A fluorescent naphthalenediimide-alkoxyfuroxan photoinduced nitric oxide donor

Article abstract of DOI:10.1246/bcsj.20180219

We describe the design and facile synthesis of a fluorescent alkoxyfuroxan naphthalenediimide (NDI) hybrid nitric oxide (NO) donor molecule which releases NO under spatiotemporal control when irradiated with visible light (420600 nm). The hybrid also demonstrates cellular uptake made possible by its fluorescence capability. This research consolidates our recent work into visible light absorbing photosensitised furoxan NO release by addressing the limitations of previous designs. Key points of photoinduced nitric oxide donors are highlighted such as water solubility, NO release, cellular uptake, fluorescence, and longer absorption wavelengths.

Full text of DOI:10.1246/bcsj.20180219

Advance Publication Cover Page
A Fluorescent Naphthalenediimide-Alkoxyfuroxan Photoinduced Nitric Oxide Donor
Christopher Peter Seymour, Akito Nakata, Motonari Tsubaki, Masahiko Hayashi, and Ryosuke Matsubara*
Advance Publication on the web October 13, 2018
doi:10.1246/bcsj.20180219
© 2018 The Chemical Society of Japan
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A Fluorescent Naphthalenediimide-Alkoxyfuroxan Photoinduced Nitric Oxide Donor
Christopher Peter Seymour,1 Akito Nakata,1 Motonari Tsubaki,1 Masahiko Hayashi1 and Ryosuke Matsubara*1
1Department of Chemistry, Graduate School of Science, Kobe University, Nada, Kobe 657-8501, Japan
E-mail: matsubara.ryosuke@people.kobe-u.ac.jp
Ryosuke Matsubara
Ryosuke Matsubara received Ph.D. from the University of Tokyo in 2007 under the supervision of Prof. Shu Kobayashi.
He was appointed as an assistant professor at the University of Tokyo in 2005. After post-doctoral research with Professor
Timothy F. Jamison at Massachusetts Institute of Technology for two years from 2009 to 2011, he became an associate
professor at Kobe University. His current research interest is photochemical reaction and its application.
Abstract
We describe the design and facile synthesis of a fluorescent
irradiation (Scheme 1a). Constrained with the short UV
wavelengths required we consolidated on the furoxan PINOD
chemistry by tethering a fluorofuroxan to a visible light
absorbing water-soluble anthraquinone molecule 5 (Scheme
1b).23 Compound 5 demonstrated photosensitised NO release
under spatiotemporal control. Furthermore, we showed that NO
release occurs even in the absence of added thiol under
irradiation, in contrast to the general notion that a thiol cofactor
is required for the NO release from furoxans.24 These results led
us to suspect that NO release from irradiated furoxan is due to a
combination of two distinctive reaction pathways: one is attack
of a nucleophile at the more reactive 3-X regioisomer (X =
electronegative group) then followed by NO release25 and the
other is discharge of NO from a reactive transient intermediate
formed during photochemical isomerization.
alkoxyfuroxan naphthalenediimide (NDI) hybrid nitric oxide
(NO) donor molecule which releases NO under spatiotemporal
control when irradiated with visible light (420–600 nm). The
hybrid also demonstrates cellular uptake made possible by its
fluorescence capability. This research consolidates on our recent
work into visible light absorbing photosensitised furoxan NO
release by addressing the limitations of previous designs. Key
points of photoinduced nitric oxide donors are highlighted such
as water solubility, NO release, cellular uptake, fluorescence,
and longer absorption wavelengths.
Keywords: Nitric oxide donor, Photoreaction, Furoxan
1. Introduction
Nitric oxide (NO) is a reactive gaseous radical which
mediates a variety of biological effects such as vasodilation1,
neuromodulation2 and inhibition of platelet aggregation3. The
availability of NO is implicated in a variety of diseases such as
cardiovascular disease,4 male impotence5 and arthritis.6 Since
NO has a short lifetime and area of effect the administration of
NO gas is impractical so stable NO prodrugs have been
developed to deliver NO exogenously.7 Many families of NO
donors exist like S-nitrosothiols and NONOates which have led
to reliable NO release rates related to their structures.7-9 However,
current NO donors are limited by lack of site specific release.
Control over NO release is imperative since the concentration
and location of NO release can determine the type of biological
response.10
Photoswitchable nitric oxide donors (PINODs) address the
Scheme 1. Our previous results. (a) Isomerization of furoxans
under UV irradiation and subsequent NO release. (b)
Photosensitised isomerization and following NO release from
anthraquinone-tethered fluorofuroxan under visible light
irradiation.
issues of non-specific donors by releasing NO in
a
spatiotemporally controlled manner under irradiation. Despite
recent progress, there is little variety in PINODs and their release
mechanisms, especially those utilizing irradiation wavelengths
in the visible or near-IR region.11-20 Therefore, the development
of new PINODs with novel release mechanisms are required in
pursuit of controllable discharge of NO under biologically inert
conditions.
