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Development of water-soluble phenazine-2,3diol-based photosensitizers for singlet oxygen
Cite this: DOI: 10.1039/d3ob00491k
generation†
Kazunori Yagi, Kazuki Ohira,
Keita Yamana, Keiichi Imato,Atsushi Ikeda
and Yousuke Ooyama *Riku Kawasaki,

Phenazine-2,3-diol-based dyes, KY-1Na and KY-2Na bearing one and two carboxylic acid sodium salts,
respectively, have been newly developed as water-soluble photosensitizers (PSs) possessing the ability togenerate singlet oxygen (1O2). In order to evaluate the solubility of KY-1Na and KY-2Na in water, thehydrophobicity/hydrophilicity of the two PSs was investigated by experimental measurement of the logarithms (log Po/w) of the 1-octanol/water partition coeﬃcient (Po/w) for the PS. The log Po/w values of both
KY-1Na and KY-2Na were determined to be −0.9, indicating that both the PSs are more hydrophilic thanRose Bengal (−0.6) and have hydrophilicity equivalent to methylene blue (−0.9). Both the PSs in watershow a broad photoabsorption band in the range of 500 to 600 nm. Thus, we estimated the 1O2 quantum
yields (ΦΔ) of KY-1Na and KY-2Na in water by using 9,10-anthracenediyl-bis(methylene)dimalonic acid
(ABDA) as a water-soluble 1O2 scavenger. It was found that in water the ΦΔ value (0.19) of KY-2Na ishigher than that of KY-1Na (0.06). Density functional theory (DFT) calculations suggested that the highest
occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distributions forthe molecular structure of KY-2Na are adequately separated, leading to a decrease in the energy gap(ΔEST) between the singlet state (S1) and the triplet state (T1) that causes eﬃcient intersystem crossing(ISC), compared to that for the molecular structure of KY-1Na. Indeed, time-dependent DFT (TD-DFT)
Received 31st March 2023,
Accepted 4th May 2023

calculations demonstrated that the ΔEST(S1–T1) value (0.82 eV) of KY-2Na is smaller than that (0.98 eV) of
KY-1Na, resulting in a relatively high ΦΔ value of KY-2Na. Consequently, we demonstrate that phenazine-

DOI: 10.1039/d3ob00491k

2,3-diol-based PSs bearing carboxylic acid salts possess high solubility and moderate 1O2 generation

rsc.li/obc

ability in water.

Introduction
Singlet oxygen (1O2) generated by light irradiation to photosensitizers (PSs),1 which is one of the reactive oxygen species(ROS), has gained more and more attention as an eﬀective andconvenient oxidant of alkenes and dienes for Schenck–ene
reactions and [2 + 2]- and [4 + 2]-cycloadditions in organic
synthesis,2–5 degradation of water pollutants in environmental
and quality control monitoring systems,6 inactivation of microbiological contamination in point-of-use water disinfection
systems,7 and cancer treatment in photodynamic therapy(PDT).8 1O2 is generally produced through the following processes: initially, the PS (1PS) absorbs light (hν) to generate thesinglet excited state (1PS*), and then the 1PS* undergoes inter-

Applied Chemistry Program, Graduate School of Advanced Science and Engineering,
Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan.
E-mail: yooyama@hiroshima-u.ac.jp† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d3ob00491k

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system crossing (ISC) to generate the triplet excited state(3PS*). Subsequent energy transfer from the photoexcited PS(3PS*) to triplet oxygen (3O2) produces 1O2. Thus, in order toobtain a high 1O2 quantum yield (ΦΔ), it is necessary toenhance the ISC eﬃciency of PSs. Moreover, for use in water
purification systems and PDT, the PSs are required to possesshigh solubility in water as well as eﬃcient ISC. Methylene blue(MB),9 Rose Bengal (RB),10 and hydrophobically modified porphyrin derivatives11 have been used as water-soluble PSs withhigh 1O2 ΦΔ values (0.50–0.60 for MB,9b,c 0.70–0.80 for RB,9b,10b
and 0.50–0.60 for free base 5,10,15,20-tetrakis(4-carboxylatophenyl)porphyrin,11a,b respectively). Meanwhile, halogen atoms
such as iodide and bromide atoms are often introduced into
chromophores (e.g. RB) to promote ISC based on spin–orbit
coupling,11 but they cause dark cytotoxicity.12 Indeed, MB as a
halogen atom-free-heteroanthracene-based PS13 is used as not
only a reference water-soluble PS for the evaluation of ΦΔ,9b,cbut also a fungicide and antidote in aquaculture.9f,gThus, in our previous work, we have designed and developed phenazine-2,3-diol-based dyes (KO-0-3)14 as halogen

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Paperatom-free-heteroanthracene-based PSs and demonstrated thatthe modification of the phenazine-2,3-diol chromophore withformyl groups promotes ISC, leading to eﬃcient 1O2 generation (Fig. 1a). According to the El-Sayed rule, formyl and carbonyl substituents in a chromophore skeleton facilitate the
ISC from the singlet (Sn) to triplet (Tn) states, that is, by thechange in the molecular orbital type such as transitions of1(nπ*) to 3(ππ*) and 1(ππ*) to 3(nπ*). In fact, KO-1-3 exhibited
moderate 1O2 generation ability (ΦΔ = 0.22–0.41) in THF.
In this work, in order to attain the solubility of phenazine2,3-diol-based PSs in water, we have developed KY-1Na and
KY-2Na bearing one and two carboxylic acid sodium salts,
respectively, as the water-soluble derivatives of KO-2 exhibitinga moderate ΦΔ value (0.17) in DMSO (Fig. 1b). Actually, inorder to evaluate the solubility of KY-1Na and KY-2Na in water,
the hydrophobicity/hydrophilicity of the two PSs was investigated by experimental measurement of the logarithms(log Po/w) of the 1-octanol/water partition coeﬃcient (Po/w) for
the PS. Both the PSs in water show a broad photoabsorption
band in the range of 500 to 600 nm. It was found that in water
KY-2Na exhibits a moderate ΦΔ value, which is higher thanthat of KY-1Na. Density functional theory (DFT) and timedependent DFT (TD-DFT) calculations were performed to gain
insight into the 1O2 generation properties of KY-1Na and
KY-2Na based on the ISC eﬃciency. Herein we report that the
introduction of carboxylic acid salts into the phenazine-2,3diol chromophore is eﬀective in yielding solubility in waterand a small energy gap (ΔEST) between the S1 state and the T1state for eﬃcient ISC, leading to the development of watersoluble halogen-atom-free-heteroanthracene-based PSs.

