Photoactivatable HNO-releasing compounds using the retro-Diels-Alder reaction

Article abstract of DOI:10.1039/b811985f

We synthesized hetero-Diels-Alder cycloadducts from acyl nitroso derivatives and 9,10-dimethylanthracene, to be photo-inducible HNO-releasing agents and found that introduction of conjugated nitroaromatic groups effectively enhanced the responsiveness of HNO release to UV-A irradiation; we confirmed photoinduced HNO formation by EPR and GCMS analysis. The Royal Society of Chemistry.

Full text of DOI:10.1039/b811985f

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Photoactivatable HNO-releasing compounds using the retro-Diels–Alder
reactionw
Yusuke Adachi, Hidehiko Nakagawa,* Kazuya Matsuo, Takayoshi Suzuki and
Naoki Miyata*
Received (in Cambridge, UK) 14th July 2008, Accepted 2nd September 2008
First published as an Advance Article on the web 23rd September 2008
DOI: 10.1039/b811985f
We synthesized hetero-Diels–Alder cycloadducts from acyl
nitroso derivatives and 9,10-dimethylanthracene, to be photo-
inducible HNO-releasing agents and found that introduction of
conjugated nitroaromatic groups effectively enhanced the
responsiveness of HNO release to UV-A irradiation; we con-
firmed photoinduced HNO formation by EPR and GCMS
analysis.
The potential pharmacological activity of nitroxyl (HNO) in
Scheme 1 HNO release through a photoinduced retro-DA reaction.
relation to nitric oxide (NO) has recently received much
attention. While NO is known to act as a vasodilator via
HNO via acyl nitroso formation. If HNO release could be
activation of soluble guanylate cyclase, HNO is reported to
regulated by photoirradiation, the controlled release would be
induce a variety of physiological effects such as positive
very useful for biological applications. We have already
inotropy, vasodilation, and cardioprotection through mecha-
nisms different from those of NO.1,2 Very recently, HNO was
developed photoinduced NO donors via a unique photo-
chemical reaction.11 For the purpose of improving the
also reported as an anticancer agent.3 In view of these benefits,
hetero-Diels–Alder cycloadducts formed from acyl nitroso
HNO donors are expected to be potentially useful as ther-
derivatives and 9,10-dimethylanthracene (9,10-DMA), we
apeutic agents in the treatment of cardiovascular diseases, and
designed and synthesized compounds bearing conjugated aro-
also as experimental pharmacological agents. Despite the
matic groups which would readily induce the retro-Diels–
potential activity of HNO, its detailed mechanisms and appli-
Alder (retro-DA) reaction by photoirradiation (Scheme 1).
cations have scarcely been studied since this compound is
The synthesized compounds were confirmed by spectral
difficult to handle due to its chemical instability.4 Because of
analyses, including 1H- and 13C-NMR, IR, and MS, and
this, HNO donors are indispensable for both HNO investiga-
tion and the design of therapeutic agents. Some compounds
We first evaluated photoinduced enhancement of HNO
elemental analysis (see ESIw).
such as Angeli’s salt, Piloty’s acid and cyanamide (NH2CN)
release from a known hetero-Diels–Alder cycloadduct (1)
have been reported to generate HNO under certain condi-
bearing a 4-nitrophenyl substituent (Chart 1). Direct detection
tions.5–7 However, their release of HNO is dependent on the
of HNO has not been established as yet due to its complicated
spontaneous degradation of the donors, which is not a con-
chemical properties. In this study, we adopted several indirect
trolled event. In addition, in some cases, the by-products have
methods to estimate photoinduced HNO generation. Firstly,
distinct biological activities of their own. Hence, the applica-
photoinduced retro-DA reactions were examined by measur-
tion of these compounds to biological systems has been quite
ing absorption spectra and proton nuclear magnetic resonance
restricted. It is clear that a variety of novel HNO donors is
(1H-NMR) spectra. By monitoring the unique absorption of
needed to aid in the research of HNO and its applications.
It is known that hydrolysis of acyl nitroso compounds
9,10-DMA at 398 nm, it was found that the formation of
9,10-DMA from 1 in acetonitrile–water solution (9 : 1) was
generates HNO, but these highly reactive forms must be
accelerated by ultraviolet-A (UV-A, 330–380 nm) irradiation
(Fig. 1). This acceleration was dependent on the light intensity.
generated in situ.8 Based on in situ acyl nitroso compound
formation, King et al. developed hetero-Diels–Alder cycload-
ducts.9,10 These are known to thermally decompose to release
1
It was also clarified by H-NMR analysis that photoirradia-
tion converted 1 into 9,10-DMA and 4-nitroaniline (Fig. 2, I).