Recently our research group have made use of the NO
donor furoxan and synthesised a series of PINODs.21,22 4-Fluoro-
and 4-alkoxy-substituted furoxan derivatives displayed
isomerization and photoswitchable release of NO under UV
In retrospect of this research we encountered a few
limitations with the design and focused our efforts on the
synthesis of an improved molecule. Previously, we were unable
to determine whether or where compound 5 was localized within
a cell culture system and 5 was only sparingly soluble in
phosphate buffer solution. Furthermore, its synthesis presented
restrictions due to the labile nature of fluorofuroxan requiring

mild conditions during the synthesis scheme. We also decided
that although 400 nm irradiation wavelength is largely
biologically benign, the absorption wavelength of the sensitiser
should be even longer to assist with tissue penetration.
With these previous limitations in mind we sought for an
improved sensitiser-furoxan hybrid molecule having the
following desirable properties;
comparable quantities of NO as the lone fluorofuroxan moiety
does under UV irradiation.21,23 Therefore, we assumed this may
be the case for an NDI-furoxan molecule as well. We screened
potential furoxans by irradiating them with 300–400 nm light in
50 mM pH 7.4 phosphate buffer and compared the NO release
after 2 hours of irradiation (Table 1). With simplicity in mind we
selected furoxans that would be stable to nucleophilic aromatic
substitution conditions required for attachment to the NDI core.
1. It is cellular membrane-permeable,
2. It absorbs light of wavelength longer than 400 nm,
3. It is fluorescent,
Table 1. NO release of furoxan after 2 hours of irradiation (300–
4. It has improved water solubility,
400 nm) in 50 mM pH 7.4 phosphate buffer solution.
5. It shows photoswitchable NO release,
6. Its synthesis is facile not limited by furoxan lability.
The ability for a molecule to both fluoresce and undergo
intersystem crossing for triplet sensitization may initially seem
counterintuitive since the quantum yield of sensitization would
surely decrease. However, a balance can be obtained whereby
part of the absorbed energy is fluoresced and part is used for
sensitised NO release. This theranostic property is highly sought
after in phototherapeutic applications.26
One such sensitiser that displays fluorescent and triplet
sensitising character is the naphthalenediimide (NDI). NDIs are
attractive because their absorption and fluorescence can be fine-
tuned based on lateral core functionalisation.27 NDIs find
Substrate
NO2– released
(μM) a)
28
31
17b)
10c)
7
Entry
R1
R2
F
OMe
NMe2
CN
nC6H13
CF3
1
2
3
4
5
6
7
8
p-MeC6H4
p-MeC6H4
p-MeC6H4
p-MeC6H4
p-MeC6H4
p-MeC6H4
Napthalen-2-yl
Napthalen-2-yl
2
application in
a variety of fields such as fluorescence
OMe
NMe2
12
12
spectroscopy, electronic devices, supramolecular structures and
ion and pH sensors.28 Recently water-soluble, core-substituted
NDIs have been used as visible light absorbing theranostic
sensitisers and demonstrated the generation of singlet oxygen
within a cell culture system reducing cancer cell viability by up
to 40%.29,30
Encouraged by the ability of core-substituted NDIs to
sensitise oxygen and our previous findings into triplet state
sensitised furoxans, we envisioned NDI-mediated furoxan
sensitised NO release by connecting a furoxan to an NDI core
(Figure 1).
–
a) NO2 release determined by Griess test. b) 22% after 5 h
irradiation. c) 14% after 5 h irradiation.
We began with 4-fluoro-3-tolylfuroxan as a control which
released 28 μM of NO (Table 1, Entry 1). Changing the fluoro to
methoxy group yielded a slightly higher NO release (Table 1,
Entry 2). Dimethylaminofuroxan released a reasonable quantity
of NO and reached 22 M after 5 h of irradiation, the lower
quantity and slower rate relative to alkoxyfuroxan is possibly
due to increased electron donation into the furoxan ring (Table 1,
Entry 3). A cyanofuroxan released a moderate quantity of NO
(Table 1, Entry 4).31 n-Hexylfuroxan released low quantities of
NO (Table 1, Entry 5) and trifluoromethylfuroxan released
negligible quantities (Table 1, Entry 6).32 Increasing the
conjugation from tolyl to napthyl reduced the NO release in both
alkoxy and amino derivatives possibly due to the formation of a
more stable delocalized transient intermediate during
isomerization (Entries 7 and 8).