Results and discussion
Synthesis
Phenazine-2,3-diol-based PSs, KY-1 and KY-2 bearing one andtwo carboxylic acids, respectively, and their sodium salt derivatives KY-1Na and KY-2Na, were synthesized by a stepwise synthetic protocol (Scheme 1). We prepared compounds 1 and 2,15
respectively, for the construction of the phenazine skeleton

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Scheme 1 Synthetic route to phenazine-2,3-diol-based PSs KY-1,
KY-2, KY-1Na, and KY-2Na.

and the introduction of the formylthiophene unit on thechromophore by the Stille coupling (see the ESI†). Phenazine2,3-diol chromophores 3 and 4 were prepared by the cyclodehydration of 2,5-dibromo-3,6-dihydroxy-p-quinone with methyl
3,4-diaminobenzoate or compound 1, respectively, and thenconverted into methoxymethyl (MOM)-protected compounds 5
and 6, respectively. The Stille coupling reaction of 5 or 6 withcompound 2 gave compounds 7 and 8, respectively.
Compounds 7 and 8 were hydrolyzed by treatment with a baseto generate compounds 9 and 10, respectively, and then thehydrolysis of 9 and 10 with an acid gave the carboxylic acids
KY-1 and KY-2, respectively. Finally, KY-1Na and KY-2Na wereobtained by treatment of KY-1 and KY-2, respectively, with abase.
In order to evaluate the hydrophobicity/hydrophilicity of
KY-1Na and KY-2Na, the logarithms (log Po/w)16 of the
1-octanol/water partition coeﬃcient (Po/w) for the PS were
measured experimentally. The log Po/w values of both KY-1Na
and KY-2Na were determined to be −0.9, indicating that boththe PSs are more hydrophilic than RB (−0.6)10c and have hydrophilicity equivalent to MB (−0.9).9d,e Incidentally, KO-2 exhibitshydrophobicity with a log Po/w value of 1.3. Thus, this fact indicates that KY-1Na and KY-2Na possess high solubility in water.
Optical properties

Fig. 1 Chemical structures of phenazine-2,3-diol-based PSs, (a) KO-03 in our previous study and (b) KY-1, KY-2, KY-1Na, and KY-2Na in thisstudy.

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The photoabsorption and fluorescence spectra of KY-1Na and
KY-2Na in DMSO and in water (D2O) are shown in Fig. 2 withthose of KO-2, KY-1, and KY-2 in DMSO as references for thecharacterization of the photoabsorption properties of KY-1Na
and KY-2Na, and their optical data are summarized in Table 1.
All the five dyes show an intense photoabsorption maximum(λabsmax ) at around 400–450 nm with a relatively high molar
extinction coeﬃcient (εmax = 25 000–30 000 M−1 cm−1), which

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Fig. 2 (a) Photoabsorption and ﬂuorescence spectra (λex = 532 nm) of
KO-2, KY-1, KY-2, KY-1Na, and KY-2Na (5.0 × 10−5 M) in DMSO. (b)Photoabsorption and ﬂuorescence spectra (λex = 532 nm) of KY-1Na
and KY-2Na (5.0 × 10−5 M) in D2O.

an 1O2 scavenger. It is well known that 1,3-diphenylisobenzofuran (DPBF) acts as an eﬃcient 1O2 scavenger in organic solvents to produce its oxidized product, o-dibenzoylbenzene.18a,b
Meanwhile, 9,10-anthracenediyl-bis(methylene)dimalonic acid
(ABDA), which can react rapidly and irreversibly with 1O2 to
producethecorresponding
endoperoxide(endoperoxideABDA), is widely used as a water-soluble 1O2
scavenger.18b,c Therefore, 1O2 generation by the five PSs wasinvestigated by monitoring the changes in the photoabsorption spectra of DPBF in the DMSO solution of each PS
and ABDA in the aqueous solution (D2O/DMSO 99 : 1, v/v) of
KY-1Na and KY-2Na under photoirradiation (Fig. 3a–d for
KY-1Na and KY-2Na, and Fig. S45† for KO-2, KY-1, KY-2, andRB in DMSO). DMSO and D2O/DMSO were bubbled with air

Table 1 Optical data and 1O2 quantum yields (ΦΔ) of KO-2, KY-1, KY-2,
KY-1Na and KY-2Na

Dye
KO-2
KY-1
KY-2
KY-1NaKY-2NaKY-1NaKY-2Naλabs
max /nm(εmax/M−1 cm−1)a522 (11 500)
534 (7700)a534 (6000)a
486shoulder (21 500)a
486shoulder (15 900)a
500shoulder (8100)b
500shoulder (7100)bε/M−1 cm−1
@λabs = 532 nma

11 300
7700a
6000a
9600a
7700a
6100b
4500b

λflmax /nm(Φfl )cΦΔa634 (0.03)
625 (<0.02)a631 (<0.02)a611 (0.02)a603 (0.02)a611 (<0.02)b611 (<0.02)b

