Photoinduced nitric oxide release from nitrobenzene derivatives

Article abstract of DOI:10.1021/ja0512024

A new type of photoinduced nitric oxide (NO) donors was designed from nitrobenzene derivatives. Visible-light irradiation of 2,6-dimethylnitrobenzenes bearing extended π-electron systems at the 4-position revealed efficient NO release using ESR analysis a

Full text of DOI:10.1021/ja0512024

Published on Web 07/28/2005
Photoinduced Nitric Oxide Release from Nitrobenzene
Derivatives
Takayoshi Suzuki,† Osamu Nagae,† Yuka Kato,† Hidehiko Nakagawa,†
Kiyoshi Fukuhara,‡ and Naoki Miyata*,†
Contribution from the Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity,
3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan, and the DiVision of Organic
Chemistry, National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan
Received February 25, 2005; E-mail: miyata-n@phar.nagoya-cu.ac.jp
Abstract: A new type of photoinduced nitric oxide (NO) donors was designed from nitrobenzene derivatives.
Visible-light irradiation of 2,6-dimethylnitrobenzenes bearing extended π-electron systems at the 4-position
revealed efficient NO release using ESR analysis and the Griess assay. Computational study and ultraviolet
spectrum analysis suggested that the NO-releasing activity was closely related to the conformation of the
nitro group, the absorption intensity, and the length of the conjugated π-electron system. Employing the
photodependent cytotoxicity of compound 14 against HCT116 human colon cancer cells, it was demonstrated
that 4-substituted-2,6-dimethylnitrobenzene analogues are useful NO donors for the time- and site-controlled
NO treatment.
Introduction
and potential therapeutic application, it appeared desirable to
liberate NO in living systems in a time- and site-controlled
Nitric oxide (NO), a simple diatomic free radical, has proven
in recent years to be involved in the maintenance and regulation
of vital functions1 and is one of the most fascinating and studied
compounds in biological chemistry, although for decades it was
merely viewed as an environmental pollutant. The development
and use of NO donors have played important roles in research
on NO physiology,2 and the significance of these donors has
recently been reaffirmed not only from the perspective of
reagents for biological studies but also with a view to their
application as pharmaceuticals.3 To date, a number of NO donors
have been developed2,4 and utilized. However, many of the
currently used NO donors such as 1-hydroxy-2-oxo-3-(ami-
noalkyl)-1-triazenes (NOCs)5 and 4-alkyl-2-hydroxyimino-5-
nitro-3-hexenes (NORs)6 are reagents that release NO by
spontaneous autolysis. For further research on NO physiology
manner. This concept led to the identification of several
photochemically triggered NO donors such as metal nitrosyl
compounds7 and some caged nitric oxides.8 The duration and
site of NO release from these compounds can be controlled by
changing the interval and position of light exposure. However,
some of these photoinducible NO donors have problems
associated with stability and toxicity. For example, the NO-
releasing rate of metal nitrosyl compounds such as dipotassium
pentachloronitrosylruthenate and sodium nitroprusside (SNP)
varies with changes in pH, and SNP has toxic consequences
attributable to the cyanide ligand.9
We previously found 6-nitrobenzo[a]pyrene (6-nitroBaP)
(Figure 1) to be a photoinducible NO-releasing agent10 whose
NO-releasing mechanism is completely different from those of
well-known photochemically triggered NO donors. 6-NitroBaP
releases NO with the concomitant formation of 6-oxylBaP radi-
cal under visible-light irradiation (Figure 1). Based on the fact
that 1-nitroBaP and 3-nitroBaP with sterically less hindered nitro
† Nagoya City University.
‡ National Institute of Health Sciences.
(1) (a) Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G.
Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9265-9269. (b) Palmer, R. M.;
Ferige, A. G.; Moncada, S. Nature 1987, 327, 524-526. (c) Stamler, J. S.;
Singel, D. J.; Loscalzo, J. Science 1992, 258, 1898-1902.
(2) For a review, see: Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.;
Cai, T.; Janczuk, A. J. Chem. ReV. 2002, 102, 1091-1134.
(3) (a) Janero, D. R.; Ewing, J. F. Free Radical Biol. Med. 2000, 29, 1199-
1221. (b) Buergler, J. M.; Tio, F. O.; Schulz, D. G.; Khan, M. M.; Mazur,
W.; French, B. A.; Raizner, A. E.; Ali, N. M. Coron. Artery Dis. 2000, 11,
351-357.
(6) (a) Thomas, G.; Ramwell, P. W. Biochem. Biophys. Res. Commun. 1989,
164, 889-893. (b) Kato, M.; Nishino, S.; Ohno, M.; Fukuyama, S.; Kita,
Y.; Hirasawa, Y.; Nakanishi, I.; Takasugi, H.; Sakane, K. Bioorg. Med.
