Photochemical generation of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical from caged nitroxides by near-infrared two-photon irradiation and its cytocidal effect on lung cancer cells

Article abstract of DOI:10.3762/bjoc.15.84

Novel caged nitroxides (nitroxide donors) with near-infrared two-photon (TP) responsive character, 2,2,6,6-tetramethyl-1-(1-(2-(4-nitrophenyl)benzofuran-6-yl)ethoxy)piperidine (2a) and its regioisomer 2b, were designed and synthesized. The one-photon (OP) (365 ± 10 nm) and TP (710–760 nm) triggered release (i.e., uncaging) of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical under air atmosphere were discovered. The quantum yields for the release of the TEMPO radical were 2.5% (2a) and 0.8% (2b) in benzene at ≈1% conversion of 2, and 13.1% (2a) and 12.8% (2b) in DMSO at ≈1% conversion of 2. The TP uncaging efficiencies were determined to be 1.1 GM at 740 nm for 2a and 0.22 GM at 730 nm for 2b in benzene. The cytocidal effect of compound 2a on lung cancer cells under photolysis conditions was also assessed to test the efficacy as anticancer agents. In a medium containing 100 μg mL^?1 of 2a exposed to light, the number of living cells decreased significantly compared to the unexposed counterparts (65.8% vs 85.5%).

Full text of DOI:10.3762/bjoc.15.84

Photochemical generation of the 2,2,6,6-tetramethylpiperidine-
-oxyl (TEMPO) radical from caged nitroxides by
1
near-infrared two-photon irradiation and its
cytocidal effect on lung cancer cells
Ayato Yamada1, Manabu Abe*1,2,3, Yoshinobu Nishimura*4, Shoji Ishizaka1,2,
Masashi Namba2,5, Taku Nakashima2,5, Kiyofumi Shimoji5 and Noboru Hattori*2,5
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 863–873.
1
Department of Chemistry, Graduate School of Science, Hiroshima
University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima
39-8526, Japan, 2Hiroshima Research Centre for
Photo-Drug-Delivery Systems (HiU-P-DDS), Hiroshima University,
-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan,
JST-CREST, K’s Gobancho 6F, 7, Gobancho, Chiyoda-ku, Tokyo
02-0075, Japan, 4Graduate School of Pure and Applied Sciences,
doi:10.3762/bjoc.15.84
7
Received: 01 February 2019
Accepted: 16 March 2019
Published: 10 April 2019
1
3
1
This article is part of the thematic issue "Reactive intermediates part I:
radicals".
University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8571,
Japan and 5Department of Molecular and Internal Medicine, Graduate
School of Biomedical & Health Sciences, Hiroshima University, 1-2-3
Kasumi, Minami-ku, Hiroshima, Hiroshima 734-8551, Japan
Guest Editor: T. P. Yoon
©
2019 Yamada et al.; licensee Beilstein-Institut.
Email:
License and terms: see end of document.
Manabu Abe* - mabe@hiroshima-u.ac.jp; Yoshinobu Nishimura* -
nishimura@chem.tsukuba.ac.jp; Noboru Hattori* -
nhattori@hiroshima-u.ac.jp
*
Corresponding author
Keywords:
caged compound; nitroxide; photolysis; radical; theranostics;
two-photon
Abstract
Novel caged nitroxides (nitroxide donors) with near-infrared two-photon (TP) responsive character, 2,2,6,6-tetramethyl-1-(1-(2-(4-
nitrophenyl)benzofuran-6-yl)ethoxy)piperidine (2a) and its regioisomer 2b, were designed and synthesized. The one-photon (OP)
(
365 ± 10 nm) and TP (710–760 nm) triggered release (i.e., uncaging) of the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical
under air atmosphere were discovered. The quantum yields for the release of the TEMPO radical were 2.5% (2a) and 0.8% (2b) in
benzene at ≈1% conversion of 2, and 13.1% (2a) and 12.8% (2b) in DMSO at ≈1% conversion of 2. The TP uncaging efficiencies
were determined to be 1.1 GM at 740 nm for 2a and 0.22 GM at 730 nm for 2b in benzene. The cytocidal effect of compound 2a on
lung cancer cells under photolysis conditions was also assessed to test the efficacy as anticancer agents. In a medium containing
1
00 μg mL−1 of 2a exposed to light, the number of living cells decreased significantly compared to the unexposed counterparts
(
65.8% vs 85.5%).
863

Beilstein J. Org. Chem. 2019, 15, 863–873.