0.17d
0.14d
0.17d
0.13d
0.32d
0.06e
0.19e

In DMSO. b In D2O. c Fluorescence quantum yields (Φfl ) were determined using a calibrated integrating sphere system (λex = 532 nm).
d1
O2 quantum yields (ΦΔ) based on the relative decomposition rate of
DPBF using RB as the standard PS (ΦΔ = 0.76 in DMSO,9a see
Fig. S44a†) and DPBF as the 1O2 scavenger under irradiation withmonochromatic light (532 nm, 300 μW cm−2) in DMSO. e ΦΔ based on
the relative decomposition rate of AMDA using RB as the standard PS(ΦΔ = 0.75 in water,9b see Fig. S44b†) and AMDA as the 1O2 scavenger
under irradiation with monochromatic light (532 nm, 300 μW cm−2) in
D2O/DMSO (99 : 1, v/v).
acan be assigned to the π → π* transition of the phenazine skeleton containing two formylthiophene units.14 In addition, for
KO-2, KY-1, and KY-2, a broad photoabsorption band (λabsmax =
520–535 nm) with a moderate εmax value (6000–12 000) was also
observed in the range of 500 to 650 nm, which is ascribable tothe formation of phenoxide ions by the partial deprotonation of
the hydroxyl groups.14 On the other hand, for KY-1Na and
KY-2Na both in DMSO and in water, the photoabsorption
spectra in the range of 500 to 600 nm are significantly broadened, and exhibit a hypsochromic shift and become a shoulder
band, compared to those of KO-2, KY-1, and KY-2 in DMSO.
This fact indicates that KY-1Na and KY-2Na existed as carboxylate anion (–COO−) species in DMSO as well as in water.17 In thecorresponding fluorescence spectra, all the five dyes show afeeble fluorescence band with a fluorescence maximum wavelength (λflmax ) at 600–635 nm. In fact, their fluorescencequantum yields (Φfl ) were significantly low (≤0.03).
1O2 generation

We evaluated the 1O2 generation ability of the phenazine-2,3diol-based PSs KO-2, KY-1, KY-2, KY-1Na, and KY-2Na by using

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Fig. 3 Photoabsorption spectra for the photooxidation of DPBF (5.0 ×
10−5 M) in the presence of (a) KY-1Na and (b) KY-2Na under photoirradiation with 532 nm (300 μW cm−2) in DMSO. Insets are magniﬁcationsof maxima in the spectra at around 415 nm. Photoabsorption spectra forthe photooxidation of ABDA (5.0 × 10−5 M) in the presence of (c) KY-1Na
and (d) KY-2Na under photoirradiation with 532 nm (300 μW cm−2) in
D2O/DMSO (99 : 1, v/v). Insets are magniﬁcations of maxima in the
spectra at around 400 nm. (e) Plots of ΔOD at around 415 nm for thephotooxidation of DPBF in the presence of KO-2, KY-1, KY-2, KY-1Na,
KY-2Na, and RB against the photoirradiation time (532 nm, 300 μW
cm−2) in DMSO. (f ) Plots of ΔOD at around 400 nm for the photooxidation of ABDA in the presence of KY-1Na, KY-2Na, and RB against thephotoirradiation time (532 nm, 300 μW cm−2) in D2O/DMSO (99 : 1, v/v).

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for 15 min prior to preparing solutions. Air-saturated DMSO
solutions containing DPBF or aqueous solutions containing
ABDA and each PS were irradiated with monochromatic lightat 532 nm (300 μW cm−2) that was obtained by the passage ofa xenon light source through a monochromator. In all the
DMSO solutions, the photoabsorption of DPBF at around
415 nm decreased with the increase in the photoirradiation
time (Fig. 3a, b and S45†), which indicates that DPBF reactedwith 1O2 generated by the photosensitization of the PSs. In
order to make clear the diﬀerence in 1O2 generation ability
between the five PSs, the changes in the optical density (ΔOD)
at around 415 nm of DPBF are plotted against the photoirradiation time (Fig. 3e), and the slope (msam) is used to estimate
the 1O2 quantum yield (ΦΔ): the ΦΔ values for PSs were determined by the relative method using the slope value (mref =−0.0100) of the plot for RB (ΦΔ = 0.76 in DMSO)9a as a standard (Table 1). Indeed, the correlation coeﬃcient (R2) values
for the calibration curves of the five PSs are 0.860–0.996, whichindicates good linearity, and the msam values become steeper
in the following order: KY-1Na (−0.0018) < KY-1 (−0.0033) ≈KY-2 (−0.0037) < KO-2 (−0.0048) < KY-2Na (−0.0058). It was
found that the ΦΔ values (0.13–0.17) of KY-1, KY-2, andKY-1Na are equivalent to that of KO-2 (ΦΔ = 0.17), and surprisingly KY-2Na exhibited the highest 1O2 generation ability (ΦΔ =0.32). Moreover, for the aqueous solutions of KY-1Na and
KY-2Na, the photoabsorption of ABDA at around 400 nm
decreased with the increase in the photoirradiation time
(Fig. 3c and d), indicating ABDA reacted with 1O2 generated by
the photosensitization of the PSs. The plots of ΔOD at around
400 nm of ABDA versus the photoirradiation time revealed thatthe calibration curve for KY-2Na shows good linearity with an
R2 value of 0.956, but that for KY-1Na has a mediocre R2 value(0.756) which may be due to a small change in the ΔOD(Fig. 3f ). In fact, the msam value (−0.00043) for KY-2Na is larger
than that (−0.00007) for KY-1Na. Thus, the ΦΔ values for PSs
were determined by the relative method using the slope value(mref = −0.00084) of the plot for RB (ΦΔ = 0.75 in water)9b as astandard. It is worth noting here that in water the ΦΔ value(0.19) of KY-2Na is much higher than that of KY-1Na (0.06),
indicating that KY-2Na bearing two carboxylic acid sodium
salts is superior to KY-1Na bearing one carboxylic acid sodium
salt for 1O2 generation. Meanwhile, both the Φfl and ΦΔ values
for phenazine-2,3-diol-based photosensitizers are quite low
which indicates a large loss of the excited energy. Thus, we
performed time-resolved fluorescence spectroscopy to determine the fluorescence lifetime (τfl ), radiative rate constant (kr= Φfl/τfl ), and nonradiative rate constant (knr = (1 − Φfl )/τfl ) for
KY-2Na in DMSO. The τfl, kr, and knr values for KY-2Na are 2.17
ns, 9.20 × 106 s−1, and 4.51 × 108 s−1, respectively. The ratio of
the nonradiative constant to radiative constant (knr/kr) is estimated to be 49, indicating that the knr value is significantly
larger than the kr value. Thus, this result suggests that for thephenazine-2,3-diol-based photosensitizers, the accelerated
nonradiative decay of the excited state may be the main reason
for the large loss of the excited energy. Nevertheless, thephotodynamic activities of KY-1Na and KY-2Na for PDT in