Chem. Lett. 1996, 6, 33-38. (c) Fukuyama, S.; Hirasawa, Y.; Kato, Y.;
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(5) (a) Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.; Saavedra,
J. E.; Hoffmann, A.; Bove, A. A.; Isaac, L.; Hrabie, J. A.; Keefer, L. K. J.
Med. Chem. 1991, 34, 3242-3247. (b) Hrabie, J. A.; Klose, J. R.; Wink,
D. A.; Keefer, L. K. J. Org. Chem. 1993, 58, 1472-1476. (c) Saavedra, J.
E.; Shami, P. J.; Wang, L. Y.; Davies, K. M.; Booth, M. N.; Citro, M. L.;
Keefer, L. K. J. Med. Chem. 2000, 43, 261-269. (d) Davies, K. M.; Wink,
D. A.; Saavedra, J. E.; Keefer, L. K. J. Am. Chem. Soc. 2001, 123, 5473-
5481.
(8) (a) Makings, L. R.; Tsien, R. Y. J. Biol. Chem. 1994, 269, 6282-6285.
(b) Kwon, N. S.; Lee, S. H.; Choi, C. S.; Kho, T.; Lee, H. S. FASEB J.
1994, 8, 529-533. (c) Singh, R. J.; Hogg, N.; Joseph, J.; Kalyanaraman,
B. FEBS Lett. 1995, 360, 47-51. (d) Namiki, S.; Arai, T.; Fujimori, K. J.
Am. Chem. Soc. 1997, 119, 3840-3841. (e) Namiki, S.; Kaneda, F.;
Ikegami, M.; Arai, T.; Fujimori, K.; Asada, S.; Hama, H.; Kasuya, Y.;
Goto, K. Bioorg. Med. Chem. 1999, 7, 1695-1702.
(9) Shishido-Silvia, M.; Ganzarolli de Oliveira, M. Prog. React. Kinet. 2001,
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9
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J. AM. CHEM. SOC. 2005, 127, 11720-11726
10.1021/ja0512024 CCC: $30.25 © 2005 American Chemical Society

Photoinduced Nitric Oxide Release
A R T I C L E S
the 4-position may increase the NO-releasing ability by stabiliz-
ing the oxylradical which is speculated to be formed concomi-
tantly with the generation of NO.10 Compounds with one methyl
group (12) and without a methyl group (13) were also prepared
as reference compounds.
ESR Analysis. Detection of NO released from nitrobenzene
derivatives was carried out by an ESR spin-trapping method
with N-methyl-D-glucamine dithiocarbamate (MGD)-Fe2+ com-
plex, which reacts with NO to give a [(MGD)2-Fe2+-NO]
stable paramagnetic complex.12 On ESR spectroscopy, the com-
plex is observed as a broadened three-line spectrum consisting
of aN ) 1.25 mT and giso ) 2.04. The prepared 2, 6-dimeth-
ylnitrobenzene derivatives were photoirradiated in aqueous
DMSO and subsequently subjected to ESR spectroscopy.
No ESR signal was detected with compounds 1, 2, and 3
bearing an OH and the NMe2 group at the 4-position of 2,6-
dimethylnitrobenzene, respectively (data not shown), whereas
a 5-min photoirradiation13 of compound 4 bearing a styrenyl
group at the 4-position exhibited a characteristic three-line
spectrum on ESR (Figure 4A), which indicates that NO was
released from compound 4 by photoirradiation. NO-releasing
activity was distinctly dependent on the two methyl groups
introduced at the ortho position to the nitro group on the phenyl
ring. The lack of the methyl groups (12 and 13) led to a much
less potent NO donor and a compound devoid of the ability to
release NO, respectively (Figure 4). We next evaluated the ESR
signal intensity of compound 4 and its derivatives 5-11 after
1-, 3-, and 5-min photoirradiation (Figure 5). All of the tested
compounds were shown to release NO during these photoirra-
diation periods. Time-dependent increases in the ESR signal
intensity indicated the photolytic generation of NO from these
compounds. With the extension of the conjugated π-electron
system (see 5-9) the NO-releasing activity increased except
for that of compound 7, which was less potent than 4. Com-
pounds with longer conjugated π-electron systems showed
greater activity when comparing 4 or 8 with 9. Next, we studied
the effect of substituents at the other phenyl ring of compound
4. It was shown that 3′,5′-dimethoxy compound 11 was more
potent, while 2′,6′-dimethyl analogue 10 significantly reduced
the activity.
Figure 1. 6-NitroBaP as a photoinducible NO-releasing agent.
Figure 2. Structures of 1-, 3-, and 6-nitroBaP.
groups did not release NO (Figure 2) and the results of
computational studies by our group10 as well as another,11 it
was suggested that the nonplanar torsional conformation of the
nitro substituent in relation to the aromatic ring is important
for the light-induced release of NO. In view of this finding,
regulation of the nitro group conformation could give simpler
nitrobenzene derivatives the ability to release NO. Contributing
to this line of thought, we report that some nitrobenzene
derivatives with extended π-electron conjugations have potent
NO-releasing activity in response to visible-light irradiation. A
simulation study and ultraviolet spectrum analysis of these
compounds suggested that the NO-releasing activity was closely
related to the conformation of the nitro group, the absorption
wavelength, and the length of the conjugated π-electron system.
Employing the photodependent cytotoxicity of compound 14
against HCT116 human colon cancer cells, it was demonstrated
that nitrobenzene derivatives are useful as time- and site-
controlled NO donors.