Based on the above results, alkoxyfuroxan was selected in
this work as the furoxan counterpart to the NDI sensitiser due to
its admirable NO release and stability under nucleophilic
substitution conditions. The selection of alkoxyfuroxan further
improves upon our previous fluorofuroxan-anthraquinone
hybrid since the alkoxyfuroxan enables derivatization in the
form of ether linkers, whereas the fluoro-functionality
effectively blocks one of the two available functionalizable sites
on the furoxan ring.
Synthesis of NDI-alkoxyfuroxan hybrid molecule. With
the alkoxyfuroxan selected as the NO donor partner of the NDI
sensitiser we designed a fluorescent, sensitizing and water-
soluble PINOD (Scheme 2). The desired compound 6 contains a
bromine atom at one of the NDI core substitution points, this
enables the utilization of spin orbit coupling to induce sensitised
triplet energy transfer to the alkoxyfuroxan. The amino core-
substitution was carefully selected so as to absorb light in the
visible region yet still be able to undergo intramolecular charge
transfer, fluoresce and sensitise the furoxan to induce NO release.
Compound 6 also contains two terminal quaternary ammonium
salts which serve a dual function; quaternarization prevents
Figure 1. Envisioned working molecule, a water soluble and
theranostic core-substituted NDI tethered to a furoxan molecule
via heteroatom linker.
In this article we detail the design and synthesis of a
fluorescent water-soluble NDI-tethered alkoxyfuroxan,
highlight the photophysical properties and demonstrate
photoswitchable NO release in pH 7.4 phosphate buffer and
cellular uptake.
2. Results and Discussion
Screening of furoxans for NO releasing ability. We
began our investigation by selecting a suitably substituted
furoxan to be connected to an NDI core. Previously we have
shown that sensitised NO release of anthraquinone-tethered
fluorofuroxans under visible light irradiation generates

4).34,35 Dibromination of dianhydride 11 was made possible by
the use of dibromoisocyanuric acid in concentrated sulphuric
acid. Due to the insoluble nature of the product the crude mixture
was used following filtration and washing with water and hot
methanol.
Imidization
with
N,N-
dimethylpropylenediaminegranted the NDI core 8 in moderate
yield following silica chromatography and filtration after stirring
the product in hot ethanol.
Scheme 4. Synthesis of NDI 8. Reagents and conditions: (i)
dibromoisocyanuric acid, H2SO4, 130 °C, 18 h, quant.; (ii) N,N-
dimethylpropylenediamine, AcOH, 130 °C, 0.5 h, 35%.
Scheme 2. Retrosynthesis of designed PINOD 6.
With both amino alkoxyfuroxan and NDI core in hand we
synthesised the hybrid molecule (Scheme 5). To our delight the
alkoxyfuroxan was stable to the reaction conditions and
connection with 8 granted core-substituted hybrid molecule 7 in
high yield following silica chromatography. Simple
quaternarization of the pendant amines with iodomethane
completed the synthesis of 6.
amine from quenching the excited state NDI via single electron
transfer and secondly the charged ammonium moieties would
enhance water solubility.
Compound 6 is accessed by methylation of the terminal
amines of 7 which in turn is synthesised by nucleophilic
aromatic substitution of NDI
8
with amino-pendant
alkoxyfuroxan 9. NDI 8 is synthesised by imidization of
dibromo dianhydride 10 with N,N-dimethylpropylenediamine.
Compound 10 can be constructed by dibromination of
commercially available dianhydride 11. Furoxan 9 is synthesised
by Boc deprotection of 12 previously furnished by nucleophilic
aromatic substitution of the nitrofuroxan 14 with N-Boc
protected 2-aminoethanol 13. Nitrofuroxan 14 is synthesised by
nitration of 4-methylstyrene (15) under acidic conditions. This
straightforward and simple synthesis takes advantage of the
relative stability of alkoxyfuroxan (in comparison to
fluorofuroxan) which enables the key nucleophilic aromatic
substitution of NDI 8 to take place without affecting the furoxan
ring.
Scheme 5. Synthesis of NDI-alkoxyfuroxan hybrid 6. Reagents
and conditions: (i) DMF, 60 °C, 14 h, 83%; (ii) MeI, MeCN,
35 °C, 24 h, 99%.
Our convergent synthesis of 6 began with the construction
of furoxan intermediate 9 (Scheme 3). Acidic Wieland
conditions granted the nitrofuroxan 14 in 27% yield following
chromatography and recrystallization.33 Nucleophilic aromatic
substitution of the nitrofuroxan with N-Boc protected
ethanolamine constructed the protected alkoxyfuroxan 12
followed by TFA deprotection to 9 in high yields.
PINOD capability of hybride molecule 6. Gratifyingly,
compound 6 presented sufficient water-solubility (the solubility
is >100 M) so we obtained the absorption and fluorescence of
Absorption
10000
Fluorescence
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
Scheme 3. Synthesis of furoxan 9. Reagents and conditions: (i)
NaNO2, AcOH, DCM, rt, 2 h then 2 M HCl, rt, 14 h, 27%; (ii)
N-Boc ethanolamine, NaOH, THF, 45 °C, 22 h, 83%; (iii) TFA,
DCM, -15 °C to rt, 14 h, 74%.