Org. Biomol. Chem.

Organic & Biomolecular Chemistrymurine colon carcinoma cells (Colon26)19 under visible lightirradiation (>510 nm) were investigated. Colon26 cells wereincubated with KY-1Na or KY-2Na for 24 h. The cells were irradiated for 30 min (9 mW), and cell viability was determined
after 24 h of incubation using a WST-8 assay. Both under dark
and irradiation conditions, unfortunately, the cell viabilitychanged little despite the increasing concentration of KY-1Na
and KY-2Na (Fig. S46†). In order to investigate the degree ofcellular uptake of KY-2Na in Colon26 cells, we performed confocal laser scanning microscopy (CLSM) for live cell fluorescence imaging. As is expected, the fluorescence image originating from the photosensitizer KY-2Na was not observed(Fig. S47†). These low photodynamic activities indicate that
KY-1Na and KY-2Na with high solubility in water (log Po/w = –0.9) are impermeable to the cell membrane of Colon26.20
Consequently, these results suggested that the 1O2 generation
ability of phenazine-2,3-diol-based PSs is attributed to thefacilitated ISC from the singlet to triplet states by the carbonylgroups based on El-Sayed’s rule which allows the change inthe molecular orbital type such as transitions of 1(nπ*) to3(ππ*) and 1(ππ*) to 3(nπ*). Moreover, it was found that KY-2Na
possesses moderate 1O2 generation ability not only in DMSO
but also in water, which is attributed to the eﬃcient ISC
characteristics, as discussed later in the next section.
Theoretical calculations
Density functional theory (DFT) and time-dependent DFT(TD-DFT)
calculations21
atthe
B3LYP/6-311G(d,p)/
DMSO-IEFPCM level for KO-2, KY-1, and KY-2, the B3LYP/6311+G(d,p)/DMSO-IEFPCM level for KY-1Na and KY-2Na in carboxylate anion forms, and the B3LYP/6-311+G(d,p)/WaterIEFPCM level for KY-1Na and KY-2Na in carboxylate anion
forms were performed to gain insight into the 1O2 generation
properties of these PSs based on the ISC eﬃciency (Fig. 4).
There is little diﬀerence in the highest occupied molecularorbital (HOMO) and the lowest unoccupied molecular orbital(LUMO) energy levels among the five PSs. Moreover, the DFT
calculations indicate that for KO-2, KY-1, KY-2, and KY-1Na,
the HOMOs are delocalized over the phenazine-2,3-diol skeleton and formylthiophene units. On the other hand, for
KY-2Na, the HOMO is delocalized on the phenazine-2,3-diolskeleton containing the carboxylate anion. Meanwhile, the
LUMOs for KO-2, KY-1Na, and KY-2Na are delocalized over thephenazine-2,3-diol skeleton and formylthiophene units, but
those for KY-1 and KY-2 are delocalized mainly on the phenazine-2,3-diol skeleton and localized partially on formylthiophene units. Thus, DFT calculations suggested that the HOMO
and LUMO distributions for the molecular structure of KY-2Na
are adequately separated, leading to a decrease in the energygap (ΔEST) between the S1 state and the T1 state that causes
eﬃcient ISC,22,23 compared to that for the molecular structuresof KO-2, KY-1, KY-2, and KY-1Na. Indeed, the TD-DFT calculations demonstrated that the ΔEST (S1–T1) values (0.80 eV in
DMSO and 0.82 eV in water) of KY-2Na are smaller than those(0.96 eV in DMSO and 0.98 eV in water) of KY-1Na, that (0.85
eV) of KY-1 and that of (0.81 eV) of KY-2 as well as that (0.93This journal is © The Royal Society of Chemistry 2023

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Fig. 4 Energy level diagram, HOMO, and LUMO of (a) KO-2, KY-1, and KY-2 at the B3LYP/6-311G(d,p)/DMSO-IEFPCM level, KY-1Na and KY-2Na as
carboxylate anions at the B3LYP/6-311+G(d,p)/DMSO-IEFPCM level, and (b) KY-1Na and KY-2Na at the B3LYP/6-311+G(d,p)/Water-IEFPCM level
using DFT calculations.

eV) of KO-2, resulting in a relatively high ΦΔ value of KY-2Na(Table S7, ESI†). Consequently, we demonstrated that phenazine-2,3-diol chromophore-bearing carboxylic acid salts can act
as water-soluble halogen-atom-free-heteroanthracene-based
PSs for 1O2 generation.