Griess Assay. For further confirmation of NO production
via the photolysis of nitrobenzene derivatives, the Griess
-
method14 was used to evaluate production by detecting NO2
resulting from NO autoxidation in aqueous solution. The visible
absorption at 546 nm of the red color formed upon diazo
coupling of the Griess reagents allowed for evaluation of the
NO formed after photolysis of the nitrobenzene derivatives.
After photoirradiation for 1 and 2 h, the absorbance at 546 nm
was measured.
Results
Molecular Design. As NO donor candidates, we focused on
2,6-dimethylnitrobenzene derivatives, which are expected to
have nonplanar conformations of the nitro group in their simple
structures (Figure 3). Electron-donating groups (2 and 3) and
olefins (4-11, and 14) were chosen as substituents at the
4-position so that the derivatives would have maximum absorp-
tion at a longer wavelength, because it is desirable that
compounds release NO under visible-light irradiation without
affecting the living organisms. In addition, the extension of
conjugated π-electron systems by the introduction of olefins at
The Griess assay was used to investigate the NO-releasing
efficiency of 4, 5, 8, and 9, positive compounds in the ESR
assay, and 12 and 13, negative compounds in the ESR assay
(Figure 6). Compounds 4, 5, 8, and 9 bearing two methyl groups
at the ortho positions of the nitro group released NO in a time-
(12) (a) Komarov, A.; Mattson, D.; Jones, M. M.; Singh, P. K.; Lai, C. S.
Biochem. Biophys. Res. Commun. 1993, 195, 1191-1198. (b) Pieper, M.
G.; Lai, C. S. Biochem. Biophys. Res. Commun. 1996, 219, 584-590.
(13) Compounds prepared for this study were irradiated using a 300-W
photoreflector lamp that produces light having a wavelength range of >300
nm.
(14) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J.
S.; Tannenbaum, S. T. Anal. Chem. 1982, 54, 131-138.
(11) Glenewinkel-Meyer, T.; Crim, F. F. J. Mol. Struct. 1995, 337, 209-224.
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A R T I C L E S
Suzuki et al.
Figure 3. Structures of nitrobenzene derivatives.
Figure 4. ESR spectra of [(MGD)2-Fe-NO] complex after photoirradia-
tion in the presence of compounds 4, 12, and 13. Samples contained 1 mM
4 (A), 12 (B), or 13 (C), 75 mM MGD, and 20 mM FeSO4 in distilled
water (containing 25% DMSO); ESR spectra were recorded after photoir-
radiation for 5 min with a modulation amplitude of 2.0 G and a microwave
power of 16 mW.
dependent manner under visible light, while an extreme reduc-
tion in the NO-releasing activity was observed with compounds
having a single methyl group (12) and lacking a methyl group
(13). On comparing the four positive compounds (4, 5, 8, and
9), the longer their conjugated π-electron systems, the better
NO donors they were. The results from the Griess assay were
consistent with those obtained by ESR analysis.
To determine the chemical yield of the NO-releasing reaction,
compound 9, the most active compound in both the ESR and
Griess assays was subjected to a longer period of photoirradia-
tion and the NO-release rate was measured by the Griess method
(Figure 7). The generation of NO from compound 9 increased
linearly until 6 h of photoirradiation and then reached a plateau.
The yield of NO formation for the photochemical reaction
determined by the Griess method was 55%.
Figure 5. ESR signal intensities of [(MGD)2-Fe-NO] complex after
photoirradiation in the presence of compounds 4-11. Samples contained 1
mM 4-11, 75 mM MGD, and 20 mM FeSO4 in water (containing 25%
DMSO); ESR spectra were recorded after photoirradiation for 1, 3, or 5
min with a modulation amplitude of 2.0 G and a microwave power of 16
mW.
induced from nitrobenzene derivatives by photoirradiation can
function as a cytotoxic agent. For the application of our NO
donors in a biological study, we prepared compound 14. In the
ESR and the Griess assays, the NO-releasing activity of
compound 14 was comparable to that of compound 9 (see
Supporting Information), the most potent compound among all
those prepared for this study. Compound 14 has the advantage
of solubility in aqueous media compared with compound 9. It
Cytotoxicity Against Cancer Cells. It has been reported that
NO is a mediator of the cytotoxic action of macrophages toward
tumor cells through inhibition of mitochondrial enzyme activity
and DNA synthesis.15 Therefore, we determined whether NO
(15) Stuehr, D. J.; Gross, S. S.; Sakuma, I.; Levi, R.; Nathan, C. F. J. Expl.
Med. 1989, 169, 1011-1020.
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Photoinduced Nitric Oxide Release
A R T I C L E S
Figure 6. Griess assay results for compounds 4, 5, 8, 9, 12, and 13 (n )
3, mean (SD).
Figure 8. Cytotoxic activity of NO released from compound 14 under
photoirradiation. Representative phase-contrast images of cells within the
irradiation area in the absence (a) and the presence (c) of P450 nor are
shown. Cells at 3.5 mm away from the irradiation center in the absence (b)
and the presence (d) of P450 nor are also presented. The experiments were
duplicated, and at least 4 areas were observed in each culture plate. In all
experiments, the same results were obtained.
indicating that the stable products after liberation of NO or
photoinduced decomposition are nontoxic.