300
350
400
450
500
550
600
650
700
wavelength/ cm-1
Figure 2. Absorption and fluorescence of 6 (100 M) in 50 mM
pH 7.4 phosphate buffer solution at 25 °C. Fluorescence
measurement was conducted with λex of 500 nm.
The NDI core was synthesised from commercially
available dianhydride according to literature protocol (Scheme

6 in 50 mM pH 7.4 phosphate buffer solution (Figure 2).
Compound 6 showed a broad absorption above 400 nm; the
intramolecular charge transfer was observed as an intense broad
peak with a λmax at 533 nm. The fluorescence spectrum has a λmax
of 581 nm (ex = 500 nm) and extends up to 650 nm which is
useful for identifying cellular uptake locations. The fluorescence
quantum yield of 6 in air-saturated water was determined to be
0.10, which is bright enough for the biological experiment.
fluorescence spectrum underwent a hypsochromic shift from
λmax 581 nm to λmax 521 nm with a significant increase in
fluorescence intensity under prolonged irradiation (Figure 4b).
Despite the possibility of limiting NO release, this can be a
useful property since the molecule behaves as a fluorometric
indicator to signal when the NO release event has finished. The
change in absorption of compound 6 with irradiation shows an
overall decrease in intensity at both 533 nm and 371 nm with
prolonged irradiation time. The decrease at 371 nm is attributed
to a perturbation of the NDI core conjugation, perhaps due to
decomposition of the diimide moieties. The decrease at 533 nm
could also be attributed to cleavage of the core substituted NDI-
amino bond shutting down the intramolecular charge transfer.
The NO releasing ability of compound
6 under
photoirradiation was next tested. Compound 6 (100 M) was
dissolved in 50 mM pH 7.4 phosphate buffer and irradiated
continuously at 37 C for 4 hours with either 420–600 nm or
500–600 nm light. Aliquots were taken at intervals and the NO%
release determined spectroscopically by treatment with Griess
reagent (Figure 3). NO was released up to 16 M after 2 hours
of irradiation with 420–600 nm light. When irradiating with a
longer wavelength (500–600 nm) the NO release rate is
comparatively more gradual, reaching 10 M over 4 hours.
Under ambient lighting conditions there is no significant NO
release observed after 4 hours.
We have attempted to directly confirm the photoinduced
generation of NO radical from compound 6 using a spin-trapping
method with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-
oxide (PTIO) as a radical-trapping reagent,36 but failed to detect
the EPR signal of the corresponding imino nitroxide product.
Therefore, it is of note that at present we have not confirmed that
the NO species detected by the Griess test are only in the NO
redox form, it may be possible that NO2– or NO– are also released.
The mechanism of NO release is currently thought to be
similar to previous anthraquinone-tethered fluorofuroxan 5,23
that is, due to the energy transfer from the excited NDI to the
alkoxy furoxan that causes rapid isomerization of the excited
furoxan leading to discharge of NO either via solvent molecule
interaction with the transient intermediate or an at present
unknown photochemical pathway. Another possible mechanism,
the electron transfer, is considered. The reduction (Ered) and
oxidation potentials (Eox) of 4-methoxy-3-tolylfuroxan were
determined to be –1.35 V and +1.60 V vs Fc/Fc+, respectively.
According to the literature,27 Ered and Eox of the NDI having the
bromo and amino groups at the 2,6-positions are approximately
–1.0 V and +1.2 V vs Fc/Fc+, respectively. Therefore, Ered and
Eox of the NDI excited state (NDI*) can be speculated to be
<+1.2 V and >–1.0 V vs Fc/Fc+, respectively. Based on the above
data, neither the electron transfer from NDI* to alkoxy furoxan
nor from alkoxy furoxan to NDI* is likely.
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
(a)
0 h
1 h
2 h
3 h
5 h
300
350
400
450
500
550
600
650
700
wavelength / nm
(b)
3 h
2 h
1 h
0 h
100
80
60
40
20
0
420
470
520
570
620
670
wavelength / nm
Figure 4. Photobleaching of compound 6 under 420–600 nm
continuous irradiation in 50 mM pH 7.4 phosphate buffer
solution at 37 °C. At intervals a 2 mL aliquot was taken. (a)
Change in the absorption spectra measured at 25 °C. (b) Change
in fluorescence spectra measured at 25 °C with λex of 420 nm.