Introduction
Nitroxides (aminoxyl radicals) possess a delocalized unpaired
electron and exhibit negligible dimerization reactivity, making
them persistent open-shell species [1-4]. In addition to their
ease of handling, nitroxides are highly sensitive to electron
paramagnetic resonance (EPR) spectroscopy and redox reac-
tions. Therefore, nitroxides have been developed and utilized in
diverse and crucial applications, not only in chemistry, but also
in biology, physiology, and energy sciences. These applications
include spin-labels [5-7], fluorophore-nitroxide probes [8],
contrast agents in magnetic resonance imaging (MRI) [9], po-
larization transfer agents for nuclear magnetic resonance
(
NMR) [10-13], and radical batteries [14,15]. Furthermore, the
efficient synthesis of polymers with narrow molecular mass dis-
tributions has been accomplished using nitroxides as a medi-
ator, i.e., so-called nitroxide-mediated polymerization (NMP)
Scheme 1: Photochemical generation of TEMPO radical.
[
16-20], and the nitroxide-mediated synthesis of ketones from
alcohols is also well utilized in organic synthesis [21-25]. The
huge number of studies concerning nitroxides clearly indicates excitation technique [44], in which a molecule is electronically
the importance of new methods of generating nitroxides for the excited to the same state generated by one-photon (OP) excita-
future development of science and technology. Notably, in tion in the UV–vis region [45]. In addition to the advantages of
physiological studies [26-32], spatiotemporal control of TP excitation, three-dimensional control of the electronic exci-
nitroxide generation is a key approach for investigating the role tation is possible because the probability of TP excitation is
of redox-active nitroxides in mediating oxidative stress in proportional to the square of the light intensity [46]. The light-
organisms [27-32].
induced generation of nitroxides using the TP excitation tech-
nique, i.e., the concentration jump of nitroxides, is one promis-
In 1997, Scaiano and co-workers reported the triplet-xanthone ing method of exploring the role of these species in life phe-
sensitized generation of the 2,2,6,6-tetramethylpiperidine-1- nomena [47-54] and of promoting site-selective chemical reac-
oxyl (TEMPO) radical from alkoxyamine 1 under ultraviolet tions such as polymerization. Very recently, Guillaneuf and
(
355 nm) irradiation (Scheme 1) [33]. De-aerated conditions are co-workers reported the two-photon-induced release of nitrox-
necessary for the triplet-sensitized generation of TEMPO due to ides in a materials science study [55].
the triplet quenching ability of O2. The polymerization reac-
tions were initiated via photochemical reaction [34-36]. For In the last decade, we developed a TP-responsive photo-labile
physiological studies, however, the photochemical release of protecting group [56-58] with simple cyclic stilbene structures
nitroxides should be achieved in the presence of O2. Thus, the such as 2-(4-nitrophenyl)benzofuran (NPBF) that absorb in the
triplet sensitized method may not be useful for physiological NIR region of 710–760 nm for the uncaging of bioactive
studies. The application of alkoxyamines as theranostic agents substances such as glutamate and Ca2+ [59-64]. Herein, we
[
37-40] has been proposed and reported by Brémond and report the synthesis of new caged nitroxides (nitroxide donors)
co-workers [41,42].
2a and 2b having the TP-responsive NPBF chromophore and
the NIR TP-triggered generation of the 2,2,6,6-tetramethylpi-
Near-infrared (NIR) photons are excellent light sources in phys- peridine-1-oxyl (TEMPO) radical under atmospheric condi-
iological studies as this wavelength of light is less harmful to tions using these species (Scheme 1). Because free radicals are
living tissue than ultraviolet irradiation. Deeper penetration of cytotoxic due to their strong DNA-damaging activity [65], they
NIR photons into biological samples is possible using NIR radi- play important roles as anticancer therapeutic agents [66].
ation with wavelengths of 650–1050 nm (= 27–44 kcal mol−1). Among the free radicals, nitroxides including the TEMPO
However, in general, chromophores do not absorb at such long radical have unique properties, where they can act not only as
wavelengths and the photon energy is too low for bond- radical scavengers, but also as anticancer agents [67]. Due to
cleavage reactions to generate (i.e., uncage) functional mole- the unique properties described above, nitroxides are not toxic
cules. For example, the bond-dissociation energy of the weak to normal host cells and exhibit toxicity only to tumor cells.
PhCH2–OPh, linkage is reported to be 52.1 kcal mol−1 [43]. Thus, nitroxides are ideal candidates as anticancer therapeutic
These issues can be solved by using the NIR-two-photon (TP) agents. Based on this knowledge, the cytocidal effect of the
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Beilstein J. Org. Chem. 2019, 15, 863–873.
radical released from compound 2a on lung cancer cells was pounds 2 and 5 were observed at ≈370 nm with a molar extinc-
tested in vitro, in addition to the fundamental study.