Discussion
MM2 calculations and the ultraviolet-visible-light absorption
spectra measurements were carried out to determine which
characteristic is necessary for NO generation under visible-light
irradiation (Table 1).
Figure 7. NO-release rate of compound 9 (n ) 3, mean (SD).
It is assumed that a nitroarene gives rise to NO via
nitritearene, which is produced from the nitroarene by an
intramolecular rearrangement mechanism (Figure 9), and the
nonplanar torsional conformation of the nitro group in relation
to the aromatic ring gives an advantage in the nitro-to-nitrite
rearrangement.10,11 Therefore, the dihedral angle between the
plane of the nitro group and the phenyl ring (∆O1N2C3C4) for
compounds 1-13 was initially calculated to clarify the effect
of the two methyl groups on the conformation of the nitro group.
The dihedral angles for dimethyl compounds 1-11 were around
45°, while those of monomethyl 12 and no-methyl 13 were 0°.
Considering this calculation result and the fact that compounds
12 and 13 did not generate NO efficiently, it was suggested
that two methyl groups at the ortho positions of the nitro group
are indispensable for inclining the nitro group to the plane of
the phenyl ring and for the subsequent intramolecular rear-
rangement from nitro to nitrite.
was more easily available for the cellular experiments as a
DMSO solution. In the presence of a 2.5 µM concentration of
compound 14, light of 330-380 nm wavelength was shed for
1 min on a fixed area of a culture plate containing human colon
cancer HCT116 cells.16 The cells were observed after a 24-h
incubation at 37 °C under dark conditions. As shown in Figure
8, the cells had detached and appeared dead within the area of
irradiation (Figure 8a), which indicates that the cytotoxicity was
quite dependent on the photoirradiation. Treatment with con-
pound 14 without photoirradiation had no effect on the cells in
24 h. The photoirradiation alone without compound 14 also had
no effect. This cytotoxic effect was diminished in the presence
of NADH and P450 nor, an NO reductase (Figure 8c), which
confirmed that NO was mainly responsible for the cytotoxicity
against the HCT 116 cells. There were no changes at a position
3.5 mm from the center of irradiation, i.e., outside the photo-
irradiation area, regardless of the absence (Figure 8b) or the
presence of P450 nor (Figure 8d). Preirradiation to the solution
of compound 14 for 30 min abolished its cytotoxic effects,
Another important factor is the intensity of light energy
absorption, which is considered to be essential for the excitation
of compounds or further torsional conformation change of the
nitro group.11 Indeed, compounds with a longer conjugated
π-electron system, which were expected to have maximum
absorption at a longer wavelength, were inclined to enhance
(16) NO release from compound 14 under assay conditions was confirmed by
the Griess Method (see Supporting Information).
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J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11723

A R T I C L E S
Suzuki et al.
Table 1. Results of MM2 Calculations and Ultraviolet Absorption
Measurements
Longer conjugated π-electron systems can also contribute to
the resonance stabilization of the oxylradicals generated by N-O
bond fission of the nitrite. The destabilization of the oxylradical
caused by the lack of a π-conjugated system may be the reason
that compounds 3, 7, and 10 did not have a significant ability
to release NO.
Unfortunately, the oxylradical, which is considered to be
formed concomitantly with the generation of NO, could not be
detected because of its instability. However, the result of the
cellular assay using compound 14 indicated that the degradation
products derived from the oxylradical did not appear to interfere
with the biological NO experiment. Therefore, nitrobenzene
derivatives may be useful NO donors, with the advantage that
the time of light exposure, the duration of NO release, and the
site of NO action can be controlled.
O1N2C3C4 a
(deg)
C5C6C7C8 b
(deg)
λ
(nm)
max
ꢀ
max
ESR signalc
1
compds
(×
104 M-1 cm-
)
(×
102 au)
1
2
3
4
5
6
7
8
44.8
43.6
44.4
44.6
44.6
44.7
44.6
44.6
44.6
44.7
44.7
0.l
255
354
396
307
329
320
389
289
332
287
308
343
349
0.14
0.14
0.65
2.4
2.0
2.7
1.1
1.4
4.3
1.6
2.6
2.2
2.6
nd
nd
nd
30.3
50.3
30.3
79.8
2.0
2.8
3.6
0.4
2.5
6.0
0.6
3.0
0.4
nd
All the synthesized compounds were very stable in crystal
forms under a dark condition. It was also confirmed that the
photoinduced cytotoxic effect of compound 14 was maintained
at least for 4 weeks when stored at -20 °C as a concentrated
solution in DMSO. This stability of the compounds in storage
appears to give usefulness in the biological applications as
NO-releasing reagents.
9
28.5
66.7
32.1
30.0
30.0
10
11
12
13
0.0
a Dihedral angle (deg) between the planes of the nitro group and the
phenyl moiety. b Dihedral angle (deg) between the ethylene moiety and the
aromatic ring. c ESR signal intensity of [(MGD)2-Fe-NO] complex after
5-min irradiation. nd ) not detected.
Conclusion
In the present study, we characterized several 4-substituted-
2,6-dimethylnitrobenzenes as a new type of NO donor. The
computational study and ultraviolet-visible-light spectrum
analysis also highlighted the significance of the conformation
of the nitro group, the intensity of light energy absorption, and
the length of the conjugated π-electron system. More impor-
tantly, one of our compounds demonstrated its activity as an
NO donor in a biological study.