With photoswitchable NO release confirmed we tested the
cellular uptake of 6 into HeLa cancer cells. Previous research
had shown cell membrane permeability for the NDI molecules
with tri- and tetrasubstituted quaternary ammonium
moieties.30,37 We were pleased to see that hybrid molecule 6,
even with the dual quaternary ammonium ions and the tethered
furoxan molecule, permeated the cell membrane (Figure 5).
Compound 6 was uniformly observed in the cytoplasm and
sparsely in the nucleus, and may be localized in Golgi apparatus
Figure 3. NO-release curve of 6 (100 μM) in 50 mM phosphate
buffer under 420–600 nm, 500−600 nm or ambient light
irradiation at 37 °C.
Compound 6 underwent photobleaching when irradiated;
almost full photobleaching was observed within 3 hours of
continuous irradiation (Figure 4a). Furthermore, the
Figure 5. Fluorescence imaging of compound 6 in cultured
living HeLa cells. Left: bright field, right: fluorescence image.

slightly more than other organelles. Although the uptake
mechanism is not fully understood, it has been suggested that
similarities between tetracationic porphyrins and NDIs indicate
endocytosis as a possible pathway.30
reported literature protocols.29, 33
Measurement of NO-releasing ability of furoxans with
UV-irradiation (Table 1). A 100 M stock solution of furoxan
was prepared by adding 100 μL of a 10 mM DMSO solution of
furoxan into a Pyrex vial containing 10 mL of 50 mM phosphate
buffer solution (pH 7.4). The Pyrex vial was placed in a 37 °C
oil bath and then irradiated using 300–400 nm light (a 300W
Xenon lamp, Asahi Spectra MAX-303 equipped with a 300- to
600-nm ultraviolet-visible module and a 400-nm short-pass
filter). At intervals, aliquots (0.5 mL) were taken out, to which
the Griess reagent (40 μL) was added. After the samples were
stood for 30 min, the absorbance of the samples at 520 nm was
We determined the amount of singlet oxygen (1O2) that was
catalytically generated by compound 6 under the non-deaerated
1
aqeuous conditions. The quantification of O2 conducted by an
absorption
spectroscopic
analysis
using
1,3-
diphenylisobenzofuran (DPBF), a 1O2 scavenger,38 revealed that
three orders of magnitude more 1O2 than NO is released.
Although this result indicates that compound 6 may be a good
candidate of a photodynamic therapy reagent for cancer disease
because both 1O2 and NO have anticancer capabilities,39 the
practical biological use of compound 6 as a pure NO donor is
limited at present. Nevertheless, there is still a large hope that
the ratio of NO to 1O2 may be improved by taking advantage of
intramolecularity of energy transfer from NDI* to the furoxan
moiety in contrast to the intermolecular sensitization of
molecular oxygen by NDI. The modification of molecular
structure such as the tether length and furoxan substituent would
dramatically change the sensitization kinetics. Thus, based on
this proof-of-concept work, a better NO donor can be designed
on demand in the future.
−
measured. The percent nitrite (NO2 ) (mol/mol) was determined
from a calibration curve prepared in advance by using NaNO2
standard solutions (20−100 μM) treated with the Griess reagent.
The Griess reagent was prepared by diluting a mixture of 1
g of sulfanilamide, 50 mg of N-naphthylethylenediamine
dihydrochloride, and 2.5 mL of 85% phosphoric acid with
distilled water to a final volume of 25 mL.
Absorption and fluorescence spectra. UV–visible
spectra were recorded on a Shimadzu UV-1800 spectrometer
with
a
quartz absorption cuvette (light path:
spectra were recorded
1
on
cm).
a
Photoluminescence
spectrofluorometer (Jasco FP-6500) with a quartz absorption
cuvette (light path: 1 cm).
3. Conclusion
Determination of fluorescence quantum yield of 6. The
fluorescence quantum yield of 6 in air-saturated water was
determined via the relative determination method with
fluorescein as a reference. See the supporting information for the
details.
Cellular uptake (Figure 5). HeLa cells (Riken
BioResource Center) were cultured in high glucose DME
medium (Sigma-Aldrich) containing 10 %(v/v) FBS, 1 %(v/v)
Penicillin-Streptomycin (P/S) (Sigma-Aldrich) at 37 °C in a CO2
incubator (5% CO2). Then, the HeLa cells were seeded at a
density of 1  105 cells/mL in DME medium in a 35 mm glass
bottom dish. After 24 hours of culture, the cells were incubated
with 20 μM 6 for 20 min in a CO2 incubator (5 % CO2) at 37 °C.
After the incubation, the cells were washed thrice with PBS
buffer. The fluorescence derived from 6 in the cells was then
We have consolidated on visible light-absorbing
photoswitchable furoxan NO donors by addressing the
limitations of previous designs. We synthesised a water soluble
fluorescent NDI-alkoxyfuroxan hybrid molecule that released
NO under in a photoswitchable mannerat longer absorption
wavelengths than previous models. Furthermore, the molecule
demonstrated cellular uptake into HeLa cells and was observable
due to the fluorescence capability. Facile synthesis was made
possible by use of a terminal amine-tethered alkoxyfuroxan
which was inert to aromatic nucleophilic substitution conditions.