tion coefficient ε ≈20000 M−1 cm−1 in both benzene and
DMSO. The emission profile showed a significant solvent
effect. The fluorescence quantum yields in DMSO of 5a and 5b
Results and Discussion
The caged-TEMPOs 2a and 2b were synthesized as shown in were determined to be 16.1 and 8.6%, respectively, although no
Scheme 2. The new compounds, 5-ethyl- and 6-ethyl-2-(4-nitro- emission was observed from these compounds in non-polar
phenyl)benzofuran (5a and 5b), were synthesized from benzene, indicating that the excited state has zwitterionic char-
1
-ethynyl-4-nitrobenzene (4) that was prepared from the com- acter. The charge transfer transition was supported by time-de-
mercially available 1-iodo-4-nitrobenzene (3) [68]. The pendent density functional theory (TD-DFT) calculations for 5a
TEMPO moiety was introduced at the benzylic position of 5a at the CAM-B3LYP/6-31G(d) level of theory (Supporting
and 5b using the copper-catalyzed radical reaction in the pres- Information File 1, Figure S2). The fluorescence quantum
ence of tert-butyl hydroperoxide (TBHP) to afford 2a and 2b in yields of caged-TEMPO 2a and 2b were found to be 2.9 and
3
8% and 52% yield, respectively [69]. The caged TEMPOs 2a 2.2% in DMSO, which are much smaller than those of 5a and
and 2b were thermally stable in benzene below 320 K (47 °C), 5b, respectively, suggesting the chemical reactivity of the
as confirmed by electron paramagnetic resonance (EPR) spec- singlet excited states of 2a and 2b.
troscopic analysis. Significant thermal decomposition of 2a and
2
b was observed at ≈340 K (67 °C), as indicated by the typical Time-correlated single photon counting (TCSPC) measurement
EPR signals (see Supporting Information File 1, Figure S1).
was performed at 298 K in DMSO to estimate the fluorescence
lifetime (τ) of 2 and 5 (Table 1). Single-exponential decay
The photophysical data for the new compounds 2a,b and 5a,b curves were observed for 5a and 5b, respectively (Supporting
are summarized in Table 1. The absorption maxima of com- Information File 1, Figure S3). The lifetimes determined by
Scheme 2: Synthesis of caged nitroxides 2a and 2b.
Table 1: Photophysical data for 2a, 2b, 5a, and 5b in benzene (DMSO).
Entry
λabs [nm]a
ε [M−1 cm−1]
λem [nm]b
Φf × 102 c
τ [ps]d
3
71
375)
66
370)
72
378)
67
372)
24800
(23100)
–
≈0.0
(2.9)
–
1
2
3
4
2a
2b
5a
5b
(
(
(
(
(576)
(220, 1370)e
3
23000
(23400)
–
≈0.0
(2.2)
–
(564)
(390, 890)f
3
23800
(20000)
–
≈0.0
(16.1)
–
(577)
(1430)
3
22300
(19000)
–
≈0.0
(8.6)
–
(563)
(870)
aAbsorption maximum of 2a, 2b, 5a, 5b. bEmission maximum of 2a (1.18 × 10−6 M), 2b (1.18 × 10−6 M), 5a (1.16 × 10−6 M), 5b (1.12 × 10−6 M).
cFluorescence quantum yields. The standard sample 9,10-diphenylanthracene (Φf = 0.91) was used for determining the quantum yields.
dFluorescence lifetime monitored at 560 nm. The concentrations were the same as those used for the fluorescence measurements. eEach contribu-
tion is 57% and 43%, respectively. fEach contribution is 70% and 30%, respectively.
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Beilstein J. Org. Chem. 2019, 15, 863–873.
single-exponential fitting were 1430 (5a) and 870 ps (5b), re- cal yield of TEMPO was 80% after 10 min irradiation in
spectively (Table 1, entries 3 and 4). Double-exponential decay benzene under air atmosphere (Figure 2g). Secondary photore-
was, however, observed for the TEMPO-substituted NPBF de- action of TEMPO gradually decreased the chemical yield of
rivatives 2a and 2b, where the lifetimes were 220 (57%) and TEMPO. The quantum yield (Φ) for photochemical release of
1
370 ps (43%) for 2a, and 390 (70%) and 890 ps (30%) for 2b the TEMPO radical was 2.5% at ≈1% conversion in the photo-
Table 1, entries 1 and 2). For 2a and 2b, intermolecular charge lysis of 2a in benzene under atmospheric conditions. Similar
transfer processes induced by the TEMPO moiety may account photochemical generation of the TEMPO radical was con-
(
for the double-exponential decay curves to some extent.