Conclusions
We have designed and developed phenazine-2,3-diol-baseddyes, KY-1Na and KY-2Na bearing one and two carboxylic acidsodium salts, respectively, as water-soluble halogen-atom-freeheteroanthracene-based photosensitizers (PSs) possessing theability to generate 1O2. The logarithms (log Po/w) of the
1-octanol/water partition coeﬃcient (Po/w) demonstrated thatboth KY-1Na and KY-2Na are more hydrophilic than Rose
Bengal and have hydrophilicity equivalent to methylene blue.
Both the PSs in water show a broad photoabsorption band in
the range from 500 to 600 nm. It was found that in water
KY-2Na exhibits a moderate 1O2 quantum yield (ΦΔ = 0.19)
value, which is higher than that (0.06) of KY-1Na. Density
functional theory (DFT) calculations suggested that the HOMO
and LUMO distributions for the molecular structure of KY-2Na
are adequately separated, leading to a decrease in the energygap (ΔEST) between the S1 state and the T1 state that causes
eﬃcient intersystem crossing (ISC), compared to that for themolecular structure of KY-1Na. Indeed, time-dependent DFT(TD-DFT) calculations demonstrated that the ΔEST (S1–T1)This journal is © The Royal Society of Chemistry 2023

value (0.82 eV) of KY-2Na is smaller than that (0.98 eV) of
KY-1Na, resulting in a relatively high ΦΔ value of KY-2Na.
Consequently, we propose that phenazine-2,3-diol chromophore-bearing carboxylic acid salts are one of the promisingwater-soluble halogen-atom-free-heteroanthracene-based PSs
for 1O2 generation. Further studies on adjustment in thehydrophobicity/hydrophilicity of phenazine-2,3-diol-based PSs
suitable for the light-induced cytotoxicity assessment are now
in progress to gain insight into the 1O2 generation under physiological conditions.

Experimental
GeneralAll solvents and reagents were used as received unless otherwise noted. Rose Bengal (RB) was purchased from Sigma
Aldrich and recrystallized from methanol twice for the use inthephotoabsorption
spectralmeasurement.
1,3Diphenylisobenzofuran (DPBF) was purchased from Tokyo
Chemical Industry and recrystallized from a mixture of dichloromethane and methanol for use in the photoabsorption
spectral measurement. High-grade 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was purchased from
Funakoshi Co., Ltd. 1H NMR and 13C NMR spectra wererecorded using a Varian-500 (500 MHz) FT NMR spectrometer.
FT-IR spectra were recorded using a Shimadzu IRTracer-100
instrument. High-resolution mass spectral data were acquired

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Paperusing a Thermo Fisher Scientific LTQ Orbitrap XL mass
spectrometer. Photoabsorption spectra were recorded using
Shimadzu
andUV-3600-plus
spectrophotometers.
Fluorescence spectra were measured using a Hitachi F-4500
spectrophotometer. The fluorescence quantum yields (Φfl )were determined with a Hamamatsu C9920-01 instrument
equipped with a CCD using a calibrated integrating spheresystem. Fluorescence decay measurement was performed on a
HORIBA DeltaFlex modular fluorescence lifetime system,
using a Nano LED pulsed diode excitation source (451 nm).
The irradiance of monochromatic and continuous light forphotosensitizing reactions was adjusted using a Newport
1918-C optical power meter.
Synthesis
Methyl 6,9-dibromo-7,8-dihydroxyphenazine-2-carboxylate(3). A solution of bromanilic acid (2.98 g, 10.0 mmol) andmethyl 3,4-diaminobenzoate (1.66 g, 10.0 mmol) in ethanol(500 mL) was stirred at 80 °C overnight. After the disappearance of the reactants, the reaction mixture was cooled
to room temperature, and the precipitate was filtered andwashed with a small amount of ethanol to obtain 3 as a redsolid (3.80 g, 89% yield); m.p. over 300 °C; IR (ATR): ν̃ = 3204,
2957, 1709, 1624, 1609, 1572, 1551 cm−1; 1H NMR (500 MHz,DMSO-d6): δ = 8.61 (br, 1H, aromatic), 8.20 (d, J = 8.60 Hz, 1H,
aromatic), 8.12 (d, J = 8.70 Hz, 1H, aromatic) ppm; the 13C
NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (APCI): m/z found 424.87924 [M −H]−, calculated for C14H7O4N2Br2 [M − H]−: 424.87671.
Dimethyl 6,9-dibromo-7,8-dihydroxyphenazine-2,3-dicarboxylate (4). A solution of bromanilic acid (1.28 g, 4.29 mmol) and
1 (0.961 g, 4.29 mmol) in ethanol (350 mL) was stirred at 80 °C
overnight. After the disappearance of the reactants, the reaction mixture was cooled to room temperature, and the precipitate was filtered and washed with a small amount of ethanol
to obtain 4 as a red solid (1.59 g, 76% yield); m.p. over 300 °C;
IR (ATR): ν̃ = 3204, 2951, 1701, 1649, 1622, 1611, 1587, 1572,
1557, 1508 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 8.36 (br,2H, aromatic), 3.89 (s, 6H, CH3) ppm; the 13C NMR spectrum
could not be obtained because of the low solubility in any solvents; HRMS (APCI): m/z found 484.89825 [M + H]+, calculated
for C16H11O6N2Br2 [M + H]+: 484.89784.
Methyl 6,9-dibromo-7,8-bis(methoxymethoxy)phenazine-2carboxylate (5). A solution of sodium hydride abt. 60% oil suspension (328 mg, 20 eq.) and 3 (172 mg, 0.404 mmol) in THF(100 mL) was stirred at 0 °C for 30 min. Then, chloromethyl
methyl ether (608 µL, 8.08 mmol) was added to the solution,and the mixture was stirred at 0 °C overnight. After concentrating under reduced pressure, the residue was chromatographedon silica gel (ethyl acetate/dichloromethane = 1/3 as an eluent)
to obtain 5 as a yellow solid (155 mg, 75% yield);m.p. 160–161 °C; IR (ATR): ν̃ = 2924, 2839, 1709, 1591 cm−1; 1H
NMR (500 MHz, CDCl3): δ = 9.11 (s, 1H, aromatic), 8.45–8.39(m, 2H, aromatic), 5.45–5.44 (s, 4H, CH2), 4.05 (s, 3H, CH3),
3.75 (s, 6H, CH3) ppm; the 13C NMR spectrum could not be
obtained because of the low stability in CDCl3 and acetone-d6;

Org. Biomol. Chem.