The obtained results represent the proof-of-concept of our initial
working hypothesis. For the practical biological use, improving
the photostability, rate and quantity of NO release and further
lengthening the wavelength of sensitiser absorption is necessary
and part of the ongoing research within our laboratory.
analyzed using
a
fluorescence microscope (BZ-X700;
KEYENCE) equipped with BZ-X filter GFP and a 40x objective
lens with the exposure time of 1/3 second.
4. Experimental
General. Unless otherwise noted, all reactions were
carried out in well cleaned glasswares with magnetic stirring.
Operations were performed under an atmosphere of dry argon
using Schlenk and vacuum techniques, unless otherwise noted.
All starting materials were obtained from commercial sources or
were synthesised using standard procedures. Melting points
were measured on a Yanaco MP-500D and are not corrected. 1H
and 13C NMR (400 and 100 MHz, respectively) were recorded
on a Bruker Avance III HD 400 using TMS (0 ppm) and CDCl3
(77.0 ppm) as an internal standard. The following abbreviations
are used in connection with NMR; s = singlet, d = doublet, t =
triplet, q = quartet, quin = quintet, sep = septet, and m = multiplet.
The X-band EPR measurements were carried out using a Bruker
EMX system. Mass spectra were measured using a JEOL JMS-
T100LP (DART method, ambient ionization) or a LTQ Orbitrap
Elite (Thermo Fisher Scientific, Brehmen, Germany) with an
electrospray ionization (ESI) ion source. Preparative column
chromatography was performed using Kanto Chemical silica gel
60 N (spherical, neutral), Fuji Silysia BW- 4:10MH silica gel or
YMC_GEL Silica (6 nm I-40−63 μm). Thin layer
chromatography (TLC) was carried out on Merck 25 TLC silica
gel 60 F254 aluminum sheets. Nitrofuroxan 14 and
naphthalenediimide 8 were prepared according to previously
Measurement of the NO-releasing ability of compound
6 with visible light-irradiation (Figure 3). A 100 μM solution
of 6 was prepared by adding 10 mL of 50 mM phosphate buffer
(pH 7.4) into a Pyrex vial containing 6, which was placed in a
37 °C oil bath and then irradiated using either 420−600 nm or
500–600 nm light (a 300W Xenon lamp, Asahi Spectra MAX-
303 equipped with a 300- to 600-nm ultraviolet-visible module,
and
a 420-nm long-pass and 500-nm long-pass filter,
respectively). At intervals, two aliquots (0.5 mL) were taken, to
one of which the Griess reagent (40 μL) was added. After the
samples were stood for 30 min, the difference in absorbance of
−
the samples at 520 nm was measured. The percent nitrite (NO2 )
(mol/mol) was determined from a calibration curve prepared in
advance by using NaNO2 standard solutions (20−100 μM)
treated with the Griess reagent.
Measurement of the NO-releasing ability of compound
6 without visible light-irradiation (Figure 3). A 100 μM
solution of 6 was prepared by adding 10 mLof 50 mM phosphate
buffer (pH 7.4) into a Pyrex vial containing 6, which was placed
in a 37 °C oil bath. At intervals, two aliquots (0.5 mL) were taken
out, to one of which the Griess reagent (40 μL) was added. After
the samples were stood for 30 min, the difference in absorbance
of the samples at 520 nm was measured. The percent nitrite

−
(NO2 ) (mol/mol) was determined from a calibration curve
797, 732 cm-1; HRMS (DART) m/z [M+H]+ Calcd for
C11H14N3O3 236.1034; Found 236.1035; mp 97–99 °C.
prepared in advance by using NaNO2 standard solutions (20−100
μM) treated with the Griess reagent.
Synthesis of alkoxyfuroxan tethered NDI 6 (Scheme 5).
N,N ’ -Bis(3-(dimethylamino)propylamino)-2-bromo-6-(2-
{[3-(4-methylphenyl)furoxan-4-yl]oxy}ethylamino)-1,4,5,8-
naphthalenetetracarboxylic bisimide (7): Compound 7 was
synthesised according to the reported method.37 NDI 8 (1.0 g,
1.68 mmol) and furoxan 9 (514.5 mg, 2.19 mmol) were stirred
in DMF (0.02 M) at 60 °C for 14 h. The reaction mixture was
cooled and poured into Na2CO3 (pH 8) basified water, diluted
with EtOAc and washed gently 5 times with an excess of aq.