ducted with 2b (5 mM, Supporting Information File 1,
Figure S5 and Figure 2d,h). The clean generation of the
OP photolysis of 2a (5 mM) was first conducted in benzene at TEMPO radical was also observed during photolysis under
298 K using 365 nm light (6.02 × 1015 photons s−1) under 365 nm irradiation in benzene at ≈298 K under atmospheric
≈
atmospheric conditions (Figure 1). Clean release of the TEMPO conditions, although the reaction was slower than that of 2a,
radical was confirmed by measuring the electron paramagnetic k = 5.5 x 10–6 s–1; Φ = 0.8% at ≈1% conversion of 2b. Howev-
resonance (EPR) signals of the typical nitroxide, AN = 15.5 G er, the chemical yield of TEMPO was also high (81% after
(
g = 2.00232, Figure 1 and Figure 2c). The first-order rate con- 20 min irradiation under the same conditions), although slow
stant for generation of TEMPO in the bulk photoreaction was photochemical decomposition of TEMPO was observed with
found to be k = 1.6 × 10–5 s−1. The amount of photochemically prolonged irradiation (Figure 2h). In DMSO, the quantum yield
released TEMPO radical was determined by comparing the EPR
intensity with the calibration curve of the standard TEMPO
sample (Supporting Information File 1, Figure S4). The chemi-
Figure 2: Time profile for photochemical generation of TEMPO radical
from 2 (5 mM) at ≈298 K in benzene: (a) from 2a under degassed
Figure 1: Photochemical generation of TEMPO from 2a and 2b. EPR
spectra acquired during the photolysis of 2a (5 mM) in benzene using
conditions, (b) from 2b under degassed conditions, (c,g) from 2a under
air conditions, (d,h) from 2b under air conditions, (e) from 2a under O2,
(f) from 2b under O2.
365 nm LED light under air atmosphere.
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Beilstein J. Org. Chem. 2019, 15, 863–873.
for the formation of TEMPO increased significantly to 13.1% 730, 740, 750, and 760 nm near infrared light from a
(
from 2a) and 12.8% (from 2b) at ≈1% conversion of 2 under Ti:sapphire laser (pulse width 100 fs, 80 MHz) emitting at an
atmospheric conditions (Figure 1). The notable effect of the sol- average of 700 mW (Figure 3 for 2a and Supporting Informa-
vent on the TEMPO generation may be due to the increase in tion File 1, Figure S6 for 2b). The typical EPR signals of
the lifetime of the excited states. Photochemical decomposition TEMPO were also observed after TP excitation of 2a and 2b
of TEMPO in DMSO was found to be faster than that in (Supporting Information File 1, Figure S7). The formation of
benzene, but the chemical yield of TEMPO (56% from 2a and TEMPO at 740 nm, k740 = 4.9 × 10−6 s−1 in the bulk photoreac-
5
8% from 2b after 40 s irradiation) was found to be lower than tion, was the fastest in the TP-uncaging reaction of 2a
that obtained in benzene (Figure 1).
(Figure 3). For the uncaging reaction of 2b, the rate of
consumption under 730 nm irradiation, k730 = 1.6 × 10–6 s−1,
To obtain insight into the mechanism of generation of the was larger than those at other wavelengths (Supporting Infor-
TEMPO radical, the photolysis of 2 was conducted under mation File 1, Figure S6).
degassed conditions using the freeze-pump-thaw (FPT) method
(
Figure 2a,b). Interestingly, the generation of the TEMPO
radical was highly suppressed under the photolysis conditions
Figure 2a,b). Under air conditions, however, the photochemi-
(
cal release of TEMPO was detected in benzene, as shown in
Figure 2c,d. Faster formation of TEMPO was observed when
O2 atmosphere was used instead of an air atmosphere
(
Figure 2e,f). Therefore, the O2 molecule may play an impor-
tant role in clean generation of the TEMPO radical during pho-
tolysis. Indeed, the compounds oxidized at the benzylic carbon,
6
and 7, were isolated in 15% (15%) and 56% (42%) yield in
the photolysis of 2a and 2b under atmospheric conditions, re-
spectively (Scheme 3), indicating that under degassed condi-
tions, the photochemically generated radical pair returns to the
starting compound 2 with rapid radical recombination. Over
7
0% of the caged TEMPO 2a and ≈85% of 2b were recovered
after 2 h of irradiation under degassed conditions. The retarded
formation of TEMPO after 5 min of irradiation is due to the de-
crease in the relative absorbance of 2a to those of primary pho-
toproducts (Figure 2c,e).
Figure 3: Time profile, ln([2a]/[2a]0) versus irradiation time, of two-
photon uncaging reaction of TEMPO in the photolysis of 2a in
benzene, at wavelengths of 710–760 nm and power of 700 mW.
The TP action spectra of 2a and 2b in benzene are shown in
The TP photolysis of 2a (10 mM) and 2b (10 mM) was carried Figure 4, where the spectra were obtained by extrapolation from
out in benzene under atmospheric conditions using 710, 720, the absolute TP cross-section of the parent NPBF (18 GM) at
Scheme 3: Photochemical generation of TEMPO radical and photoproducts 6 and 7 under air atmosphere.
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Beilstein J. Org. Chem. 2019, 15, 863–873.
more stable than BRb generated from 2b based on DFT calcula-
tions at the B3LYP/6-31G(d) level of theory. In DMSO, no sig-
nificant difference between 2a and 2b was observed for the
photochemical release of the TEMPO species, although the sol-
vent effect is not clearly explained.