Figure 9. Predicted mechanism for NO generation from 4-substituted-2,6-
dimethylnitrobenzene derivatives.
the NO-releasing activity. The results of the ultraviolet-visible-
light spectroscopic investigation of nitrobenzene derivatives
were in good agreement with those obtained by the ESR assay.
Specifically, all of the compounds with an adequate ability to
provide NO (4, 5, 6, 8, 9, and 11) had strong absorption in the
range of irradiating light (wavelength of >300 nm), while
compounds having weak absorption (1 and 3) under these
conditions did not generate NO although they had two methyl
groups at the ortho positions of the nitro group. These results
support the idea that NO is released from nitrobenzene deriva-
tives by light energy absorption and not by any other factor.
Experimental Section
General Methods. Melting points were determined using a
Yanagimoto micromelting point apparatus or a Bu¨chi 545 melting point
apparatus and were left uncorrected. Proton nuclear magnetic reso-
nance spectra (1H NMR) were recorded on a JEOL JNM-LA400
spectrometer with CDCl3 as the solvent. Chemical shifts (δ) were
reported in parts per million relative to the internal standard tetram-
ethylsilane. Elemental analysis was performed with a Yanaco CHN
CORDER NT-5 analyzer, and all values were within (0.4% of the
calculated values. High-resolution mass spectra (HRMS) were recorded
on a JEOL JMS-SX102A mass spectrometer. GC-MS analyses were
performed on a Shimadzu GCMS-QP2010. Ultraviolet-visible-light
spectra were recorded on a HITACHI U-3000 spectrophotometer or
an Agilent 8453 spectrophotometer. Compound 1 was purchased from
Wako Pure Chemical Industries, and MGD was obtained from
DOJINDO. All other reagents and solvents were purchased from
Aldrich, Tokyo Kasei Kogyo, Wako Pure Chemical Industries, and
Kanto Kagaku and used without purification. Flash column chroma-
tography was performed using Silica Gel 60 (particle size 0.046-0.063
mm) supplied by Merck. In ESR analysis and the Griess assay,
irradiation was performed through a Pyrex filter with a 300-W
photoreflector lamp.
However, compounds 7 and 10, which have two methyl
groups, had adequate absorption at wavelengths of >300 nm
and seemed to have long conjugated π-electron systems, hardly
having any ability to release NO. The calculated dihedral angle
between the ethylene moiety and the aromatic ring (∆C5C6C7C8)
of compounds 7 and 10 was 79.8° and 66.7°, respectively. These
values were greater than those of other compounds. This
suggested that compounds 7 and 10 have difficulty in creating
a planar structure, and therefore there is little conjugation
between the two aromatic rings. It can be concluded that the
lack of a conjugated π-electron system made it difficult for
compounds 7 and 10 to absorb the light energy necessary for
generating NO.17
3,5-Dimethyl-4-nitrophenol (2). Compound 2 was prepared by a
previously reported method:18 mp 109-111 °C; 1H NMR (CDCl3, 400
MHz, δ; ppm) 6.56 (2H, d, J ) 0.49 Hz), 5.19 (1H, s), 2.30 (6H, s).
MS (EI) m/z: 167 (M+). Anal. Calcd for C8H9NO3: C, 57.48; H, 5.43;
N, 8.38. Found: C, 57.33; H, 5.55; N, 8.61.
(17) In the case of compound 7, the absorption at 386 nm, which is due to the
anthracene ring, has little to do with NO generation since the nitrobenzene
moiety and the anthracene ring are not conjugated.
(18) Gaude, D.; Le Goaller, R.; Pierre, J. L. Synth. Commun.1986, 16, 63-68.
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11724 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005

Photoinduced Nitric Oxide Release
A R T I C L E S
(3,5-Dimethyl-4-nitrophenyl)dimethylamine (3). Compound 3 was
prepared by a previously reported method:19 mp 115-116 °C; 1H NMR
(CDCl3, 400 MHz, δ; ppm) 6.31 (3H, s), 3.01 (6H, s), 2.37 (6H, s).
MS (EI) m/z: 194 (M+). Anal. Calcd for C10H14N2O2: C, 61.84; H,
7.27; N,14.42. Found: C, 61.71; H, 7.30; N, 14.58.
6.62 (4H, m), 6.40 (1H, s), 3.83 (6H, s), 2.33 (6H, s). MS (EI) m/z:
339 (M+). Anal. Calcd for C19H19NO3: C, 73.77; H, 6.19; N, 4.53.
Found: C, 73.63; H, 6.23; N, 4.55.
Compounds 4-13 were prepared from the corresponding dieth-
ylphosphonates and aldehydes using the procedure described for 14
(step 1′).