Na2CO3. The organics were dried over Na2SO4, filtered and
solvent removed in vacuo. The crude was purified by silica
chromatography (5% MeOH, 1% Et3N, 94% CHCl3) to yield a
red solid (1.04 g, 1.39 mmol, 83%). 1H NMR (400 MHz, CDCl3)
δH(ppm) 10.44 (1H, t, J = 7.2 Hz), 8.84 (1H, s), 8.28 (1H, s),
7.89 (2H, d, J = 8.4 Hz), 7.15 (2H, d, J = 8.4 Hz), 4.86 (2H, t, J
= 5.2 Hz), 4.24–4.14 (6H, m), 2.46 (4H, q, J = 6.8 Hz), 2.33 (3H,
s), 2.27 (6H, s), 2.26 (6H, s), 1.95–1.86 (4H, m); δC (100 MHz,
CDCl3) 166.0, 161.9, 161.7, 161.7, 161.2, 151.7, 141.0, 138.5,
129.5, 128.4, 127.5, 126.0, 123.6, 123.4, 121.6, 121.1, 119.6,
119.1, 107.6, 101.1, 68.7, 57.1, 45.3, 45.2, 41.5, 39.8, 38.9, 25.8,
25.6, 21.6; IR (neat) 2937, 2812, 2759, 1707, 1686, 1662, 1633,
1604, 1580, 1439, 1312, 1256, 1256, 1218, 1178, 1162, 1016,
817, 791 cm-1; HRMS (ESI) m/z [M+H]+ Calcd for
C35H39BrN7O7 750.2068; Found 750.2076; mp 163 °C
(decomposed).
N,N’ -Bis(3-(trimethylamino)propylamino)-2-bromo-6-
(2-{[3-(4-methylphenyl)furoxan-4-yl]oxy}ethylamino)-
1,4,5,8-naphthalenetetracarboxylic bisimide diiodide (6): To
a solution of compound 7 (75.0 mg, 100 mol) in MeCN (0.01
M) was added iodomethane (14.3 L, 230 μmol) and the reaction
heated to 35 °C for 24 h. Upon completion the solvent was
removed in vacuo and the solid filtered and washed with hexane,
Et2O and CHCl3 to yield a magenta solid (102.4 mg, 99.2 mol,
99%). 1H NMR (400 MHz, D2O) δH(ppm) 8.41 (1H, s), 7.97 (1H,
s), 7.37 (2H, d, J = 8.2 Hz), 6.78 (2H, d, J = 8.2 Hz), 4.91–4.88
(2H, m), 4.21–4.15 (4H, m), 4.04 (2H, apparent t, J = 7.2 Hz),
3.56–3.52 (4H, m), 3.22 (9H, s), 3.19 (9H, s), 2.28–2.15 (4H, m),
2.09 (3H, s); δC (100 MHz, acetonitrile-d3) 166.9, 163.2, 162.9,
162.7, 162.2, 153.4, 141.9, 138.1, 130.1, 129.5, 128.2, 126.7,
124.6, 124.4, 122.6, 121.4, 120.4, 108.7, 101.5, 70.7, 65.2, 65.0,
53.9, 42.0, 38.7, 37.9, 22.8, 22.5, 21.4; IR (neat) 3435, 2954,
2917, 2847, 1709, 1670, 1637, 1584, 1517, 1475, 1444, 1311,
1267, 1241, 1191, 1163, 1124, 960, 785, 738, 720 cm-1; HRMS
(ESI) m/z [M–2I]2+ Calcd for C37H44BrN7O7 389.6227; Found
389.6232; mp 188 °C (decomposed).
Synthesis of naphthalenediimide 8 (Scheme 4). 2,6-
Dibromonaphtalene-1,4,5,8-tetracarboxylic
dianhydride
(10): Compound 10 was synthesised according to the reported
method.35 To a solution of dibromoisocyanuric acid (9.56 g, 33.3
mmol) in concentrated H2SO4 (0.3 M) was added portionwise
1,4,5,8-naphthalenetetracarboxylic dianhydride (4.47 g, 16.7
mmol) at room temperature, following complete addition the
reaction was heated to 130 °C for 18 h. Upon completion the
reaction was cooled, poured into ice water and stirred until
yellow precipitate formed. The precipitate was filtered and
washed with water and hot MeOH then dried to yield a yellow
crude mixture containing 10 (7.37 g, 17.3 mmol, quant.). Due to
the insoluble nature of 10, the mixture is used without further
purification.