As mentioned above, the spatiotemporally controlled genera-
tion of the radical pair of TEMPO and BR was confirmed in the
photolysis of compounds 2a and 2b. Because free radicals play
important roles as anticancer therapeutic agents, the cytocidal
effect of the radical released from compound 2a was also tested
in vitro using lung cancer cells. One hundred thousand Lewis
lung carcinoma (LLC) cells were seeded into 24-well plates
(
medium: DMEM) and incubated overnight at 37 °C under an
atmosphere of 95% air and 5% CO2. The medium was replaced
with fresh phenol-red free DMEM containing various concen-
trations of 2a (0, 10, 100 μg mL−1) and further incubated for 4 h
under the same conditions. Without exposure to light, 2a
itself exhibited slight cytotoxicity based on trypan blue exclu-
sion, and ≈80–90% living cells remained in the medium con-
taining of 100 μg mL−1 of 2a (Supporting Information File 1,
Figure S8).
Figure 4: ESR spectra acquired during the photolysis of 2a (5 mM) in
benzene using 365 nm light.
The cytocidal effect of the radicals released from compound 2a
on LLC cells was also tested. Four hours after 1 min exposure
7
20 nm [58]. The TP cross-section of 2a was higher than that of to 360 nm light in various concentrations of 2a-containing me-
b by ≈15 GM. This higher GM value may be due to the dium, the number of living cells decreased in a 2a concentra-
2
stronger donor–acceptor character of 2a relative to that of 2b, tion-dependent manner (Supporting Information File 1, Figure
because the electron-donating alkyl group is located at the para- S9). After exposure of the cells in the medium containing
position of the p-nitrophenyl group in 2a.
100 μg mL−1 of 2a, the number of living cells decreased signifi-
cantly compared to that without exposure (66.5% vs 87.8%,
As observed in the OP uncaging reaction at 365 nm, the effi- Supporting Information File 1, Figure S10). An irradiation-
ciency of the TP-induced TEMPO uncaging reaction of 2a was time-dependent decline in the viability of the LLC cells was
almost three times higher than that of 2b in benzene. This is at- also observed (Figure 5). To evaluate whether the cytocidal
tributed to the substituent effect of the meta-alkoxy group on effect was due to photochemical radical generation, cells
the reactivity in the electronically excited states [70]. Moreover, exposed to 360 nm light for 1 min and the unexposed congeners
the relative stability of radicals BRa and BRb generated by the were stained by using a ROS-ID oxidative stress detection kit
photolysis of 2a and 2b had an important impact on the (Enzo Life Sciences, Farmingdale, NY, U.S.A.). Reactive
uncaging efficiency. The isodesmic reaction shown in Scheme 4 oxygen species (ROS) were detected in the cells irradiated in
suggests the radical BRa derived from 2a was 2.04 kcal mol−1 the 2a-containing medium, but not in the non-irradiated cells in
Scheme 4: Isodesmic reaction from BRa and 5b to 5a and BRb.
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Beilstein J. Org. Chem. 2019, 15, 863–873.
and 0.8% in benzene, respectively. The quantum yields in
DMSO were found to be higher than those in benzene, 13.1%
and 12.8%, respectively. The OP-uncaging efficiency (ε × Φ)
was found to be 480 and 175 for 2a and 2b, respectively, at
3
60 ± 10 nm, in benzene, and 3026 and 2995 in DMSO, respec-
tively. The TP efficiency of the TEMPO uncaging reaction was
found to be 1.1 GM at 740 nm for 2a and 0.22 GM at 730 nm
for 2b in benzene. The TP-induced clean release of the TEMPO
radical is expected to be applicable to further physiological
studies and site-selective polymerization reactions.
Experimental
All reagents were purchased from commercial sources and were
used without additional purification, unless otherwise mention-
ed. Caged nitroxides 2a and 2b were prepared according to the
methods described previously (Scheme 2) and were isolated by
silica gel column chromatography and GPC. 1H and 13C NMR
spectra were reported in parts per million (δ) by using CDCl3.
IR spectra were recorded with a FTIR spectrometer. UV–vis
spectra were taken by a SHIMADZU UV-3600 Plus spec-
trophotometer. Mass spectra were measured by a Mass Spectro-
metric Thermo Fisher Scientific LTQ Orbitrap XL, performed
by the Natural Science Center for Basic Research and Develop-
Figure 5: Irradiation time-dependent decline in viability of LLC cells
with compound 2a.