1,3-Dimethyl-2-nitro-5-[(1E,3E)-4-(3,5-dimethoxyphenyl)-1,3-
butadienyl]benzene (14). Step 1: Preparation of 3,5-Dimethyl-4-
nitrobenzylbromide. To phosphorus tribromide (1.16 g, 4.29 mmol)
was added 3,5-dimethyl-4-nitrobenzyl alcohol20 (489 mg, 2.70 mmol)
under argon with cooling by an ice bath, and the solution was stirred
for 5 h at room temperature. The reaction mixture was poured into
water and extracted with CHCl3. The CHCl3 layer was separated,
washed with brine, and dried over Na2SO4. Filtration and concentration
in vacuo and purification by silica gel flash chromatography (n-hexane/
AcOEt ) 9/1) gave 610 mg (93%) of 3,5-dimethyl-4-nitrobenzylbro-
1
(E)-3,5-Dimethyl-4-nitrostilbene (4): mp 101-103 °C; H NMR
(CDCl3, 400 MHz, δ; ppm) 7.52 (2H, d, J ) 7.3 Hz), 7.38 (2H, dd, J
) 7.1, 7.8 Hz), 7.32-7.28 (1H, m), 7.25 (2H, s), 7.14 (1H, d, J )
16.3 Hz), 7.02 (1H, d, J ) 16.3 Hz), 2.35 (6H, s). MS (EI) m/z: 253
(M+). Anal. Calcd for C16H15NO2: C, 75.87; H, 5.97; N, 5.53. Found:
C, 75.83; H, 6.12; N, 5.62.
1-[(E)-2-(3,5-Dimethyl-4-nitrophenyl)ethenyl]naphthalene (5): mp
1
164-165 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 8.20 (1H, d, J )
8 Hz), 7.94-7.83 (3H, m), 7.74 (1H, d, J ) 7.3 Hz), 7.57-7.49 (3H,
m), 7.34 (2H, s), 7.07 (1H, d, J ) 16.1 Hz), 2.39 (6H, s). MS (EI) m/z:
303 (M+). Anal. Calcd for C20H17NO2: C, 79.19; H, 5.65; N, 4.62.
Found: C, 79.30; H, 5.67; N, 4.66.
1
mide as a pale yellow solid: mp 49-50 °C; H NMR (CDCl3, 400
MHz, δ; ppm) 7.15 (2H, s), 4.40 (2H, s), 2.31 (6H, s). MS (EI) m/z:
243 (M+, 79Br), 245 (M+, 81Br).
2-[(E)-2-(3,5-Dimethyl-4-nitrophenyl)ethenyl]naphthalene (6): mp
147-149 °C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.88-7.82 (4H,
Step 2: Preparation of Diethyl 3,5-Dimethyl-4-nitrobenzylphos-
phonate. A mixture of 3,5-dimethyl-4-nitrobenzylbromide (694 mg,
2.84 mmol) obtained above, tetrabutylammonium iodide (77 mg, 0.21
mmol), and triethyl phosphite (596 mg, 3.59 mmol) was stirred for 6
h at 120 °C. The reaction mixture was subjected to silica gel flash
chromatography (CHCl3/MeOH ) 100/1) to give 873 mg (q.y.) of 3,5-
dimethyl-4-nitrobenzylphosphonate as a pale yellow oil: 1H NMR
(CDCl3, 400 MHz, δ; ppm) 7.06 (2H, d, J ) 2.4 Hz), 4.10-4.02 (4H,
m), 3.09 (2H, d, J ) 22.2 Hz), 2.30 (6H, s), 1.28 (6H, t, J ) 7.1 Hz).
MS (EI) m/z: 301 (M+). HRMS: calcd for C13H20NO5P, 301.108;
found, 301.108.
1
m), 7.72 (1H, dd, J ) 1.7, 8.8 Hz), 7.52-7.46 (2H, m), 7.31 (1H, d,
J ) 16.3 Hz), 7.30 (2H, s), 7.14 (1H, d, J ) 16.34), 2.37 (6H, s). MS
(EI) m/z: 303 (M+). Anal. Calcd for C20H17NO2: C, 79.19; H, 5.65;
N, 4.62. Found: C, 78.96; H, 5.70; N, 4.62.
9-[(E)-2-(3,5-Dimethyl-4-nitrophenyl)ethenyl]anthracene (7): mp
219-221 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 8.44 (1H, s), 8.31-
8.28 (2H, m), 8.05-8.02 (2H, m), 7.98 (1H, d, J ) 16.6 Hz), 7.52-
7.46 (4H, m), 7.42 (2H, s), 6.90 (1H, d, J ) 16.6 Hz), 2.41 (6H, s).
MS (EI) m/z: 353 (M+). Anal. Calcd for C24H19NO2: C, 81.56; H,
5.42; N, 3.96. Found: C, 81.70; H, 5.75; N, 3.86.
Step 1′: Preparation of 3-(3,5-Dimethoxyphenyl)acrylonitrile. To
a suspension of sodium hydride (60%, 1.27 g, 31.8 mmol) in THF (30
mL) was added a solution of cyanomethylphosphonic acid diethyl ester
(500 mg, 2.82 mmol) in THF (10 mL) under argon with cooling by an
ice bath, and the solution was stirred for 15 min at room temperature.