Organic & Biomolecular Chemistry
HRMS (APCI): m/z found 536.92700 [M + Na]+, calculated for
C18H16O6N2Br2Na [M + Na]+: 536.92673.
Dimethyl 6,9-dibromo-7,8-bis(methoxymethoxy)phenazine2,3-dicarboxylate (6). A solution of sodium hydride abt. 60%
oil suspension (307 mg, 15 eq.) and 4 (250 mg, 0.514 mmol) in
dry DMF (250 mL) was stirred at 0 °C for 20 min under a N2
atmosphere. Then, bromomethyl methyl ether (610 µL,
7.71 mmol) was added to the solution, and the mixture wasstirred at 0 °C for 90 min. The reaction was quenched withwater, and then the solution was extracted with ethyl acetate
and hexane (=1/4). After concentrating under reduced pressure,
the residue was chromatographed on silica gel (ethyl acetate/dichloromethane = 1/3 as an eluent) to obtain 6 as a yellow
solid (123 mg, 42% yield); m.p. 166–168 °C; IR (ATR): ν̃ = 2953,
1722 cm−1; 1H NMR (500 MHz, CDCl3): δ = 8.78 (s, 2H, aromatic), 5.46 (s, 4H, CH2), 4.02 (s, 6H, CH3), 3.75 (s, 6H, CH3)
ppm; the 13C NMR (125 MHz, CDCl3) δ = 166.96, 153.54,
142.60, 141.08, 133.27, 131.80, 117.03, 100.47, 58.96,
53.25 ppm; HRMS (ESI): m/z found 572.95044 [M + H]+, calculated for C20H19O8N2Br2 [M + H]+: 572.95027.
Methyl7,8-bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)thiophen-2-yl)phenazine-2-carboxylate (7). A solution of 5 (235 mg, 0.455 mmol), 2 (567 mg,
1.51 mmol), and Pd(PPh3)4 (89 mg, 0.0770 mmol) in toluene(110 mL) was stirred at 110 °C for 24 h. After concentrating
under reduced pressure, the residue was chromatographed on
silica gel (ethyl acetate/hexane = 1/3 as an eluent) to obtain 7
as a red solid (292 mg, 82% yield); m.p. 104–106 °C; IR (ATR):ν̃ = 2955, 2872, 1726, 1622, 1580, 1537 cm−1; 1H NMR(500 MHz, CDCl3): δ = 8.87 (s, 1H, aromatic), 8.38 (d, J = 1.90
Hz, 1H, aromatic), 8.30 (d, J = 1.90 Hz, 1H, aromatic), 7.81(d, J= 3.75 Hz, 1H, aromatic), 7.78 (d, J = 3.75 Hz, 1H, aromatic),
7.37–7.20 (m, 2H, aromatic), 6.31 (s, 1H, CH), 6.30 (s, 1H, CH),
5.30 (s, 4H, CH2), 4.03 (s, 3H, CH3), 3.30 (s, 6H, CH3),
1.36–1.33 (m, 24H, CH3) ppm; 13C NMR (125 MHz, CDCl3): δ =166.63, 151.96, 151.27, 146.58, 146.50, 143.18, 141.24, 140.96,
140.83, 133.88, 133.85, 133.03, 131.31, 131.28, 131.20, 129.97,
129.09, 125.26, 125.22, 125.05, 124.83, 99.80, 99.76, 97.20,
97.18, 83.28, 83.26, 58.06, 58.05, 52.74, 24.37, 24.34, 22.28,
22.27 ppm; HRMS (ESI): m/z found 801.24866 [M + Na]+, calculated for C40H46O10N2NaS2 [M + Na]+: 801.24861.
Dimethyl 7,8-bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)thiophen-2-yl)phenazine-2,3-dicarboxylate (8). A solution of 6 (55.0 mg, 0.0960 mmol), 2 (144 mg,
0.380 mmol), and Pd(PPh3)4 (50 mg, 0.0428 mmol) in toluene(50 mL) was stirred at 110 °C for 24 h. After concentrating
under reduced pressure, the residue was chromatographed on
silica gel (ethyl acetate/dichloromethane = 1/3 as an eluent) toobtain 8 as a red solid (71.0 mg, 89% yield); m.p. 61–63 °C; IR(ATR): ν̃ = 2978, 1728 cm−1; 1H NMR (500 MHz, acetone-d6): δ =8.56 (s, 2H, aromatic), 7.81–7.75 (m, 2H, aromatic), 7.35–7.29(m, 2H, aromatic), 6.31 (s, 2H, CH), 5.31 (s, 4H, CH2), 3.98 (s,6H, CH3), 3.30 (s, 6H, CH3), 1.34 (m, 24H, CH3) ppm; 13C NMR(125 MHz, acetone-d6): δ = 167.61, 153.49, 148.20, 142.63,
142.05, 134.31, 133.61, 132.28, 132.21, 126.01, 125.72, 100.77,
97.81, 83.75, 58.18, 53.39, 24.70, 22.53 ppm; HRMS (APCI): m/zThis journal is © The Royal Society of Chemistry 2023