The formation of 4-nitroaniline was suppressed by the
Graduate School of Pharmaceutical Sciences, Nagoya City University,
3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8673, Japan.
E-mail: deco@phar.nagoya-cu.ac.jp.
E-mail: miyata-n@phar.nagoya-cu.ac.jp; Fax: +81-52-836-3407;
Tel: +81-52-836-3408
w Electronic supplementary information (ESI) available: Details of the
synthesis of 1–6 and results of H-NMR, UV/Vis, EPR, and GC
analysis. See DOI: 10.1039/b811985f
Chart 1 Designed and synthesized molecules.
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Table 1 N2O formation
Conditions
N2O formationa/mmol
hn
2-MEb
10 min
20 min
30 min
Compounds
Angeli’s salt
1
—
—
+
+
—
+
+
—
+
+
—
—
—
+
—
—
+
—
—
+
0.254
0.203
0.261
NDc
0.081
0.223
NDc
0.085
0.122
NDc
0.424
0.373
0.484
NDc
0.198
0.364
NDc
0.215
0.260
NDc
0.533
0.472
0.594
NDc
0.329
0.484
NDc
0.346
0.426
NDc
5
6
a
Fig. 1 Conversion of cycloadducts in acetonitrile–water (9 : 1) was
calculated from the increase in the absorption of 9,10-DMA at
398 nm.
Calculation based on Angeli’s salt (1.5 mmol) decomposition accord-
ing to Hughes and Wimbledon.13 b 2-Mercaptoethanol (10 equiv.).
c
N2O was not detected.
addition of 2-mercaptoethanol (2-ME), probably because of a
nucleophilic attack on the carbonyl carbon of an acyl nitroso
derivative. These outcomes supported the photoinduced retro-
DA reaction of 1. Secondly, HNO trapping-electron para-
magnetic resonance (EPR) spectroscopy and gas chromato-
graphic (GC) analysis were performed to confirm HNO
generation.8–10,12 EPR spectra of a mixture of 1 and hemin
in dimethyl sulfoxide (DMSO)–water solution (9 : 1) showed
the typical three-line EPR signal of a ferrous nitrosyl complex
under anaerobic conditions. The reductive nitrosylation of a
ferric heme by HNO provides evidence for the release of HNO
from 1. GC analysis of the reaction headspace under anaerobic
conditions showed that photoirradiation of 1 in DMSO–water
(9 : 1) gave N2O formed via HNO dimerization and dehydra-
tion (Table 1).4 The amount of the formed N2O was calculated
and expressed as the equivalent amount of Angeli’s salt
(1.5 mmol). Although we also tried direct calibration of N2O
formation by using authentic N2O gas injection, it was difficult
to obtain reproducible quantitative results due to the water-
soluble property of N2O and the difficulty of the accurate
dilution of N2O gas.
The N2O formation was enhanced by photoirradiation and
was significantly reduced by the addition of 2-ME as an HNO
scavenging agent. Thus, it was revealed that 1 was able
to undergo a photoinduced retro-DA reaction to yield
HNO. However, 1 was unfortunately not stable even under
dark conditions at ambient temperature, and the photo-
enhancement effect of HNO release was small compared with
the amount of thermal release.
To improve the photo-enhancement effect, we evaluated the
substituent effect on the stability of cycloadducts under dark
conditions as well as photoinduced conversion by synthesizing
2–4. Although the UV-A irradiation might not be directly
applicable to cardiovascular disorders, this evaluation would
be helpful to confirm the efficiency of our strategy, and to
obtain basic data for improving the compounds to be acti-
vated by longer wavelengths. Nonetheless, these compounds
would be applicable to in vitro or cellular experiments, or
hopefully to skin cancer.
Fig. 2 I. 1H-NMR spectral change by photoinduced conversion of 1
in DMSO–water (9 : 1). Spectrum A was obtained just after preparing
the solution, and spectrum B shows the same solution after 10 min of
UV-A irradiation. Spectrum C shows the solution under the same
conditions as those for B except for the addition of 2-ME. Each peak
was labelled with a letter such as x, compound 1; y, 9,10-DMA; and z,
4-nitroaniline. Assignment of those peaks is shown in the ESI.w Peaks
labelled with an asterisk are the reaction product of compound 1 (or
acyl nitroso derivative) and 2-ME. II. EPR spectra of
5 in
DMSO–water (9 : 1) with hemin under anaerobic conditions (arrowed
signals). Spectra were measured either under dark conditions (top) or
under UV-A irradiation (bottom) in the presence of manganese as an
external standard.