To the mixture was added to 3,5-dimethoxybenzaldehyde (656 mg, 3.95
mmol), and the reaction mixture was stirred for 17 h at room
temperature. The mixture was poured into water and extracted with
CHCl3. The CHCl3 layer was separated, washed with brine, and dried
over Na2SO4. Filtration and concentration in vacuo and purification
by silica gel flash chromatography (n-hexane/AcOEt ) 4/1) gave 833
mg (q.y.) of 3-(3,5-dimethoxyphenyl)acrylonitrile as a white solid; 1H
NMR (CDCl3, 400 MHz, δ; ppm) 7.32 (1H, d, J ) 16.5 Hz), 6.57
(2H, d, J ) 2.2 Hz), 6.53 (1H, t, J ) 2.2 Hz), 5.85 (1H, d, J ) 16.6
Hz), 3.81 (6H, s).
1,3-Dimethyl-2-nitro-5-(1,3-petadienyl)benzene (8): yellow oil; 1H
NMR (CDCl3, 400 MHz, δ; ppm) 7.09 (2H, s), 6.80-6.73 (1H, m),
6.33 (1H, d, J ) 15.8 Hz), 6.25-6.18 (1H, m), 5.94-5.88 (1H, m),
2.31 (6H, s), 1.83 (3H, d, J ) 6.8 Hz). MS (EI) m/z: 217 (M+).
HRMS: Calcd for C13H15NO2, 217.110; found, 217.110.
1,3-Dimethyl-2-nitro-5-[(1E,3E)-4-phenyl-1,3-butadienyl]ben-
zene (9): mp 126-130 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.45
(2H, d, J ) 7.1 Hz), 7.35 (2H, dd, J ) 7.3, 7.8 Hz), 7.26 (1H, m), 7.17
(2H, s), 7.104-6.903 (2H, m), 6.73 (1H, d, J ) 14.6 Hz), 6.57 (1H, d,
J ) 14.9 Hz), 2.33 (6H, s). MS (EI) m/z: 279 (M+). Anal. Calcd for
C18H17NO2: C, 77.40; H, 6.13; N, 5.01. Found: C, 77.17; H, 6.29; N,
5.03.
5-[(E)-2-(2,6-Dimethylphenyl)ethenyl]-1,3-dimethyl-2-nitroben-
zene (10): mp 147-149 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm)
7.88-7.82 (4H, m), 7.72 (1H, dd, J ) 1.7, 8.8 Hz), 7.52-7.46 (2H,
m), 7.31 (1H, d, J ) 16.3 Hz), 7.30 (2H, s), 7.14 (1H, d, J ) 16.3 Hz),
2.37 (6H, s). MS (EI) m/z: 303 (M+). Anal. Calcd for C20H17NO2: C,
79.19; H, 5.65; N, 4.62. Found: C, 78.96; H, 5.70; N, 4.62.
5-[(E)-2-(3,5-Dimethoxyphenyl)ethenyl]-1,3-dimethyl-2-nitroben-
zene (11): mp 104-105 °C; 1H NMR (CDCl3, 400 MHz, δ; ppm) 7.24
(2H, s), 7.07 (1H, d, J ) 16.4 Hz), 6.99 (1H, d, J ) 16.4 Hz), 6.67
(2H, d, J ) 2.2 Hz), 6.43 (1H, t, J ) 2.2 Hz), 3.85 (6H, s), 2.35 (6H,
s); MS (EI) m/z: 313 (M+); Anal. Calcd for C18H19NO4: C, 68.99; H,
6.11; N, 4.47. Found: C, 68.79; H, 6.11; N, 4.57.
Step 2′: Preparation of 3-(3,5-Dimethoxyphenyl)propenal. To a
solution of 3-(3,5-dimethoxyphenyl)acrylonitrile (1.31 g, 7.56 mmol)
obtained above in toluene (30 mL) was added diisobutyl aluminum
hydride (1.0 M, 15.1 mL, 15.1 mmol) under argon at -50 °C, and the
solution was stirred for 22 h at room temperature. The mixture was
poured into 1 N aqueous HCl and extracted with AcOEt. The AcOEt
layer was separated, washed with brine, and dried over Na2SO4.
Filtration and concentration in vacuo and purification by silica gel flash
chromatography (n-hexane/AcOEt ) 4/1) gave 378 mg (26%.) of
3-(3,5-dimethoxyphenyl)propenal as
a
pale yellow crystal; 1H
(E)-3-Methyl-4-nitrostilbene (12): mp 85-87 °C; 1H NMR (CDCl3,
400 MHz, δ; ppm) 8.03 (1H, d, J ) 8.5 Hz), 7.54 (2H, dd, J ) 1.5,
7.3 Hz), 7.48-7.38 (4H, m), 7.35-7.31 (1H, m), 7.23 (1H, d, J )
16.6 Hz), 7.09 (1H, d, J ) 16.6 Hz), 2.66 (3H, s). MS (EI) m/z: 239
(M+). Anal. Calcd for C15H13NO2: C, 75.30; H, 5.48; N, 5.85. Found:
C, 75.14; H, 5.44; N, 6.03.
NMR (CDCl3, 400 MHz, δ; ppm) 9.70 (1H, d, J ) 10.9 Hz), 7.41
(1H, d, J ) 15.9 Hz), 6.71-6.65 (3H, m), 6.55 (1H, t, J ) 2.3 Hz),
3.83 (6H, s).