2
a-containing medium or the irradiated cells without 2a-con- ment (N-BARD), Hiroshima University.
taining medium (Figure 6). Thus, the preliminary analyses indi-
cated that the photochemical generation of radicals from 2a in- Preparation of caged compounds 2a and 2b
duced cancer cell death in vitro, although no in vivo study was 6-Ethyl-2-(4-nitrophenyl)benzofuran (5a). 4-Nitro-1-iodo-
performed because of the low water solubility of 2a. At this benzene (16.3 g, 65.5 mmol), Pd(dppf)Cl2 (0.97 g, 1.3 mmol),
point, we cannot rule out generate of ROS by photosensitiza- PPh3 (recrystallized, 0.51 g, 1.9 mmol) and CuI (0.25 g,
tion of the chromophore in the presence of O2 for the cytotoxic- 1.3 mmol) were added under N2 atmosphere followed by tolu-
ity.
ene (97 mL) and iPr2NH (49 mL, 359 mmol). The mixture was
stirred for 10 min, and TMSA (11.5 mL, 81.6 mmol) in toluene
(64 mL) was added at room temperature. It was stirred until all
Conclusion
In the present study, novel caged nitroxides 2a and 2b having a iodobenzene was consumed (20 min). TBAF 1 M in THF
TP-responsive chromophore were synthesized, and OP- and (100 mL, 100 mmol) was added followed by 5-ethyl-2-
TP-induced generation of the TEMPO radical with these species iodophenol (21.6 g, 86.9 mmol). The temperature was in-
was examined. The quantum yields for generation of the creased to 80 °C, and the mixture was stirred for 21.5 h. The
TEMPO radical from 2a and 2b were determined to be 2.5% reaction was quenched with 10% aqueous citric acid (400 mL)
Figure 6: Detection of intracellular ROS only in irradiated LLC cells with 2a-containing medium.
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Beilstein J. Org. Chem. 2019, 15, 863–873.
and extracted with DCM. The combined extracts were washed extracted with DCM. Combined extracts were washed with
with 10% aqueous NaOH (400 mL), water and dried with an- 10% aqueous NaOH (666 mL), water and dried with anhydrous
hydrous MgSO4. The solvent was removed by rotary evapora- MgSO4. The solvent was removed by rotary evaporation and
tion and the crude product was purified by silica gel column the crude product was purified silica gel column chromatogra-
chromatography (hexane/EtOAc 10:1, v/v) to give 6-ethyl-2-(4- phy (hexane/EtOAc 10:1, v/v) to give 5-ethyl-2-(4-
nitrophenyl)benzofuran (5a, 10.0 g, 57.3%). mp 114 °C; IR nitrophenyl)benzofuran (5b, 12.2 g, 67.6%). mp 129 °C; IR
(
KBr, cm−1): 3429, 2968, 1601, 1520, 1344, 1108, 828, 825, (KBr, cm−1): 2922, 1601, 1514, 1340, 1194, 853, 811, 754, 690;
7
9
7
1
54, 692; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.30 (d, J = 1H NMR (400 MHz, CDCl3) δ (ppm) 8.30 (d, J = 9.0 Hz, 2H),
.0 Hz, 2H), 7.98(d, J = 9.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 9.2 Hz, 1H), 7.44 (s, 1H),
.39 (s, 1H), 7.20 (d, J = 0.9 Hz, 1H), 7.14 (dd, J = 8.0, 1.4 Hz, 7.21 (dd, J = 8.4, 1.8 Hz, 1H), 7.19 (d, J = 0.8 Hz, 1H), 2.76 (q,
H), 2.80 (q, J = 7.6 Hz, 2H), 1.32 (t, J = 7.6 Hz, 3H); J = 7.6 Hz, 2H) 1.30 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
1
3C NMR (100 MHz, CDCl3) δ (ppm) 156.05 (C), 152.86 (C), CDCl3) δ (ppm) 154.12 (C), 153.38 (C), 147.21 (C), 139.74
1
1
47.12 (C), 143.06 (C), 136.56 (C), 126.43 (C), 125.01 (CH), (C), 136.47 (C), 128.79 (C), 126.24 (CH), 125.12 (CH), 124.29
24.33 (CH), 124.07 (CH), 121.22 (CH), 110.42 (CH), 105.12 (CH), 120.14 (CH), 111.12 (CH), 105.04 (CH), 28.83 (CH2),
(
CH), 29.25 (CH2), 15.84 (CH3); HRMS–ESI (m/z): [M−] calcd. 16.15 (CH3); HRMS–ESI (m/z): [M−] calcd. for C16H13NO3,
for C16H13NO3, 267.09009; found, 267.09064. 267.09009; found, 267.09030.