N,N’-Bis((dimethylamino)propylamino)-2,6-
dibromonaphthalene-1,4,5,8- tetra-carboxylic acid bisimide
(8): Compound 8 was synthesised according to the reported
method.34 To a solution of compound 10 (7.00 g, 16.4 mmol) in
acetic acid (210 mL) was added dropwise N,N-
dimethylpropylenediamine (5.17 mL, 41.1 mmol) and the
reaction heated to 130 °C for 1 h. Upon reaction completion the
mixture was poured into ice water, basified with K2CO3 (pH 8)
and extracted 6 times with CHCl3. The organics were dried over
Na2SO4, filtered and solvent removed in vacuo. The crude solid
was partially purified by silica chromatography (5% MeOH, 1%
Et3N in CHCl3). The product was then stirred in EtOH (400 mL)
at 60 °C for 30 min and allowed to cool then filtered under
vacuum and dried to yield 8 as an orange solid (3.46 g, 5.82
1
mmol, 35%). The H and 13C NMR spectra were in accordance
with the reported data.
Synthesis of furoxan 9 (Scheme 3). tert-Butyl 2-{[3-(4-
methylphenyl)furoxan-4-yl]oxy}ethylcarbamate (12): To a
solution of 3-(4-methylphenyl)-4-nitrofuroxan (1.0 g, 4.52
mmol) in THF (0.5 M) was added N-Boc-ethanolamine (948.1
mg, 5.88 mmol) followed by NaOH (235 mg, 5.88 mmol) and
stirred at 45 °C for 22 h. Upon reaction completion the mixture
was diluted with water and extracted thrice with EtOAc. The
organics were dried over Na2SO4, filtered and solvent removed
in vacuo, the crude was purified by silica chromatography
(CH2Cl2) to yield 12 as an off white solid (1.25 g, 3.75 mmol,
1
83%). H NMR (400 MHz, CDCl3) δH(ppm) 8.01 (2H, d, J = 8
Hz), 7.32 (2H, d, J = 8 Hz), 4.84 (1H, br s), 4.55 (2H, t, J = 5.2
Hz), 3.67 (2H, apparent q, J = 5.2 Hz), 2.42 (3H, s), 1.44 (9H,
s); δC (100 MHz, CDCl3); 162.2, 155.7, 141.0, 129.6, 126.1,
119.4, 107.7, 80.0, 70.0, 39.5, 28.4, 21.6; IR (neat) 3349, 2983,
2927, 1681, 1604, 1556, 1537, 1519, 1481, 1442, 1383, 1359,
1332, 1314, 1287, 1274, 1253, 1163, 1115, 1046, 1030, 998, 966,
860, 839, 822, 631 cm-1; HRMS (DART) m/z [M+H]+ Calcd for
C16H22N3O5 336.1566; Found 336.1560; mp 157–159 °C.
4-(2-Aminoethoxy)-3-(4-methylphenyl)furoxan (9): A
stirred solution of 12 (1.15 g, 3.45 mmol) in CH2Cl2 (0.1 M) was
cooled to –15 °C and added to it TFA (7.5 mL) dropwise and the
reaction allowed to warm to rt over 14 h. The solvent was
removed in vacuo and the mixture was diluted with K2CO3
basified water (pH 8) and extracted thrice with EtOAc. The
organics were dried over MgSO4, filtered and solvent removed
in vacuo to yield an orange solid (598.3 mg, 2.54 mmol, 73%).
The product was used without further purification. 1H NMR (400
MHz, CDCl3) δH(ppm) 8.01 (2H, d, J = 8.4 Hz), 7.32 (2H, d, J =
8.4 Hz), 4.53 (2H, t, J = 5.2 Hz), 3.23 (2H, t, J = 5.2 Hz), 2.41
(3H, s); δC (100 MHz, CDCl3) 162.4, 141.0, 129.6, 126.1, 119.4,
107.7, 72.8, 40.8, 21.6; IR (neat) 3386, 2954, 2922, 2862, 1691,
1597, 1550, 1516, 1441, 1396, 1310, 1159, 1123, 990, 857, 818,
Acknowledgement
We thank Prof. Atsunori Mori, Prof. Kentaro Okano, Mr.
Tatsuhi Yabuta, Dr. Takehiro Watanabe and Suntory foundation
for life sciences for their help on mass analysis. We thank Ms.
Rebecca Sewell for her assistance with the graphical abstract and
Mr. Rei Toda in Osaka university for his fruitful discussion on
the cellular uptake experiments. CPS wishes to thank the MEXT
for the Japanese Government Scholarship Program. We also
thank Mr. Tomoaki Kaiba, Prof. Yasuhiro Kobori and Prof.
Takashi Tachikawa for their help on fluorescence quantum yield
measurement. Prof. Yasuhiro Kobori and Dr. Hiroki Nagashima
are appreciated for the EPR measurement. This work was
financially supported by JSPS KAKENHI Grant Numbers
JP16K18844 and JP17J00025, Futaba Electronics Memorial
Foundation, Suzuken Memorial Foundation, Inamori
Foundation, and Daiichi Sankyo Foundation of Life Science.
Supporting Information

Method for the quantum yield determination and copies of
the 1H and 13C NMR spectra of all new compounds are available.
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