Step 3: Preparation of 1,3-Dimethyl-2-nitro-5-[(1E,3E)-4-(3,5-
dimethoxyphenyl)-1,3-butadienyl]benzene (14). Compound 14 was
prepared from diethyl 3,5-dimethyl-4-nitrobenzylphosphonate obtained
in step 2 and 3-(3,5-dimethoxyphenyl)propenal obtained above using
the same procedure described in step 1′ in a 55% yield: mp 132-133
1
(E)-4-Nitrostilbene (13): mp 161-162 °C; H NMR (CDCl3, 400
MHz, δ; ppm) 8.22 (2H, ddd, J ) 2, 2.4, 6.8 Hz), 7.64 (2H, ddd, J )
2, 2.4, 8.8 Hz), 7.56 (2H, d, J ) 7.1 Hz), 7.42-7.39 (2H, m), 7.36-
7.31 (1H, m), 7.28 (1H, d, J ) 16.3 Hz), 7.15 (1H, d, J ) 16.3 Hz).
MS (EI) m/z: 225 (M+). Anal. Calcd for C14H11NO2: C, 74.65; H,
4.92; N, 6.22. Found: C, 74.65; H, 4.92; N, 6.22.
1
°C; H NMR (CDCl3, 400 MHz, δ; ppm) 7.16 (2H, s), 6.93 (2H, m),
(19) Jones, M. E.; Taft, R. W.; Kamlet, M. J. J. Am. Chem. Soc. 1977, 99,
8452-8453.
9
J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11725

A R T I C L E S
Suzuki et al.
ESR Analysis. The Fe2+ complex of MGD [Fe2+-MGD2, (Fe-
MGD)] was used to trap NO. Fresh stock solutions of Fe-MGD (1:4)
were prepared by adding ferrous ammonium sulfate to an aqueous
solution of MGD. A sample containing 1 mM of compounds 1-14
and 20 mM Fe-MGD in distilled water (25% DMSO) was introduced
to a quartz flat curette cell. ESR spectra were recorded after light
irradiation at a distance of 10 cm with a JES-RE 2X spectrometer (JEOL
Co. Ltd., Tokyo, Japan). The spectrometer settings were modulation
frequency, 100 kHz; modulation amplitude, 2.0 G; scan time, 4 min;
microwave power, 16 mW; and microwave frequency, 9.42 GHz.
Griess Assay. The Griess reagents were prepared by mixing acetic
acid (5 mL), sulfanilic acid (500 mg), N-1-naphthylethylenediamine
dihydrochloride (50 mg), and distilled water (95 mL). A sample
containing 200 µM compounds 4, 5, 8, 9, 12, or 13 in o-dichlorobenzene
(2 mL) and distilled water (1 mL) was photoirradiated for the specified
times at a distance of 12 cm. The mixture was treated with the Griess
reagents (1 mL), and it was agitated for 15 min. The aqueous layer
was separated with a fixed angle centrifuge for 5 min at 5000 rpm,
and the absorbance at 546 nm was measured. The results were expressed
as the percentile of the conversion ratio of the testing compounds.
Cytotoxicity Against HCT116 Cells. HCT116 human colon cancer
cells were purchased from American Type Culture Collection (ATCC,
Manassas, VA) and cultured in McCoy5A culture medium containing
penicillin and streptomycin, supplemented with fetal bovine serum as
described in the ATCC instructions. The cells were maintained at 37
°C in a humidified 5% (v/v) CO2 incubator under sub-confluent
conditions. For the experiments, the cells were plated at 2.5 × 105
cells per 6-cm culture dish 2 days before treatments and incubated at
37 °C. On the day of the experiment, the culture medium was refreshed
and reduced to 2 mL per 6-cm culture dish. The cells were then treated
with compound 14 in 2 µL of DMSO giving a final concentration of
2.5 µM, which resulted in a final DMSO concentration in the culture
media of less than 0.1% (v/v), at which concentration there were no
apparent effects from the DMSO. The cells were also treated with
compound 14 in the same manner described above in the presence of
40 nM P450 nor (Wako Pure Chemical Industry Co. Ltd, Osaka, Japan)
and 2 µM NADH. The treated cells were subsequently subjected to
the photoirradiation for 1 min by using the light-source (100 W mercury
lamp) of a fluorescence microscope (Olympus BX60/BX-FLA with
UplanFl 10X objective lens) with a WU filter (330-380 nm band-
pass filter). The irradiation area was set as a circular area with a diameter
of around 3 mm. After 24 h incubation, the cells were observed under
an inverted phase-contrast microscope, and the images were taken with
a digital camera and processed with a personal computer.
MM2 Calculations. Force-field (MM2) minimizations of compounds
1-13 were performed using Macromodel 8.0 software.21 All structures
were fully optimized with each parameter set as follows: force field,
MM2*; method, LBFGS; max no. iterations, 10 000; converge on,
gradient; convergence threshold, 0.05.
Supporting Information Available: Ultraviolet-visible-light
spectra of compounds 1-14 and the ESR and Griess assay
results of compound 14 are reported. This material is available
free of charge via the Internet at http://pubs.acs.org.
JA0512024
(20) Goldstein, S. L.; McNelis E. J. Org. Chem. 1984, 49, 1613-1620.
(21) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
1990, 11, 440-467.
9
11726 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005