2
,2,6,6-Tetramethyl-1-(1-(2-(4-nitrophenyl)benzofuran-6- 2,2,6,6-Tetramethyl-1-(1-(2-(4-nitrophenyl)benzofuran-5-
yl)ethoxy)piperidine (2a). Under air, TEMPO (0.23 g, yl)ethoxy)piperidine (2b). Under air, TEMPO (46.8 mg,
1
4
0
.5 mmol), 6-ethyl-2-(4-nitrophenyl)benzofuran (5a, 1.26 g, 0.3 mmol), 5-ethyl-2-(4-nitrophenyl)benzofuran (5b, 267 mg,
.72 mmol), Cu(OAc)2 (16.5 mg, 0.092 mmol), bpy (13.8 mg, 1 mmol), Cu(OAc)2 (3.6 mg, 0.02 mmol), bpy (3.1 mg,
.094 mmol), TBHP (aqueous 70%, 0.41 mL, 2.9 mmol) were 0.02 mmol), TBHP (aqueous 70%, 0.086 mL, 0.6 mmol) were
added into a two-necked flask in the dark. The reaction was added into a Schlenk tube in the dark. The reaction was stirred
stirred at 60 °C for 15 h. Upon completion, the mixture was at 60 °C for 16.5 h. Upon completion, the mixture was purified
purified silica gel column chromatography (hexane/ether 15:1, by silica gel column chromatography (hexane/ether 15:1, v/v) to
v/v) to give 2a (236 mg, 37.8%). mp 109 °C; IR (KBr, cm−1): give 2b (66 mg, 52%). mp 144 °C; IR (KBr, cm−1): 2922, 1602,
3
429, 2934, 1600, 1521, 1345, 1062, 825; 1H NMR (400 MHz, 1520, 1342, 1108, 852, 746; 1H NMR (400 MHz, CDCl3) δ
CDCl3) δ (ppm) 8.31 (d, J = 9.0 Hz, 2H), 7.99 (d, J = 9.0 Hz, (ppm) 8.31 (d, J = 9.0 Hz, 2H), 7.99 (d, J = 9.0 Hz, 2H), 7.57
2
1
1
1
H), 7.57 (d, J = 8.1 Hz, 1H), 7.54 (s, 1H), 7.25 (dd, J = 8.1, (d, J = 1.6 Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.36 (dd, J = 8.6,
.2 Hz, 1H), 7.21 (d, J = 0.7 Hz, 1H), 4.91 (q, J = 6.7 Hz, 1H), 1.7 Hz, 1H), 7.23 (s, 1H), 4.88 (q, J = 6.6 Hz, 1H), 1.53 (d, J =
.54 (d, J = 6.6 Hz, 3H), 1.51 (s, 3H), 1.32 (s, 6H), 1.20 (s, 3H), 6.7 Hz, 3H), 1.51 (s, 3H), 1.33 (s, 6H), 1.19 (s, 3H), 1.02 (s,
.05 (s, 3H), 0.66 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 3H), 0.60 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm) 154.75
(
(
ppm) 155.69 (C), 153.25 (C), 147.18 (C), 144.70 (C), 136.50 (C), 153.43 (C), 147.24 (C), 141.55 (C), 136.47 (C), 128.34 (C),
C), 127.39 (C), 125.08 (CH), 124.33 (CH), 122.65 (CH), 125.15 (CH), 125.04 (CH), 124.32 (CH), 119.50 (CH), 110.98
1
21.04 (CH), 109.47 (CH), 105.13 (CH), 83.28 (CH), 59.79 (CH), 105.39 (CH), 83.13 (CH), 59.76 (C), 40.42 (CH2), 34.37
C), 40.43 (CH2), 34.23 (CH3) , 23.82 (CH2), 20.40 (CH3), (CH3), 23.78 (CH2), 20.38 (CH3), 17.24 (CH3); HRMS–ESI
7.24 (CH3); HRMS–ESI (m/z): [M + H]+ calcd. for (m/z): [M + H]+ calcd. for C25H30N2O4; 423.22783; found
C25H30N2O4, 423.22783; found, 423.22754. 423.22757.
(
1
5
-Ethyl-2-(4-nitrophenyl)benzofuran (5b). 4-Nitro-1-iodo- 1-(2-(4-Nitrophenyl)benzofuran-6-yl)ethan-1-ol (6a). mp
benzene (16.8 g, 67.5 mmol), Pd(dppf)Cl2 (1.0 g, 1.3 mmol), 133 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.31 (d, J =
PPh3 (recrystallized, 0.53 g, 2.0 mmol) and CuI (0.26 g, 9.0 Hz, 2H), 7.99 (d, J = 9.0 Hz, 2H), 7.61 (d, J = 7.7 Hz, 1H),
1
.3 mmol) were added under N2 atmosphere followed by tolu- 7.61 (s, 1H), 7.30 (dd, J = 8.1, 1.3 Hz, 1H), 7.22 (s, 1H), 5.06
ene (100 mL) and iPr2NH (50.5 mL, 370 mmol). The mixture (m, 1H), 1.90 (d, J = 3.6 Hz, 1H), 1.57 (d, J = 6.5 Hz, 3H);
was stirred for 10 min, and TMSA (1.75 mL, 12.5 mmol) in tol- 13C NMR (100 MHz, CDCl3) δ (ppm) 155.72 (C), 153.61 (C),
uene (10 mL) was added at room temperature. It was stirred 147.24 (C), 144.47 (C), 136.26 (C), 127.96 (C), 125.16 (CH),
until all iodobenzene was consumed (20 min). TBAF 1 M in 124.34 (CH), 121.56 (CH), 121.34 (CH), 108.33 (CH), 104.97
THF (100 mL, 100 mmol) was added followed by 4-ethyl-2- (CH), 70.50 (CH), 25.58 (CH3).
