOH may occur much easier than that
•
•
•
in NO
2
PyOH . Furthermore, PyOH is significantly bent as
indicated by the out-of-plane N–O bond bending angle (a) of
◦
•
◦
1
52 , whereas NO
2
PyOH is relatively flat (a = 169 ). Similar
results have been reported for the N–O bond fragmentation in
19
N-methoxy-substituted aromatic compounds. The N–O bond
cleavage requires mixing of p* and r* orbitals, which is achieved
by bending the N–O bond out of the plane of the aromatic ring.
In conclusion, photoreduction of a simple pyridine N-oxide
by BNAH in deaerated aprotic medium resulted in generation
Scheme 1
•
ethanol, which is a OH scavenger, resulted in a decrease in the
yield of three oxidized SA products (Fig. 1b).
Photoirradiation of BNAH is known to give the singlet excited
state, BNAH*. The fluorescence of BNAH* is quenched effi-
ciently by the addition of PyO in deaerated MeCN. The quench-
ing rate constant is determined as 1.4 × 10 mol dm s , which
is close to the diffusion limited value in MeCN. Nanosecond
laser excitation of a deaerated MeCN solution of PyO and
•
of OH, which can oxidize SA to 2,3-DHBA, 2,5-DHBA, and
1
5
catechol. The electron-withdrawing group such as NO on the
2
aromatic ring can significantly stabilize the radical anion of
1
0
−1
3
−1
•
pyridine N-oxide, precluding the subsequent OH release. We are
currently investigating the detailed effects of various substituents
on the photoreactivities of pyridine N-oxides in the presence of
various reducing agents.
•
−
BNAH results in the formation of PyO (kmax = 540 nm)
•
+
16
and BNAH (kmax = 380 nm) (see Electronic Supplementary
Information, Fig. S1†).
Acknowledgements
The reaction mechanism of electron-transfer reduction of
PyO by BNAH is shown in Scheme 1. Photoinduced electron
transfer from BNAH* to PyO takes place to produce a radical
This work was partially supported by Grant-in-Aids for Scien-
tific Research (A) (No. 16205020) and for Young Scientist (B)
•
+
•−
ion pair of BNAH and PyO , since the oxidation potential
(
No. 17790044) from the Ministry of Education, Culture, Sports,
0
17
of BNAH* (E ox = − 2.65 V vs. SCE) determined in DMF
Science and Technology, Japan and by the Budget for Nuclear
Research of the Ministry of Education, Culture, Sports, Science
and Technology, Japan based on the screening and counselling
Atomic Energy Commission.
is more negative than the reported reduction potential of PyO
0
18
•+
(
E
red = − 2.30 V) in DMF. Proton transfer from BNAH ,
•
−
•
•
thus produced, to PyO gives PyOH and BNA . The resulting
•
•
PyOH then undergoes the N–O bond cleavage to produce OH
and pyridine. Judging from the more positive oxidation potential
•
0
17
of BNA (E ox = − 1.05 V) inDMF than the reduction potential
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0
18
•
of PyO (E red = − 2.30 V), no electron transfer from BNA to
1
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•
of BNA may occur to give a dimer (BNA)
2
.
3
2 6 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 2 6 3 – 3 2 6 5

View Article Online
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0
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0
•
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9
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