Multiple bond-conjugated photoinduced nitric oxide releaser working with two-photon excitation

Article abstract of DOI:10.1016/j.bmcl.2009.10.120

Four novel nitric oxide (NO) releasers working via two-photon excitation (TPE), based on an acceptor-donor-acceptor (A-D-A) molecular design, were synthesized. Their decomposition and NO release in response to one-photon excitation, and their decomposition in response to two-photon excitation were examined. Their photoinduced decomposition characteristics are discussed.

Full text of DOI:10.1016/j.bmcl.2009.10.120

Bioorganic & Medicinal Chemistry Letters 20 (2010) 302–305
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multiple bond-conjugated photoinduced nitric oxide releaser working with
two-photon excitation
b
c
a
Kazuhiro Hishikawa a, Hidehiko Nakagawa a, , Toshiaki Furuta , Kiyoshi Fukuhara , Hiroki Tsumoto ,
*
Takayoshi Suzuki a, Naoki Miyata a,c,
*
a Contribution from the Graduate School of Pharmaceutical Science, Nagoya City University, 3-1 Tanabe-dori Mizuho-ku, Nagoya, Aichi 467-8603, Japan
b Department of Biomolecular Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 247-8510, Japan
c Division of Organic Chemistry, National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel nitric oxide (NO) releasers working via two-photon excitation (TPE), based on an acceptor–
donor–acceptor (A–D–A) molecular design, were synthesized. Their decomposition and NO release in
response to one-photon excitation, and their decomposition in response to two-photon excitation were
examined. Their photoinduced decomposition characteristics are discussed.
Received 24 June 2009
Revised 24 October 2009
Accepted 27 October 2009
Available online 30 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Nitric oxide
Photoinduced release
Two photon excitation
Acceptor–donor–acceptor
Electron spin trapping
Nitric oxide (NO) is an endogenous endothelium-derived relax-
ing factor (EDRF), and plays many important biological roles in
mammals, including regulation of blood pressure, neuromodula-
tion, and biodefense.1 However, research into the functions of NO
is difficult, because NO is an unstable free radical. To overcome this
difficulty, many NO releasers have been developed,2 but most of
them release NO by spontaneous decomposition, so that it is diffi-
cult to control NO release precisely. Controllable NO releasers,
which would permit temporal and spatial control of NO release,
would be candidates for superior physiologically active reagents
and therapeutic agents. To synthesize such compounds, we
adopted photo-irradiation as the trigger for NO release.
We previously reported photoinduced NO release from 6-nitro-
benzo[a]pyrene (6-nitroBaP)3 via isomerization of the nitro group
to nitrite ester, followed by homolytic cleavage. In the case of 6-
nitroBaP, the isomerization reaction is easy, since the nitro group
is in a non-planar relationship to the pyrene group due to the
hydrogen atoms at the two peri-positions. We also found photoin-
duced NO release from 4-substituted-2,6-dimethylnitrobenzenes.4
In these nitrobenzenes, the nitro group is also non-planar with
respect to the benzene ring due to the steric effect of the two
methyl groups at the ortho-positions, and isomerization of the ni-
tro group can occur easily by photoactivation.
However, the maximum absorption band for NO release from
2,6-dimethylnitrobenzenes is in the UV–A range, which could be
harmful to living cells. To overcome this potential problem, we
adopted the two-photon excitation (TPE) technique, which offers
the advantages not only of excitation at longer wavelength, but
also high spatial resolution. Though several NO releasers utilizing
TPE have been reported,5 those molecules contain a nitrosyl-che-
lated transition metal ion, which is potentially harmful to living
cells and is usually metabolically unstable. We recently reported
the first TPE-active NO releaser without a nitroxyl moiety, which
was designed to contain the fluorescein group as the two-photon
absorbing moiety.6 Here, we report new TPE-active NO releasers
based on a different molecular design.
We designed four NO releasers based on acceptor–donor–
acceptor (A–D–A) structure (Fig. 1), which is well known to be
suitable for TPE.7 In these molecules, 2,6-dimethylnitrobenzene
moieties work as acceptors and NO releasing moieties, and 1,4-
dimethoxybenzene moieties as receptors. The two acceptor–donor
pairs were connected with a conjugated multiple bond linker. TB1-
DNB and TB2-DNB contain one and two triple bonds, respectively.
DB1-DNB and DB2-DNB contain one and two double bonds,
respectively.
The preparation of phosphate ester 5 is shown in Scheme 1. To
obtain carboxylic acid 2, 2-nitromesitylene 1 was oxidized with
chromium(VI) oxide. Reduction of 2 with sodium tetrahydroborate
gave alcohol 3. The hydroxyl group of 3 was brominated to give 4,
* Corresponding authors.
E-mail address: deco@phar.nagoya-cu.ac.jp (H. Nakagawa).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.120

K. Hishikawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 302–305
303
Me
Me
O2N
Me
O2N
Me
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
n
n
Me
Me
NO2
TB1-DNB (n=1)
TB2-DNB (n=2)
DB1-DNB (n=1)
DB2-DNB (n=2)
NO2
Me
Me
Figure 1. Chemical structures of 2,6-dimethylnitrobenzene derivatives designed and synthesized as TPE-mediated NO releasers.
Me
Me
OMe
OMe
OMe
Me
O2N
Me
OHC
OHC
OHC
O2N
Me
O2N
Me
a
b
a
b
OH
I
Me
COOH
OMe
TMS
OMe
OMe
2
3
1
Me
Me
7
9
10
O2N
Me
O2N
Me
c
d
c
Br
PO(OEt)2
4
5
Me
Me
O2N
Me
O2N
Me
OMe
Scheme 1. Reagents and conditions: (a) CrO3, AcOH; (b) NaBH4, BF3 etherate; (c)
PBr3; (d) P(OEt)3, (n-Bu)4NI, 120 °C.
OMe
OMe
d
O
Me
Me
B
OMe
O
which was converted into phosphate ester 5 by means of the Arbu-
zov reaction.
Synthesis of the intermediate 8 is shown in Scheme 2. Iodin-
ation of 2,5-dimethoxybenzaldehyde 6 gave iodide 7. Conversion
of aldehyde 7 into alkene 8 was achieved via Horner–Wads-
worth–Emmons reaction with 5.
Me
Me
11
12
Scheme 3. Reagents: (a) trimethylsilylacetylene, PdCl2, PPh3, CuI, piperidine; (b)
2 N-NaOH aq, MeOH, CHCl3; (c) 5, NaH, THF; (d) pinacolborane, Cp2ZrHCl, CH2Cl2.
Preparation of the intermediates 11 and 12 is shown in Scheme
3. Iodide 7 was converted into alkyne 9 via Sonogashira coupling.
The TMS group of 9 was removed by hydrolysis with aqueous
NaOH solution to give 10, which was converted into 11 by means
of Horner–Wadsworth–Emmons reaction with 5. Treatment of
alkyne 11 with Schwartz reagent and pinacolborane gave borate
12.
Intermediate 15 was prepared as shown in Scheme 4. After con-
version of iodide 7 into alkene 13 via the Heck reaction, 13 was
treated with N-iodosuccinimide to give iodide 14. Aldehyde 14
was converted into 15 by means of Horner–Wadsworth–Emmons
reaction with 5.
Preparation of TB1-DNB, TB2-DNB, DB1-DNB, and DB2-DNB is
shown in Scheme 5. TB1-DNB was prepared from iodide 8 and al-
kyne 11 via Sonogashira coupling. TB2-DNB was synthesized from
two molecules of 11 by the use of Glaser coupling. DB1-DNB was
prepared from 8 and 12, and DB2-DNB was prepared from 12
and 15, both via Suzuki–Miyaura coupling.
First, we measured the absorption spectra of the four
compounds in DMSO solution (Fig. 2). The absorption maximum
wavelengths of TB1-DNB and TB2-DNB were shorter than those
of DB1-DNB and DB2-DNB, probably because sp2 carbons in the
linker double bond of the latter compounds are more efficiently
conjugated with sp2 carbons in benzene rings, as compared with
the sp carbons in the former compounds (Table 1).
OMe
OMe
OMe
OHC
OHC
OHC
a
b
I
TMS
I
OMe
OMe
OMe
14
13
7
c
Me
O2N
Me
OMe
I
OMe
15
Scheme 4. Reagents: (a) vinyltrimethylsilane, Pd(OAc)2, PPh3, AgNO3, Et3N, MeCN;
(b) N-iodosuccimide, MeCN; (c) 5, NaH, THF.
To investigate NO release from the four compounds through the
single photon excitation pathway, DMSO solutions of TB1-DNB,
TB2-DNB, DB1-DNB, and DB2-DNB were irradiated with 400–
440 nm or 450–480 nm light (from a 100 W Hg lamp with suitable
band pass filters) in the presence of Fe–DETC complex, which traps
NO to yield an Fe–DETC–NO complex that shows triplet signals at
around 330 mT in 4 GHz ESR spectrometry. After irradiation, the
ESR spectra of the solutions showed typical triplet signals due to
the Fe–DETC–NO complex, suggesting that all of the compounds
released NO upon photo-irradiation (data are shown in Supple-
mentary data). The amounts of NO released from the compounds
were evaluated from the ESR spectra (Table 2). In the case of
15 min irradiation, TB1-DNB and TB2-DNB released NO in 9–10%
molar ratio, but DB1-DNB and DB2-DNB released only a little
Me
OMe
OMe
O2N
Me
OMe
OMe
OHC
OHC
b
a
I
I
OMe
OMe
7
8
6
Scheme 2. Reagents: (a) I2, AgNO3, MeOH; (b) 5, NaH, THF.