iodophenol (22.22 g, 89.6 mmol). The temperature was in-
creased to 80 °C, and the mixture was stirred for 20 h. The reac- 1-(2-(4-Nitrophenyl)benzofuran-5-yl)ethan-1-ol (6b). mp
tion was quenched with 10% aqueous citric acid (666 mL) and 149 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.30 (d, J =
870

Beilstein J. Org. Chem. 2019, 15, 863–873.
9
8
.0 Hz, 2H), 7.99 (d, J = 9.0 Hz, 2H), 7.66 (s, 1H), 7.53 (d, J = tion Kit (Enzo Life Sciences, Farmingdale, NY, USA) in
.5 Hz, 1H), 7.39 (dd, J = 8.5, 1.7 Hz, 1H), 7.22 (s, 1H), 5.04 conjunction with fluorescence microscopy. Intracellular ROS
(
q, J = 6.4 Hz, 1H), 1.88 (s, 1H), 1.57 (d, J = 6.4 Hz, 3H); was detected in the form of green fluorescence signals. Bars,
3C NMR (100 MHz, CDCl3) δ (ppm) 154.93 (C), 153.81 (C), 100 µm.
1
1
1
47.29 (C), 141.48 (C), 136.22 (C), 128.74 (C), 125.24 (CH),
24.32 (CH), 123.67 (CH), 118.28 (CH), 111.45 (CH), 105.14
Supporting Information
(
CH), 70.47 (CH), 25.64 (CH3); HRMS–ESI (m/z): [M−] calcd.
for C16H13NO4, 283.08501; found, 283.08548.
Supporting Information File 1
1
H and 13C NMR charts for new compounds and
Figures S1–S8.
https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-84-S1.pdf]
1
2
8
8
3
-(2-(4-Nitrophenyl)benzofuran-6-yl)ethan-1-one (7a). mp
14 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.35 (d, J =
.9 Hz, 2H), 8.18 (s, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.93 (dd, J =
.2, 1.3 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.29 (s, 1H), 2.70 (s,
H); 13C NMR (100 MHz, CDCl3) δ (ppm) 197.22 (C), 156.45
[
(
C), 155.12 (C), 147.82 (C), 135.52 (C), 134.84 (C), 132.93 (C), Acknowledgements
25.76 (CH), 124.41 (CH), 123.88 (CH), 121.41 (CH), 111.79 The NMR and MS measurements were performed at N-BARD,
1
(
CH), 104.85 (CH), 26.86 (CH3).
Hiroshima University. This work was supported by a Grant-in-
Aid for Scientific Research JP17H0302200 from the Ministry of
1
2
9
9
-(2-(4-Nitrophenyl)benzofuran-5-yl)ethan-1-one (7b). mp Education, Culture, Sports, Science and Technology, Japan.
29 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.34 (d, J = This work was partly supported by a research grant from Taka-
.0 Hz, 2H), 8.30 (s, 1H), 8.04 (d, J = 8.6 Hz, 1H), 8.03 (d, J = hashi Industrial and Economic Research Foundation.
.0 Hz, 2H), 7.62 (d, J = 8.7, 1H), 7.32 (s, 1H), 2.69 (s, 3H);
1
3C NMR (100 MHz, CDCl3) δ (ppm) 197.35 (C), 157.84 (C), ORCID® iDs
1
54.86 (C), 147.63 (C), 135.59 (C), 133.41 (C), 128.79 (C), Ayato Yamada - https://orcid.org/0000-0002-0631-0806
Manabu Abe - https://orcid.org/0000-0002-2013-4394
1
26.38 (CH), 125.51 (CH), 124.40 (CH), 122.86 (CH), 111.57
Yoshinobu Nishimura - https://orcid.org/0000-0002-9982-141X
(
CH), 105.38 (CH), 26.82 (CH3); HRMS–ESI (m/z): [M−]
Shoji Ishizaka - https://orcid.org/0000-0001-9866-5809
Taku Nakashima - https://orcid.org/0000-0002-0035-674X
Kiyofumi Shimoji - https://orcid.org/0000-0001-5382-4730
calcd. for C16H11NO4, 281.06936; found, 281.06970.
Photoirradiation of LLC cells with compound
2a
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BZ-9000, Keyence, Osaka, Japan), cell viability was deter-
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(https://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.15.84
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