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Organic & Biomolecular Chemistryfound 837.27246 [M + H]+, calculated for C42H49O12N2S2 [M +H]+: 837.27214.
7,8-Bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3dioxolan-2-yl)thiophen-2-yl)phenazine-2-carboxylic acid (9). A
solution of 9 (106 mg, 0.136 mmol) and NaOH (100 mg) in the
mixture of methanol (85 mL), water (17 mL), and THF (10 mL)
was stirred at 40 °C for 3 h. After concentrating under reducedpressure, the residue was neutralized with 1 N HCl aq. andextracted into dichloromethane to obtain 9 as a red solid(97 mg, 93% yield); m.p. 211–212 °C; IR (ATR): ν̃ = 2976, 2926,
2851, 1717, 1699, 1653, 1558, 1541, 1506 cm−1; 1H NMR(500 MHz, acetone-d6): δ = 8.91 (dd, J = 1.90 Hz and 0.55 Hz,
1H, aromatic), 8.42 (dd, J = 9.00 Hz and 1.90 Hz, 1H, aromatic),
8.31 (dd, J = 9.00 Hz and 0.61 Hz, 1H, aromatic), 7.82 (d, J =3.70 Hz, 1H, aromatic), 7.78 (d, J = 3.70 Hz, 1H, aromatic),
7.34–7.27 (m, 2H, aromatic), 6.31 (s, 1H, CH), 6.30 (s, 1H, CH),
5.30 (s, 4H, CH2), 3.31 (s, 6H, CH3), 1.38–1.29 (m, 24H, CH3)
ppm; the 13C NMR spectrum could not be obtained because of
the low solubility in any solvents; HRMS (ESI): m/z found765.25153 [M + H]+, calculated for C39H45O10N2S2 [M + H]+:765.25101.
7,8-Bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3dioxolan-2-yl)thiophen-2-yl)phenazine-2,3-dicarboxylic
acid(10). A solution of 8 (110 mg, mmol) and NaOH (100 mg) in
the mixture of methanol (100 mL), water (20 mL), and THF(10 mL) was stirred at 40 °C for 6 h. After concentrating under
reduced pressure, the residue was neutralized with 1 N HCl aq.
and extracted into dichloromethane to obtain 10 as a red solid(63 mg, 59% yield); m.p. over 300 °C; IR (ATR): ν̃ = 2976, 2924,
2850, 1728, 1605, 1541, 1508 cm−1; 1H NMR (500 MHz,
acetone-d6): δ = 9.24 (s, 2H, aromatic), 7.81 (d, J = 3.75 Hz, 2H,
aromatic), 7.30 (d, J = 3.70 Hz, 2H, aromatic), 6.30 (s, 2H, CH),
5.29 (s, 4H, CH2), 3.31 (s, 6H, CH3), 1.37–1.29 (m, 24H, CH3)
ppm; the 13C NMR spectrum could not be obtained because of
the low solubility in any solvents; HRMS (ESI): m/z found807.22552 [M − H]−, calculated for C40H43O12N2S2 [M − H]−:
807.22519.
6,9-Bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2-carboxylic acid (KY-1). A solution of 9 (175 mg) and 1 N HCl aq.
(0.5 mL) in the mixture of THF (100 mL) and water (100 mL)
was stirred at 70 °C overnight. The precipitate was filtered andwashed with hexane to obtain KY-1 as a black solid (70 mg,
64% yield); m.p. over 300 °C; IR (ATR): ν̃ = 3505, 3159, 2961,
2799, 2737, 2349, 2085, 1996, 1989, 1703, 1643, 1614, 1557,
1520, 1508 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 9.97 (s,1H, CHO), 9.93 (s, 1H, CHO), 8.96 (s, 1H, aromatic), 8.76 (d, J =3,20 Hz, aromatic), 8.71–8.70 (m, 1H, aromatic), 8.19–8.16 (m,2H, aromatic), 8.04–8.00 (m, 2H, aromatic) ppm; the 13C NMR
spectrum could not be obtained because of the low solubility
in any solvents; HRMS (APCI): m/z found 477.02100 [M + H]+,calculated for C23H13O6N2S2 [M + H]+: 477.02095.
6,9-Bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2,3dicarboxylic acid (KY-2). A solution of 10 (63.0 mg, mmol) and1 N HCl aq. (2 mL) in the mixture of THF (50 mL) and water(50 mL) was stirred at 70 °C overnight. The precipitate was filtered and washed with hexane to obtain KY-2 as a black solidThis journal is © The Royal Society of Chemistry 2023