304
K. Hishikawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 302–305
Table 1
Me
Absorption maximum wavelength of the four compounds and molar absorbance
coefficient at this wavelength
O2N
Me
OMe
OMe
Compounds Absorption maximum
wavelength (nm)
Molar absorbance efficient
(L molÀ1 cmÀ1
OMe
OMe
)
a
8 + 11
TB1-DNB
TB2-DNB
DB1-DNB
DB2-DNB
324, 421
323, 430
339, 445
352, 459
3.5 Â 104, 6.5 Â 104
3.1 Â 104, 7.0 Â 104
2.3 Â 104, 6.7 Â 104
1.9 Â 104, 7.7 Â 104
Me
NO2
Me
TB1-DNB
Me
O2N
Me
OMe
Table 2
Comparison of released NO
OMe
OMe
Compounds Amount of released Rate of released NO to concentration of
OMe
b
NO (
lM)
the compoundc (%)
11 + 11
23.9a
24.1a
9.2a
Me
TB1-DNB
TB2-DNB
DB1-DNB
DB2-DNB
9.7a
2.61a, 2.13b
2.40a, 1.36b
1.0a, 0.86b
0.96a, 0.54b
NO2
Me
TB2-DNB
a
b
c
Photoirradiation wavelength is 400–440 nm.
Photoirradiation wavelength is 450–480 nm.
Rate at 250 lM donor compound.
Me
O2N
Me
OMe
OMe
OMe
OMe
c
NO. According to the UV–vis spectra, the absorption coefficients of
DB1-DNB and DB2-DNB are larger than those of TB1-DNB or TB2-
DNB in this irradiation range. Low efficiency of NO release from
DB1-DNB and DB2-DNB might be due to insufficient excitation
energy to isomerize the nitro group to nitrite ester in the irradia-
tion range of 450–480 nm. It is considered that photo isomeriza-
tion of the dimethylnitrobenzene moiety would require 440 nm
or shorter wavelength photoirradiation.
We next examined TPE-based decomposition of the four com-
pounds. DMSO solutions of TB1-DNB and TB2-DNB were irradiated
at 800 nm to 860 nm and DMSO solutions of DB1-DNB and DB2-
DNB were irradiated at 800 nm to 880 nm with a pulse laser (Tsu-
nami; Spectra-Physics), and the products were analyzed by HPLC.
In this wavelength range, none of the four compounds showed con-
ventional single photon absorption. A parameter of two-photon
decomposition efficiency, the two-photon uncaging cross section
(du)8, was calculated for each compound, and the results are shown
in Figure 3. It was found that TB2-DNB was decomposed to the
greatest extent among the four compounds. Although there is little
difference between TB1-DNB and TB2-DNB in one-photon excita-
8 + 12
Me
NO2
Me
DB1-DNB
Me
O2N
Me
OMe
OMe
OMe
c
12 + 15
OMe
Me
NO2
Me
DB2-DNB
Scheme 5. Reagents: (a) Pd(PPh3)4, CuI, AgNO3, Et3N, THF; (b) Cu(OAc)2, THF–
pyridine; (c) PdCl2(PPh3)2, NaOEt, toluene.
Figure 2. UV–vis spectra of TB1-DNB, TB2-DNB, DB1-DNB, and DB2-DNB.
Concentrations of all compounds are 10
l
M in DMSO.
Figure 3. Two-photon uncaging cross section (du) spectra of the four compounds.

K. Hishikawa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 302–305
305
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research (No. 17590089 to H.N.) from the Ministry of Education,
Culture, Sports Science, and Technology, Japan, Takeda Science
Foundation (H.N.), and the Nagoya City University Special Fund
for Research Promotion (H.N.).
Figure 4. ESR spectra of [(DETC)2–Fe–NO] complex after 840 nm pulse laser
irradiation in the presence of TB2-DNB. Samples contained 250 M TB2-DNB, 8 mM
DETC, and 2 mM FeSO4 in DMSO; ESR spectra were recorded after photoirradiation
for 8 min with a modulation width of 0.5 G and a microwave power of 10 mW.
l
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.120.
tion, there is a marked difference in TPE. It seems that TB2-DNB
collects photons more efficiently than TB1-DNB because of its lar-
ger conjugation area.
We also demonstrated TPE-based NO release from TB2-DNB
which is the most decomposed compound by TPE. A DMSO solu-
tion of TB2-DNB with Fe–DETC complex was irradiated 840 nm
pulse laser and the solution was measured by ESR. The ESR spectra
of the solution showed typical triplet signals assigned as the Fe–
DETC–NO complex, suggesting that TB2-DNB released NO by TPE
(Fig. 4).
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In conclusion, we demonstrated photoinduced NO generation
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DNB and TB2-DNB, containing triple bonds, were more
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