Compounds 2 and 3 were more thermally stable than 1 and
were not activated by UV-A irradiation. As for 4, its tendency
to thermally degrade was the same as that of 1, but its
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promotion of HNO release by UV-A irradiation was signifi-
cantly lower (Fig. S1w). These results indicate that stability
largely depends on the steric hindrance of the substituent.
Introduction of conjugated aryl groups as substituents was
found to be necessary for the photoinduced retro-DA reaction.
Based on these findings, other sterically unhindered conju-
gated groups, such as nitrobiphenyl and nitrostyryl substitu-
ents, were introduced into cycloadducts 5 and 6. From
absorption studies, it was revealed that 5 was more stable
than 1 under dark conditions and that the retro-DA reaction
of 5 was facilitated by UV-A irradiation (Fig. 1). The change
in the absorption spectrum of 6 was too complicated to
determine its conversion into 9,10-DMA and the correspond-
ing amine. EPR spectra provided evidence for HNO release
from 5 and 6 (Fig. 2, II, and Fig. S3w). GC analysis indicated
that the stabilities of 5 and 6 were improved over 1 under dark
conditions and that they were significantly prompted to pro-
duce N2O by UV-A irradiation (Table 1). In particular,
photoinduced N2O formation of 5 by a 10 min irradiation
was 3-fold greater than that under dark conditions, whereas
N2O induction by photoirradiation of 1 was only 1.3-fold
greater. Moreover, the HNO-releasing ability of 5 under
photoirradiating conditions was nearly equal to that of
Angeli’s salt.
but the absorption coefficient was too low. Accordingly, the
40-nitrobiphenyl functional group was shown to be the most
suitable in view of its steric and electronic factors. To date,
there have been no observations published about photo-
activatable HNO donors or donors whose HNO-releasing
ability can be arbitrarily controlled. In this study, we assessed
the steric and electronic effects of conjugated aromatic groups
in terms of improvement of their stability under dark condi-
tions and their reactivity upon photoirradiation, and our
findings showed 5 to be a photoactivatable HNO-releasing
agent under UV-A irradiation. These results should contribute
to the development of more sophisticated HNO donors. As for
use in biological systems, the poor water solubility of the
synthesized compounds is one of the key problems to be
overcome, and further thermal stability is another one. We
are now working to improve the solubility and enhance the
stability of these compounds for future biological applications.
This work was supported in part by Grants-in-Aid for
Scientific Research (No. 17590089 for HN) from the Ministry
of Education, Culture, Sports Science, and Technology,
Japan, the Mochida Memorial Foundation for Medical and
Pharmaceutical Research (HN), the Kowa Life Science Foun-
dation (HN), and the Nagoya City University Special Fund
for Research Promotion (HN).
For 20 and 30 min irradiation, the photo-enhancement
effect became not apparent. This means that photoactivation
of the compound effectively occurred within the first 10 min,
and N2O formation through HNO was enhanced in this
duration. Photoinduced conversion of 5 into 9,10-DMA was
also nearly saturated after 10 min irradiation (Fig. S1w).
Improvement of the stability was assumed to cause an increase
of the energy of the photoinduced retro-DA reaction and a
decrease in HNO release rate. Because HNO dimerization to
form N2O is a second order reaction, N2O formation is highly
dependent on the HNO concentration. That is, the enhance-
ment effect on N2O formation was less apparent than that on
the conversion to 9,10-DMA. Factors affecting the conversion
efficiency by photoirradiation are under investigation.
Notes and references
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Nonetheless, these results demonstrated that aromatic-
group-substituted cycloadducts 1 and 4–6 were capable of
releasing HNO and that the release of HNO was accelerated
by UV-A irradiation. Among the cycloadducts, the HNO
generation of 5 was the most facilitated. These differences
probably reflect their respective absorption ranges and steric
hindrances. The local maximum absorption wavelength of 5
was 337 nm, while those of 1 and 6 were 324 and 397 nm,
respectively. Since the wavelength of the utilized UV-A ranged
from 330 to 380 nm, 5 was able to effectively absorb UV-A
energy. The maximum wavelength of 4 was similar to that of 5,
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Chem. Commun., 2008, 5149–5151 | 5151