Paper
(8.00 mg, 20% yield); m.p. over 300 °C; IR (ATR): ν̃ = 3198,
2376, 2347, 2160, 1726, 1701, 1697, 1678, 1655, 1612, 1595,
1535 cm−1; 1H NMR (500 MHz, DMSO-d6): δ = 9.95 (s, 2H,
CHO), 8.91–8.87 (m, 2H, aromatic), 8.34 (s, 2H, aromatic), 8.01
(d, J = 4.25 Hz, 2H, aromatic) ppm; 13C NMR spectrum could
not be obtained because of the low solubility in any solvents;
HRMS (ESI): m/z found 518.99603 [M − H]−, calculated for
C24H11O8N2S2 [M − H]−: 518.99513.
Sodium 6,9-bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2-carboxylate (KY-1Na). A solution of KY-1 (42 mg,
0.881 mmol) and 1 N NaOH aq. (70 µL) in water (100 mL) was
stirred at room temperature for 10 min and concentrated toobtain KY-1Na as a red solid (46% yield); m.p. over 300 °C; IR(ATR): ν̃ = 3200, 2303, 2110, 1994, 1846, 1578 cm−1; 1H NMR(500 MHz, D2O): δ = 9.51 (s, 1H, CHO), 9.36 (s, 1H, CHO), 8.71
(s, 1H, aromatic), 8.59 (s, 1H, aromatic), 8.43 (s, 1H, aromatic),
8.00 (d, J = 8.62 Hz, 1H, aromatic), 7.93 (d, J = 8.50 Hz, 1H, aromatic), 7.84 (s, 1H, aromatic), 7.68 (s, 1H, aromatic) ppm; the13
C NMR spectrum could not be obtained because of the low
solubility in any solvents; HRMS (ESI): m/z found 475.00620 [M− Na]−, calculated for C23H11O6N2S2 [M − Na]−: 475.00530.
Sodium 6,9-bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2,3-dicarboxylate (KY-2Na). A solution of KY-2 (8.00 mg,
0.0154 mmol) and 1 N NaOH aq. (31.0 µL) in water (50 mL)
was stirred at room temperature for 10 min and concentrated
to obtain KY-2Na as a red solid (10 mg, quant.); m.p. over
300 °C; IR (ATR): ν̃ = 3318, 1560 cm−1; 1H NMR (500 MHz,D2O): δ = 9.84 (s, 1H, CHO), 9.78 (s, 1H, CHO), 8.45 (m, 1H,
aromatic), 8.39 (m, 1H, aromatic), 8.27 (s, 1H, aromatic), 8.14
(s, 1H, aromatic), 8.03 (s, 2H, aromatic) ppm; the 13C NMR
spectrum could not be obtained because of the low solubility
in any solvents; HRMS (APCI): m/z found 518.99615 [M + H −2Na]−, calculated for C24H11O8N2S2 [M + H − 2Na]−:
518.99513.
n-Octanol/water partition coeﬃcient
The log Po/w values were determined from the concentration ofPSs partitioned into water (100 mL) and n-octanol (100 mL)
and the concentration of PSs partitioned into each phase.
Evaluation of the 1O2 quantum yield
1

O2 generation by phenazine-2,3-diol-based PSs KO-2, KY-1,
KY-2, KY-1Na, and KY-2Na was investigated by monitoring thechanges in the photoabsorption spectra of DPBF in the DMSO
solution of each PS and ABDA in aqueous solution (D2O/DMSO 99 : 1, v/v) of KY-1Na and KY-2Na under photoirradiation. DMSO and water were bubbled with air for 15 min prior
to preparing solutions. Air-saturated DMSO containing DPBFor aqueous solutions containing ABDA and each PS were irradiated with monochromatic light at 532 nm (300 μW cm−2)that was obtained by passage of a xenon light source(HAL-320, Asahi Spectra) through a monochromator (CMS-100,
Asahi Spectra). The concentration of DPBF and ABDA was 5 ×
10−5 M in air-saturated DMSO or aqueous solutions. The concentration of the PSs and RB was adjusted so that the absorbance was ca. 0.03 at an irradiation wavelength of 532 nm.

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Except for nonirradiated solutions, each spectrum wasmeasured immediately after photoirradiation for 1 min. The
procedure using DPBF and ABDA was promptly repeated until
the total photoirradiation time reached 10 min and 60 min,respectively. The changes in the optical density (ΔOD) of DPBF
and ABDA were plotted against the photoirradiation time toobtain the slopes (m). The ΦΔ values of KO-2, KY-1, KY-2,
KY-1Na, and KY-2Na were determined using the relative
method using RB (ΦΔ = 0.76 in DMSO9a and 0.75 in water9b) as
a standard according to the following equation:
ΦΔsam ¼ ΦΔref  ½ðmsam =mref Þ  ðLref =Lsam Þ
where ΦΔsam and ΦΔref are the 1O2 quantum yields of phenazine-2,3-diol-based PSs and RB, respectively, msam and mref areslopes in the plots of ΔOD at the photoabsorption maximum
wavelength of DPBF (415 nm) or ABDA (400 nm) against thephotoirradiation time, and Lsam and Lref are light harvesting
eﬃciencies, which are given by L = 1–10−A (“A” is the absorbance at the photoirradiation wavelength).
Evaluation of the cellular uptake of KY-2Na
Colon26 cells were seeded on glass bottom dishes at a densityof 1 × 105 cells per well and incubated overnight. The cells
were treated with KY-2Na for 24 h. The medium was collected
and the fluorescence spectrum was recorded. Then, the cells
were observed under a confocal laser scanning microscope(LSM700, Carl Zeiss, Germany).
Photodynamic activity against cancer cell lines
Colon26 cells were seeded on 48-well plates (Thermo Fischer
Science) at 1.71 × 104 cells (N = 3) and incubated overnight(approximately 18 h). The cells were exposed to KY-1Na or
KY-2Na at varying concentrations. After 24 h of incubation, thecells were washed with PBS thrice, and photoirradiation(>510 nm, 9 mW cm−2) was carried out for 30 min. Afterward,
the cells were incubated for 24 h, and the Cell Counting Kit-8
solution was added to the cells. After 30 min of incubation,
the absorbance at 450 nm was measured using a microplate
reader.
Theoretical calculations
The Gaussian 16 program21 was used for density functional
theory (DFT) calculations and time-dependent DFT (TD-DFT)
calculations. DFT and TD-DFT calculations at the B3LYP/6311G(d,p)/DMSO-IEFPCM level for KO-2, KY-1, and KY-2, theB3LYP/6-311+G(d,p)/DMSO-IEFPCM level for KY-1Na and
KY-2Na in carboxylate anion forms, and the B3LYP/6-311+G(d,p)/Water-IEFPCM level for KY-1Na and KY-2Na in carboxylateanion forms were performed. Geometrical optimizations of theS0 state at each calculation level were performed with frequency calculations. There are no imaginary frequencies for all
optimized structures. The TD-DFT calculations of both excited
singlet and triplet states were performed using optimized S0
geometries.

Org. Biomol. Chem.

Conﬂicts of interest
There are no conflicts to declare.

Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research (B) from the Japan Society for the Promotion of
Science (JSPS) KAKENHI Grant Number 22H02123 and by The
Futaba Foundation.

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